Establishing Suspension Cell Cultures for Improved Manufacturing of Oncolytic Adenovirus.

Recent clinical trials have shown the potential of oncolytic adenoviruses as a cancer immunotherapy. A successful transition of oncolytic adenovirus to clinical applications requires efficient and good manufacturing practice compatible production and purification bioprocesses. Suspension cultures are preferable for virus production as they can reduce process costs and increase product quality and consistency. This work describes the adaptation of the A549 cell line to suspension culture in serum-reduced medium validated by oncolytic adenovirus production in stirred tank bioreactor. Cell concentrations up to 3 × 106 cells mL-1 are obtained during the production process. At harvest 1.4 × 1010 infectious particles mL-1 and 6.9 ± 1.1 × 1010 viral genome mL-1 are obtained corresponding to a viral genome: infectious particles ratio of 5.2 (± 1.9): 1 confirming the virus quality. Overall, the suspension characteristics of these A549 cells support an easily scalable, less time-consuming, and more cost-effective process for expanded success in the use of oncolytic viruses for cancer therapy.

Scalable Culture Strategies for the Expansion of Patient-Derived Cancer Stem Cell Lines.

Cancer stem cells (CSCs) have recently raised great interest as a promising biological system for designing effective cancer therapies. The scarcity of CSCs in vivo and the consequent low numbers obtained from biopsies represent a major hurdle to the development of such strategies. It is therefore necessary to design robust scalable methods to enable efficient expansion of bona fide CSCs in vitro. Here, we evaluated the applicability of computer-controlled bioreactors combined with 3D aggregate culture and microcarrier technology, widely used in stem cell bioprocessing, for the expansion and enrichment of CSCs isolated from different types of solid tumors-colorectal cancer (CRC) and non-small-cell lung cancer (NSCLC) from two patients. Results show that these culture strategies improved cell expansion and CSC enrichment. Both patient-derived CSC lines were able to grow on microcarriers, the best results being achieved for PPlus 102-L, Pro-F 102-L, Fact 102-L, and CGEN 102-L beads (5-fold and 40-fold increase in total cell concentration for CRC and NSCLC cells, respectively, in 6 days). As for 3D aggregate culture strategy, the cell proliferation profile was donor dependent. NSCLC cells were the only cells able to form aggregates and proliferate, and the flat-bottom bioreactor vessel equipped with a trapezoid-shaped paddle impeller was the most efficient configuration for cell growth (21-fold increase in cell concentration achieved in 8 days). Serum-free medium promotes CSC enrichment in both 3D aggregate and microcarrier cultures. The protocols developed herein for CSC expansion have the potential to be transferred to clinical and industrial settings, providing key insights to guide bioprocess design towards the production of enriched CSC cultures in higher quantity and improved quality.

Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

INTRODUCTION: Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. METHODS: In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. RESULTS: Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. DISCUSSION: The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates.

Impact of Adenovirus infection in host cell metabolism evaluated by (1)H-NMR spectroscopy.

Adenovirus-based vectors are powerful vehicles for gene transfer applications in vaccination and gene therapy. Although highly exploited in the clinical setting, key aspects of the adenovirus biology are still not well understood, in particular the subversion of host cell metabolism during viral infection and replication. The aim of this work was to gain insights on the metabolism of two human cell lines (HEK293 and an amniocyte-derived cell line, 1G3) after infection with an adenovirus serotype 5 vector (AdV5). In order to profile metabolic alterations, we used (1)H-NMR spectroscopy, which allowed the quantification of 35 metabolites in cell culture supernatants with low sample preparation and in a relatively short time. Significant differences between both cell lines in non-infected cultures were identified, namely in glutamine and acetate metabolism, as well as by-product secretion. The main response to AdV5 infection was an increase in glucose consumption and lactate production rates. Moreover, cultures performed with or without glutamine supplementation confirmed the exhaustion of this amino acid as one of the main causes of lower AdV5 production at high cell densities (10- and 1.5-fold less specific yields in HEK293 and 1G3 cells, respectively), and highlighted different degrees of glutamine dependency of adenovirus replication in each cell line. The observed metabolic alterations associated with AdV5 infection and specificity of the host cell line can be useful for targeted bioprocess optimization.

