Juliana Fraga Vasconcelos

Effects of umbelliferone in a murine model of allergic airway inflammation

The therapeutic effects of umbelliferone (30, 60 and 90 mg/kg), a coumarin isolated from Typha domingensis (Typhaceae) were investigated in a mouse model of bronchial asthma. BALB/c mice were immunized and challenged by nasal administration of ovalbumin. Treatment with umbelliferone (60 and 90 mg/kg) caused a marked reduction of cellularity and eosinophil numbers in bronchoalveolar lavage fluids from asthmatic mice. In addition, a decrease in mucus production and lung inflammation were observed in mice treated with umbelliferone. A reduction of IL-4, IL-5, and IL-13, but not of IFN-gamma, was found in bronchoalveolar lavage fluids of mice treated with umbelliferone, similar to that observed with dexamethasone. The levels of ovalbumin-specific IgE were not significantly altered after treatment with umbelliferone. In conclusion, our results demonstrate that umbelliferone attenuates the alteration characteristics of allergic airway inflammation. The investigation of the mechanisms of action of this molecule may contribute for the development of new drugs for the treatment of asthma.

Reversion of gene expression alterations in hearts of mice with chronic chagasic cardiomyopathy after transplantation of bone marrow cells

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries, being associated with intense inflammatory response and fibrosis. We have previously shown that bone marrow mononuclear cell (BMC) transplantation improves inflammation, fibrosis, and ventricular diameter in hearts of mice with chronic Chagas disease. Here we investigated the transcriptomic recovery induced by BMC therapy by comparing the heart transcriptomes of control, chagasic, and BMC transplanted mice. Out of the 9390 unique genes quantified in all samples, 1702 had their expression altered in chronic chagasic hearts compared to those of normal mice. Major categories of significantly upregulated genes were related to inflammation, fibrosis and immune responses, while genes involved in mitochondrion function were downregulated. When BMC-treated chagasic hearts were compared to infected mice, 96% of the alterations detected in infected hearts were restored to normal levels, although an additional 109 genes were altered by treatment. Transcriptomic recovery, a new measure that considers both resotrative and side effects of treatment, was remarkably high (84%). Immunofluorescence and morphometric analyses confirmed the effects of BMC therapy in the pattern of inflammatory-immune response and expression of adhesion molecules. In conclusion, by using large-scale gene profiling for unbiased assessment of therapeutic efficacy we demonstrate immunomodulatory effects of BMC therapy in chronic chagasic cardiomyopathy and identify potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets.

Gene Expression Changes Associated with Myocarditis and Fibrosis in Hearts of Mice with Chronic Chagasic Cardiomyopathy

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries. About 30% of Trypanosoma cruzi-infected individuals develop this severe symptomatic form of the disease, characterized by intense inflammatory response accompanied by fibrosis in the heart. We performed an extensive microarray analysis of hearts from a mouse model of this disease and identified significant alterations in expression of approximately 12% of the sampled genes. Extensive up-regulations were associated with immune-inflammatory responses (chemokines, adhesion molecules, cathepsins, and major histocompatibility complex molecules) and fibrosis (extracellular matrix components, lysyl oxidase, and tissue inhibitor of metalloproteinase 1). Our results indicate potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets in chronic Chagas disease.

Transplantation of stem cells obtained from murine dental pulp improves pancreatic damage, renal function, and painful diabetic neuropathy in diabetic type 1 mouse model

Diabetes mellitus (DM) is one of the most common and serious chronic diseases in the world. Here, we investigated the effects of mouse dental pulp stem cell (mDPSC) transplantation in a streptozotocin (STZ)-induced diabetes type 1 model. C57BL/6 mice were treated intraperitoneally with 80 mg/kg of STZ and transplanted with 1 × 106 mDPSCs or injected with saline, by an endovenous route, after diabetes onset. Blood and urine glucose levels were reduced in hyperglycemic mice treated with mDPSCs when compared to saline-treated controls. This correlated with an increase in pancreatic islets and insulin production 30 days after mDPSC therapy. Moreover, urea and proteinuria levels normalized after mDPSC transplantation in diabetic mice, indicating an improvement of renal function. This was confirmed by a histopathological analysis of kidney sections. We observed the loss of the epithelial brush border and proximal tubule dilatation only in saline-treated diabetic mice, which is indicative of acute renal lesion. STZ-induced thermal hyperalgesia was also reduced after cell therapy. Three days after transplantation, mDPSC-treated diabetic mice exhibited nociceptive thresholds similar to that of nondiabetic mice, an effect maintained throughout the 90-day evaluation period. Immunofluorescence analyses of the pancreas revealed the presence of GFP+ cells in, or surrounding, pancreatic islets. Our results demonstrate that mDPSCs may contribute to pancreatic β-cell renewal, prevent renal damage in diabetic animals, and produce a powerful and long-lasting antinociceptive effect on behavioral neuropathic pain. Our results suggest stem cell therapy as an option for the control of diabetes complications such as intractable diabetic neuropathic pain.

