Diagnostic Performance of Three ELISAs for Detection of Antibodies against SARS-CoV-2 in Human Samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) is a disease with a high rate of transmission. Serological tests are important to perform surveys and to determine the immunological status of the population. Based on this, we evaluated three enzyme-linked immunoassays (ELISAs) using different antigens from SARS-CoV-2 in a cohort of 161 patients. The performance of the ELISA developed for immunoglobulin G (IgG) measurement against SARS-CoV-2 was evaluated based on sensitivity, specificity, and accuracy. We found specificities of 0.98, 0.98, and 0.99 and sensitivities of 0.99, 0.91, and 0.87 for the nucleocapsid (N) protein, spike protein, and receptor binding domain (RBD) fraction, respectively. The accuracy assessment indicated the N protein (accuracy = 0.98) as the antigen most likely to give a correct diagnosis. Overall, the antibody responses were present for all three proteins in subjects with confirmed SARS-CoV-2 infections, showing a similar pattern of antibody production for different antigens. In summary, these highly sensitive and specific ELISAs, with a more competitive price, appear to be a valid approach for the serodiagnosis of COVID-19.

Efficacy and Safety of Granulocyte-Colony Stimulating Factor Therapy in Chagas Cardiomyopathy: A Phase II Double-Blind, Randomized, Placebo-Controlled Clinical Trial.

Aim: Previous studies showed that granulocyte-colony stimulating factor (G-CSF) improved heart function in a mice model of Chronic Chagas Cardiomyopathy (CCC). Herein, we report the interim results of the safety and efficacy of G-CSF therapy vs. placebo in adults with Chagas cardiomyopathy. Methods: Patients with CCC, New York Heart Association (NYHA) functional class II to IV and left ventricular ejection fraction (LVEF) 50% or below were included. A randomization list using blocks of 2 and 4 and an allocation rate of 1:1 was generated by R software which was stratified by functional class. Double blinding was done to both arms and assessors were masked to allocations. All patients received standard heart failure treatment for 2 months before 1:1 randomization to either the G-CSF (10 mcg/kg/day subcutaneously) or placebo group (1 mL of 0.9% saline subcutaneously). The primary endpoint was either maintenance or improvement of NYHA class from baseline to 6-12 months after treatment, and intention-to-treat analysis was used. Results: We screened 535 patients with CCC in Salvador, Brazil, of whom 37 were randomized. Overall, baseline characteristics were well-balanced between groups. Most patients had NYHA class II heart failure (86.4%); low mean LVEF was 32 ± 7% in the G-CSF group and 33 ± 10% in the placebo group. Frequency of primary endpoint was 78% (95% CI 0.60-0.97) vs. 66% (95% CI 0.40-0.86), p = 0.47, at 6 months and 68% (95% CI 0.43-0.87) vs. 72% (95% CI 0.46-0.90), p = 0.80, at 12 months in placebo and G-CSF groups, respectively. G-CSF treatment was safe, without any related serious adverse events. There was no difference in mortality between both arms, with five deaths (18.5%) in treatment vs. four (12.5%) in the placebo arm. Exploratory analysis demonstrated that the maximum rate of oxygen consumption during exercise (VO2 max) showed an improving trend in the G-CSF group. Conclusion: G-CSF therapy was safe and well-tolerated in 12 months of follow-up. Although prevention of symptom progression could not be demonstrated in the present study, our results support further investigation of G-CSF therapy in Chagas cardiomyopathy patients. Clinical Trial Registration: [www.ClinicalTrials.gov], identifier [NCT02154269].

Clinical Trials Using Mesenchymal Stem Cells for Spinal Cord Injury: Challenges in Generating Evidence.

Spinal cord injury (SCI) remains an important public health problem which often causes permanent loss of muscle strength, sensation, and function below the site of the injury, generating physical, psychological, and social impacts throughout the lives of the affected individuals, since there are no effective treatments available. The use of stem cells has been investigated as a therapeutic approach for the treatment of SCI. Although a significant number of studies have been conducted in pre-clinical and clinical settings, so far there is no established cell therapy for the treatment of SCI. One aspect that makes it difficult to evaluate the efficacy is the heterogeneity of experimental designs in the clinical trials that have been published. Cell transplantation methods vary widely among the trials, and there are still no standardized protocols or recommendations for the therapeutic use of stem cells in SCI. Among the different cell types, mesenchymal stem/stromal cells (MSCs) are the most frequently tested in clinical trials for SCI treatment. This study reviews the clinical applications of MSCs for SCI, focusing on the critical analysis of 17 clinical trials published thus far, with emphasis on their design and quality. Moreover, it highlights the need for more evidence-based studies designed as randomized controlled trials and potential challenges to be addressed in context of stem cell therapies for SCI.