Human amniocyte-derived cells are a promising cell host for adenoviral vector production under serum-free conditions.

Recombinant adenovirus vectors (AdVs) have been used for the development of vaccines, as gene therapy vectors and for protein production. Currently, the production of clinical grade batches of recombinant E1-deleted adenovirus type 5 vectors is performed using human-derived HEK293 or PER.C6(®) cell lines. In this work we describe the generation of a new human amniocyte-derived cell line named 1G3 and show that it can be used as a very promising cell host for AdV production in serum-free conditions, allowing for production in high cell density cultures and avoiding the typical cell density effect observed for HEK293. By design, this cell line makes the generation of replication-competent adenovirus during production of E1-deleted AdVs very unlikely. The impact of the culture system (static versus agitated) and AdV infection parameters such as multiplicity of infection, time of harvesting and cell concentration at infection were evaluated and compared with HEK293. Using stirred tanks bioreactors, it was possible to grow 1G3 cells to cell densities of up to 9 × 10(6) cells/mL using serum-free media. Moreover, without a medium exchange step at infection, a three-fold increase in AdV volumetric titers was obtained, as no cell density effect was observed at CCI 3. Overall, our results clearly demonstrate the potential of the human amniocyte-derived newly established cell line 1G3 for AdV production in a serum-free scalable process, paving the way for further process improvements based on fed-batch or perfusion strategies.

Testing a new formulation for Peste des Petits Ruminants vaccine in Ethiopia.

In this paper extended tests on a new candidate formulation for Peste des Petits Ruminants (PPR) vaccine carried out at National Veterinary Institute (NVI) in Ethiopia are presented. This work was performed in the frame of the VACNADA project from GALVmed which aimed at procuring vaccines against neglected veterinary diseases to African vaccine producing laboratories, in particular PPR. After the eradication of Rinderpest, Peste des Petits Ruminants became the next veterinary disease on target for elimination, requiring an effective and thermostable vaccine. In this work a Tris/Trehalose formulation was evaluated in thermal stability studies in comparison to the current used formulation of the live attenuated PPR vaccine, the Weybridge medium. The extended results presented herein show an increased thermal stability of the vaccine, especially at 37 and 45 °C, as expected from previously published results (Silva A.C. et al., 2011). Furthermore, during the course of this project, the NVI teams have clearly demonstrated ability to produce higher quality PPR vaccines after a successful transfer of the technology. These results should significantly enhance the utility of the vaccine in the eradication of PPR.

Scalable production of adenovirus vectors.

Recombinant adenoviruses (AdV) are highly efficient at gene transfer for a broad spectrum of cell types and species. They became one of the vectors of choice for gene delivery and expression of foreign proteins in gene therapy and vaccination purposes. To meet the need of significant amounts of adenoviral vectors for preclinical and possibly clinical uses, scalable and reproducible production processes are required.In this chapter, we review processes used for scalable production of two types of first generation (E1-deleted) adenoviral vectors (Human and Canine) using stirred tank bioreactors. The production of adenovirus vectors using either suspension (HEK 293) or anchorage-dependent cells (MDCK-E1) are described to exemplify scalable production processes with different cell-culture types. The downstream processes will be covered in the next chapter.

¹H-NMR protocol for exometabolome analysis of cultured mammalian cells.

(1)H-Nuclear magnetic resonance ((1)H-NMR) spectroscopy is a powerful technique to analyze the composition of complex mixtures based on the particular proton fingerprint of each molecule. Here we describe a protocol for exometabolome analysis of mammalian cells using this technique, including sample preparation, spectra acquisition, and integration. The potential of this technique is exemplified by application to cultures of a Chinese hamster ovary (CHO) cell line. The average error associated to this method is under 3% and the limit of quantification for all metabolites analyzed is below 180 µM.