Granulocyte colony-stimulating factor treatment in chronic Chagas disease: preservation and improvement of cardiac structure and function

This study investigates the effects of granulocyte colony‐stimulating factor (G‐CSF) therapy in experimental chronic chagasic cardiomyopathy. Chagas disease is one of the leading causes of heart failure in Latin America and remains without an effective treatment other than cardiac transplantation. C57BL/6 mice were infected with 103 trypomastigotes of Trypanosoma cruzi, and chronic chagasic mice were treated with G‐CSF or saline (control). Evaluations following treatment were functional, immunological, and histopathological. Comparing hearts of G‐CSF‐treated mice showed reduced inflammation and fibrosis compared to saline‐treated chagasic mice. G‐CSF treatment did not alter the parasite load but caused an increase in the number of apoptotic inflammatory cells in the heart. Cardiac conductance disturbances in all infected animals improved or remained stable due to the G‐CSF treatment, whereas all of the saline‐treated mice deteriorated. The distance run on a treadmill and the exercise time were significantly greater in G‐CSF‐treated mice when compared to chagasic controls, as well as oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ration (RER) during exercise. Administration of G‐CSF in experimental cardiac ischemia had beneficial effects on cardiac structure, which were well correlated with improvements in cardiac function and whole animal performance.—Macambira, S. G., Vasconcelos, J. F., Costa, C. R. S., Klein, W., Lima, R. S., Guimaraes, P., Vidal, D. T. A., Mendez, L. C., Ribeiro‐dos‐Santos, R., Soares, M. B. P. Granulocyte colony‐stimulating factor treatment in chronic Chagas disease: preservation and improvement of cardiac structure and function. FASEB J. 23, 3843–3850 (2009). www.fasebj.org

Activity of physalin F in a collagen-induced arthritis model.

The effects of physalin F (1), a steroid derivative purified from Physalis angulata, were investigated in models of collagen-induced arthritis in DBA/1 mice and allergic airway inflammation in BALB/c mice. Oral treatment with 1 or dexamethasone caused a marked decrease in paw edema and joint inflammation when compared to vehicle-treated arthritic mice. In contrast, treatment with 1 had no effect in mice with allergic airway inflammation caused by ovalbumin immunization, whereas dexamethasone significantly reduced the number of inflammatory cells and eosinophils in the broncoalveolar lavage fluid and in lung sections of challenged mice. To further demonstrate that 1 acts through a mechanism different from that of glucocorticoids, a nuclear translocation assay was performed of the glucocorticoid receptor (GR) using COS-7 cells transfected with a plasmid encoding for a yellow fluorescent protein (YFP)-GR fusion protein. Untreated or treated cells with 1 had YFP staining mainly in the cytoplasm, whereas in dexamethasone-treated cells the YFP staining was concentrated in the nuclei. It is concluded that the mechanism of the immunosuppressive activity of physalin F is distinct from that of the glucocorticoids.

Distribution and proliferation of bone marrow cells in the brain after pilocarpine-induced status epilepticus in mice

The distribution of bone marrow cells in brain areas during the acute period after pilocarpine‐induced status epilepticus (SE) was investigated here. To achieve this, we generated chimeric mice by engrafting bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic mice. GFP+ bone marrow–derived cells were found throughout the brain, predominantly in the hippocampus. As expected, these cells exhibited the characteristics of microglia. The pattern of distribution, proliferation, and differentiation of GFP+ cells changes as a function of intensity and time following SE. This pattern is also a consequence of the inflammatory response, which is followed by the progressive neuronal damage that is characteristic of the pilocarpine model.