Acoustic detection of cell adhesion to a coated quartz crystal microbalance - implications for studying the biocompatibility of polymers.

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly-D-lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL-coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)-binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly-Arg-Gly-Asp-Ser (GRGDS) peptides were used to interfere with cell-ECM binding motifs onto FN-coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.

Acoustic detection of cell adhesion on a quartz crystal microbalance.

An acoustic quartz crystal microbalance (QCM) was used to signal and follow the cell­adhesion process of epithelial cells [human embryonic kidney(HEK) 293T and cervical cancer (HeLa) and fibroblasts [African Green Monkey kidney cells (COS-7)] onto gold surfaces. Cells were applied on the sensor and grown under serum-free and serum-supplemented culture media. The sensor resonance frequency (Δf) and motional resistance (ΔR) variations were measured during cell growth to monitor cell adhesion processes. Fingerprints of the adhesion processes, generated using the QCM signal, were found to be specific for each cell type while enabling the identification of the phases of the adhesion process. Under serum-free conditions, the deposition of HEK 293T and HeLa cells was characterized by a decrease of Δf with constant ΔR, whereas for COS­7 cells, this initial deposition was signaled by variations of ΔR at constant Δf. Toward the end of the adhesion process, fingerprints were characterized by a continuous increase of ΔR consistent with the increase in viscoelasticity. The morphology of adherent cells was visualized by fluorescent microscopy, enabling the association of the cell morphology with QCM signals.

Strategies for improved stability of Peste des Petits Ruminants Vaccine.

The main focus of this work was the improvement of the stability of the current PPRV vaccine. First, new formulations based on the Tris buffer were tested, with and without the addition of sucrose and trehalose and compared with the formulation normally used to stabilize the vaccine, the Weybridge medium. The results show a virus half-life of 21 h at 37°C and 1 month at 4°C for the Tris/trehalose liquid formulation and, in the lyophilized form, the formulation was able to maintain the viral titer above the 1 × 10(4) TCID(50)/mL (>10 doses/mL) for at least 21 months at 4°C (0.6 log lost), 144 h at 37°C (0.6 log lost) and 120 h at 45°C (1 log lost). Secondly, a strategy based on culture medium composition manipulation aiming at improving the intrinsic PPRV vaccine stability was also evaluated. The addition of 25 mM fructose resulted in a higher virus production (1log increase) with higher stability (2.6-fold increase compared to glucose 25 mM) at 37°C. Increased concentrations of NaCl, improved virus release, reducing the cell-associated fraction of the virus produced. Moreover this harvesting strategy is scalable and more suitable for a larger scale production than the freeze/thaw cycles normally used. The information gathered in this work showed that it is possible for the PPRV vaccine to have adequate short-term stability at non-freezing temperatures to support manufacturing, short-term shipping and storage. The identification of a more stable formulation should significantly enhance the utility of the vaccine in the control of a PPRV outbreak.

Adenovirus vector production and purification.

Replication deficient adenovirus vectors are frequently used tools for the delivery of transgenes in vitro and in vivo. In addition, several therapeutic products based on adenovirus are under clinical development. This review outlines adenovirus vector production discussing different vector types, available production cell lines and state of the art of production process development and purification.

Fermentation of deproteinized cheese whey powder solutions to ethanol by engineered Saccharomyces cerevisiae: effect of supplementation with corn steep liquor and repeated-batch operation with biomass recycling by flocculation.