Genetic Vaccination against Experimental Infection with Myotropic Parasite Strains of Trypanosoma cruzi

In earlier studies, we reported that a heterologous prime-boost regimen using recombinant plasmid DNA followed by replication-defective adenovirus vector, both containing Trypanosoma cruzi genes encoding trans-sialidase (TS) and amastigote surface protein (ASP) 2, provided protective immunity against experimental infection with a reticulotropic strain of this human protozoan parasite. Herein, we tested the outcome of genetic vaccination of F1 (CB10XBALB/c) mice challenged with myotropic parasite strains (Brazil and Colombian). Initially, we determined that the coadministration during priming of a DNA plasmid containing the murine IL-12 gene improved the immune response and was essential for protective immunity elicited by the heterologous prime-boost regimen in susceptible male mice against acute lethal infections with these parasites. The prophylactic or therapeutic vaccination of resistant female mice led to a drastic reduction in the number of inflammatory infiltrates in cardiac and skeletal muscles during the chronic phase of infection with either strain. Analysis of the electrocardiographic parameters showed that prophylactic vaccination reduced the frequencies of sinus arrhythmia and atrioventricular block. Our results confirmed that prophylactic vaccination using the TS and ASP-2 genes benefits the host against acute and chronic pathologies caused by T. cruzi and should be further evaluated for the development of a veterinary or human vaccine against Chagas disease.

Evaluation of Galectin-3 as a Novel Biomarker for Chagas Cardiomyopathy

Objectives: Chagas cardiomyopathy has worse long-term outcomes than other cardiomyopathies. A biomarker strategy to refer subjects for noninvasive cardiac imaging may help in the early identification of cardiac damage in subjects with Chagas disease. Galectin-3 (Gal-3) is a mediator of cardiac fibrosis shown to be upregulated in animal models of decompensated heart failure. Here we assessed the correlation of Gal-3 with myocardial fibrosis in patients with Chagas disease. Methods: This study comprised 61 subjects with Chagas disease. All subjects underwent clinical assessments, Doppler echocardiography and magnetic resonance imaging. Plasmatic Gal-3 was determined by ELISA. Results: Delayed enhancement (DE) was identified in 37 of 61 subjects (64%). The total amount of myocardial fibrosis was 9.4% [interquartile interval (IQI): 2.4-18.4]. No differences were observed in Gal-3 concentration according to the presence or absence of myocardial fibrosis, with a median Gal-3 concentration of 11.7 ng/ml (IQI: 9.4-15) in subjects with DE versus 12.9 ng/ml (IQI: 9.2-14) in subjects without DE (p = 0.18). No correlation was found between myocardial fibrosis and Gal-3 concentration (r = 0.098; p = 0.47). Conclusions: There is no correlation between the degree of myocardial fibrosis and the concentration of Gal-3 in subjects with Chagas disease.

Galectin-3 Knockdown Impairs Survival, Migration, and Immunomodulatory Actions of Mesenchymal Stromal Cells in a Mouse Model of Chagas Disease Cardiomyopathy

Therapies based on transplantation of mesenchymal stromal cells (MSC) hold promise for the management of inflammatory disorders. In chronic Chagas disease cardiomyopathy (CCC), caused by chronic infection with Trypanosoma cruzi, the exacerbated immune response plays a critical pathophysiological role and can be modulated by MSC. Here, we investigated the role of galectin-3 (Gal-3), a beta-galactoside-binding lectin with several actions on immune responses and repair process, on the immunomodulatory potential of MSC. Gal-3 knockdown in MSC did not affect the immunophenotype or differentiation potential. However, Gal-3 knockdown MSC showed decreased proliferation, survival, and migration. Additionally, when injected intraperitoneally into mice with CCC, Gal-3 knockdown MSC showed impaired migration in vivo. Transplantation of control MSC into mice with CCC caused a suppression of cardiac inflammation and fibrosis, reducing expression levels of CD45, TNFα, IL-1β, IL-6, IFNγ, and type I collagen. In contrast, Gal-3 knockdown MSC were unable to suppress the immune response or collagen synthesis in the hearts of mice with CCC. Finally, infection with T. cruzi demonstrated parasite survival in wild-type but not in Gal-3 knockdown MSC. These findings demonstrate that Gal-3 plays a critical role in MSC survival, proliferation, migration, and therapeutic potential in CCC.