The lactose in cheese whey is an interesting substrate for the production of bulk commodities such as bio-ethanol, due to the large amounts of whey surplus generated globally. In this work, we studied the performance of a recombinant Saccharomyces cerevisiae strain expressing the lactose permease and intracellular beta-galactosidase from Kluyveromyces lactis in fermentations of deproteinized concentrated cheese whey powder solutions. Supplementation with 10 g/l of corn steep liquor significantly enhanced whey fermentation, resulting in the production of 7.4% (v/v) ethanol from 150 g/l initial lactose in shake-flask fermentations, with a corresponding productivity of 1.2 g/l/h. The flocculation capacity of the yeast strain enabled stable operation of a repeated-batch process in a 5.5-l air-lift bioreactor, with simple biomass recycling by sedimentation of the yeast flocs. During five consecutive batches, the average ethanol productivity was 0.65 g/l/h and ethanol accumulated up to 8% (v/v) with lactose-to-ethanol conversion yields over 80% of theoretical. Yeast viability (>97%) and plasmid retention (>84%) remained high throughout the operation, demonstrating the stability and robustness of the strain. In addition, the easy and inexpensive recycle of the yeast biomass for repeated utilization makes this process economically attractive for industrial implementation.

Acoustic wave biosensors: physical models and biological applications of quartz crystal microbalance.

Piezoelectric sensors are acoustic sensors that enable the selective and label-free detection of biological events in real time. These sensors generate acoustic waves and utilize measurements of the variation of the wave propagation properties as a signal for probing events at the sensor surface. Quartz crystal microbalance (QCM) devices, the most widespread acoustic resonators, allow the study of viscoelastic properties of matter, the adsorption of molecules, or the motility of living cells. In a tutorial-like approach, this review addresses the physical principles associated with the QCM, as well as the origin and effects of major interfering phenomena. Special attention is paid to the possibilities offered by QCM that go beyond microweighing, and important recent examples are presented.

Scalable culture systems using different cell lines for the production of Peste des Petits ruminants vaccine.

Peste des Petits ruminants (PPR) is considered as one of the major constraints to the productivity of small ruminants in Africa and Asian countries. Currently PPR control is done by vaccination with an attenuated PPR strain (Nigeria 75/1) produced in monolayers of Vero cells grown in roller bottles or static flasks. This work focuses on the production of a PPR vaccine strain using stirred conditions as an advanced option for process scale-up. Non-porous microcarriers (Cytodex-1) were used to support Vero cell growth in suspension cultures. The use of Ex-Cell medium could improve cell specific productivities obtained with standard serum containing medium, independently of the type of system used, i.e. static as well as suspension stirred cultures. As an alternative, several cell lines adapted to grow as single cells in suspension (CHO-K1, BHK-21A and 293) and another anchorage-dependent (MRC-5) were evaluated in their capacity to produce a PPR vaccine. BHK-21A and 293 cells grown as single-cell suspension in serum free medium were both suited to produce PPR vaccine with productivities similar to Vero cells, namely 10(6)TCID(50)/mL. However, for the 293 cells, these results were only obtained 2-3 days later. CHO-K1 and MRC-5 cells have shown not to be suitable to adequately produce this virus. These results provide further insights into the feasibility of applying microcarrier cell culture technology to produce PPR vaccine in Vero cells as well as in the alternative use of single-cell suspension cultures of BHK-21A, significantly simplifying the existing production process.

Efeitos da exposição do etanol e da desnutrição sobre o córtex visual durante o desenvolvimento perinatal / Ethanol exposure and malnutrition effects on visual cortex during perinatal development

Este trabalho tem por objetivo avaliar os efeitos do etanol e da desnutrição sobre o peso corporal e encefálico, bem como as alterações histológicas do córtex visual. Ratos machos Wistar foram gerados e amamentados por matrizes submetidas a duas dietas: (1) padrão do biotério (“Labina®”- 23 por cento de proteína, grupo N); e (2) hipoprotéica (“Dieta Básica Regional” – DBR - 8 por cento de proteína, grupo D). Cada grupo foi subdividido em dois, conforme o tratamento (gavagem) com água ou etanol (E), originando quatro grupos (N, D, E e ED). Evolução ponderal - períodos avaliados: 30 (P3), 250 (P25) e 400 (P40) dias pós-natais. Os cortes histológicos encefálicos foram corados com HE, Tricrômico de Masson, Ácido Periódico de Schiff e Van Gienson. Em P3, os grupos E, D e ED tinham pesos menores que N. Em P25 e P40, ED apresentou peso menor que N, D e E. O grupo D apresentou peso médio menor que o de N nos 3 períodos. Para o peso encefálico (P40) houve diferença entre ED e D e entre D e N. Para a densidade de vasos, não houve diferença significativa entre todos os grupos. Não havia depósitos de fibras colágenas e (ou) elásticas na neurópila. N apresentou maior número de células PAS+ que os outros grupos e com distribuição regular. Conclui-se que as condições experimentais promovem redução de peso corporal e encefálico no período pós-natal precoce e no córtex visual, mas não há diferença na densidade vascular.

Encapsulation of adenoviral vectors into chitosan-bile salt microparticles for mucosal vaccination.

The objective of this study is the incorporation of adenoviral vectors into a microparticulate system adequate for mucosal delivery. Microencapsulation of the vectors was accomplished by ionotropic coacervation of chitosan, using bile salts as counter-anion. The process was optimized in order to promote high encapsulation efficiency, with a minimal loss of viral infectivity. The maintenance of sterility during all the encapsulation procedure was also taken into account. The principle relies on the simple addition of a solution containing adenoviral vectors to a solution of neutralized chitosan, under stirring. Some surfactants were added to the chitosan solution, to improve the efficiency of this process, such as Tween 80, and Pluronic F68 at 1% (w/v). Encapsulation efficiency higher than 84% was achieved with formulations containing sodium deoxycholate as counter-anion and Pluronic F68 as dispersant agent. The infectivity of the adenoviral vectors incorporated into microparticles was assessed by release assays in PBS and by direct inoculation in 293 and Caco-2 cells. The release in aqueous media was negligible but, when in contact with monolayers of the cells, an effective release of bioactive adenovirus was obtained. Our work shows that encapsulation in microparticles, not only appear to protect the adenovirus from the external medium, namely from low pH, but can also delay their release that is fully dependent on cell contact, an advantage for mucosal vaccination purposes. The formulations developed are able to maintain AdV infectivity and permit a delayed release of the bioactives that is promoted by digestion in situ of the microparticles by the cell monolayers. The onset of delivery is, that way, host-controlled. In view of these results, these formulations showed good properties for mucosal adenovirus delivery.

Screening of novel excipients for improving the stability of retroviral and adenoviral vectors.

In the past decade there has been an increase in the application of viral vectors in the laboratory and clinical trials of human gene therapy, retroviral and adenoviral vectors among the most used. However, the limited stability of these vectors creates problems in the design of experiments, transport, and storage. Vectors stored at -80 degrees C must be quickly shipped on dry ice, which is somewhat cumbersome. Alternatively, viral vectors can be preserved in a lyophilized form. However, loss of viral activity during lyophilization can also be a serious problem. In this report we identify novel candidate formulations containing new compatible solutes, ectoin, hydroxyectoin, and firoin, that allow better stability of retroviral and adenoviral vectors during storage. For retroviral vectors, the maximum stabilization for long-term storage was achieved through lyophilization followed by storage at -20 degrees C using a formulation of Tris buffer pH 7.2 containing firoin (0.5 M), a half-life of 340 days being obtained. Adenoviral vectors storage at -80 degrees C in solution using Tris buffer pH 8.0 with firoin was the best method for long-term storage, with a half-life exceeding 1 year.

Criptococose pulmonar: aspectos na tomografia computadorizada / Pulmonary cryptococcosis: computed tomography findings

A criptococose pulmonar é uma doença causada pelo Criptococcus neoformans, um fungo unimórfico que possui distribuição mundial, existindo na mesma forma tanto no seu habitat natural quanto em animais e humanos. A doença possui apresentações clínica e patológica variáveis e pode manifestar-se tanto em pacientes com a imunidade normal como em imunocomprometidos, que representam a maioria dos casos. Neste trabalho são analisados os aspectos encontrados nas tomografias computadorizadas do tórax de 14 pacientes com criptococose pulmonar confirmada. Os achados mais freqüentes na tomografia do tórax foram as massas e os nódulos pulmonares. Outros aspectos observados foram as áreas de escavação, as consolidações, o espessamento do interstício peribroncovascular e o reticulado difuso. Massa pulmonar foi o achado isolado mais comum (64,2 por cento), seguido dos nódulos isolados ou múltiplos (35,7 por cento). Doença pulmonar difusa foi vista em apenas 14,2 por cento dos casos. Os lobos superiores foram os mais comprometidos, sendo a doença mais comum nas regiões anteriores. A tomografia do tórax permitiu avaliar com precisão o grau de comprometimento do parênquima pulmonar.

Pulmonary cryptococcosis is caused by Cryptococcus neoformans, a world-wide distribution unimorphic fungus that exhibits the same form in its natural habitat, animals and humans. The disease presents variable clinical and pathologic features and occurs in immunocompetent and immunocompromised patients. We reviewed the computed tomography findings of 14 patients with confirmed pulmonary cryptococcosis. The most frequent computed tomography findings were masses and pulmonary nodules. Other abnormalities included cavities, areas of consolidation, peribronchovascular interstitial thickening and diffuse linear opacities. Pulmonary masses were the most frequent findings (64.2%), followed by nodules (35.7%) whereas diffuse pulmonary disease accounted for 14.2% of the cases. The most affected areas were the upper lobes and the anterior regions. Computed tomography scan of the chest allows a precise assessment of pulmonary parenchymal involvement in patients with pulmonary cryptococcosis.

Histoplasmose pulmonar aguda: relato de uma microepidemia / Acute pulmonary histoplasmosis: report of an outbreak

Os autores relatam uma microepidemia de histoplasmose pulmonar, com cinco crianças que desenvolveram a doença em um período de 7 a 14 dias após a limpeza de um forno desativado para produção de carvão vegetal. Todas apresentaram quadro de febre alta persistente, tosse seca, astenia e anorexia, com 28 dias de evolução. Quando buscaram atendimento médico, uma delas encontrava-se taquipnéica, febril, com hepatomegalia e palidez cutânea, estando as restantes em regular estado geral e já sem febre. As radiografias de tórax demonstravam, em todos os casos, infiltrados reticulonodulares grosseiros, difusos e bilaterais, além de linfonodomegalias hilares. As tomografias computadorizadas evidenciaram pequenos nódulos difusos, com distribuição aleatória, além das linfonodomegalias. Os diagnósticos foram confirmados por meio da imunodifusão em gel para Histoplasma capsulatum, que foi positiva em todas as amostras pareadas coletadas com 15 dias de intervalo. Apenas uma criança necessitou de internação, por causa de importante queda no estado geral, sendo realizado tratamento de suporte e observação. Todas as crianças evoluíram com melhora clínica, sem o uso de antifúngicos, e foram submetidas a tomografias de controle após cerca de 50 dias, que demonstraram importante regressão das lesões.

The authors report a pulmonary histoplasmosis outbreak occurring in five children after the cleaning of a deactivated coal furnace. The symptoms were high fever, dry cough, asthenia and anorexia. By the time parents seeked for medical care, only one child remained symptomatic with fever, tachypnea and hepatomegaly. All the patients had similar radiological findings. Chest films showed diffuse, bilateral, reticulonodular infiltrates and lymph node enlargement. Computed tomography showed small nodules with a random distribution and hilar and mediastinal lymph nodes. Diagnosis was confirmed by positive gel immunodiffusion for Histoplasma capsulatum. Only one patient needed to be hospitalized to receive supportive treatment. The use of antifungal agents was not necessary in any of the patients. After 50 days all patients were submitted to computed tomography studies that revealed significant remission of the lesions.