Immunomodulation for the Treatment of Chronic Chagas Disease Cardiomyopathy: A New Approach to an Old Enemy.

Chagas disease is a parasitic infection caused by the intracellular protozoan Trypanosoma cruzi. Chronic Chagas cardiomyopathy (CCC) is the most severe manifestation of the disease, developed by approximately 20-40% of patients and characterized by occurrence of arrhythmias, heart failure and death. Despite having more than 100 years of discovery, Chagas disease remains without an effective treatment, especially for patients with CCC. Since the pathogenesis of CCC depends on a parasite-driven systemic inflammatory profile that leads to cardiac tissue damage, the use of immunomodulators has become a rational alternative for the treatment of CCC. In this context, different classes of drugs, cell therapies with dendritic cells or stem cells and gene therapy have shown potential to modulate systemic inflammation and myocarditis in CCC models. Based on that, the present review provides an overview of current reports regarding the use of immunomodulatory agents in treatment of CCC, bringing the challenges and future directions in this field.

Therapeutic miR-21 Silencing Reduces Cardiac Fibrosis and Modulates Inflammatory Response in Chronic Chagas Disease.

Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), remains a serious public health problem for which there is no effective treatment in the chronic stage. Intense cardiac fibrosis and inflammation are hallmarks of chronic Chagas disease cardiomyopathy (CCC). Previously, we identified upregulation of circulating and cardiac miR-21, a pro-fibrotic microRNA (miRNA), in subjects with CCC. Here, we explored the potential role of miR-21 as a therapeutic target in a model of chronic Chagas disease. PCR array-based 88 microRNA screening was performed in heart samples obtained from C57Bl/6 mice chronically infected with T. cruzi and serum samples collected from CCC patients. MiR-21 was found upregulated in both human and mouse samples, which was corroborated by an in silico analysis of miRNA-mRNA target prediction. In vitro miR-21 functional assays (gain-and loss-of-function) were performed in cardiac fibroblasts, showing upregulation of miR-21 and collagen expression upon transforming growth factor beta 1 (TGFß1) and T. cruzi stimulation, while miR-21 blockage reduced collagen expression. Finally, treatment of T. cruzi-infected mice with locked nucleic acid (LNA)-anti-miR-21 inhibitor promoted a significant reduction in cardiac fibrosis. Our data suggest that miR-21 is a mediator involved in the pathogenesis of cardiac fibrosis and indicates the pharmacological silencing of miR-21 as a potential therapeutic approach for CCC.

In vitro and In Vivo Immunomodulatory Activity of Physalis angulata Concentrated Ethanolic Extract.

The need for new immunomodulatory drugs is due to the side effects associated with the prolonged use of the currently used immunomodulatory drugs. In this context, the present work aimed to investigate the immunomodulatory effect of an ethanolic concentrated extract from Physalis angulata. The cytotoxicity of samples was determined using peritoneal macrophages though the Alamar Blue assay. The immunomodulatory activity of the ethanolic extract from P. angulata on activated macrophages was determined by measurement of nitrite and cytokine production. The immunosuppressive effects of the ethanolic extract from P. angulata was evaluated on lymphocyte proliferation and cytokine production. The effects of the extract on cell cycle progression and cell death on lymphocytes were evaluated by flow cytometry. Lastly, the ethanolic extract from P. angulata was tested in vivo in toxicological tests and in models of peritonitis and delayed-type hypersensitivity response. The ethanolic extract from P. angulata decreased nitrite, interleukin-6, interleukin-12, and TNF-α production by activated macrophages without affecting the cell viability. In addition, the ethanolic extract from P. angulata inhibited lymphoproliferation and the secretion of interleukin-2, interleukin-6, and IFN-γ, and increased interleukin-4 secretion by activated splenocytes. Flow cytometry analysis in lymphocyte cultures showed that treatment with the ethanolic extract from P. angulata induces cell cycle arrest in the G1 phase followed by cell death by apoptosis. Moreover, mice treated with the extract from P. angulata at 100 or 200 mg/kg did not show signs of toxicity or alterations in serum components. Finally, the ethanolic extract from P. angulata significantly reduced neutrophil migration and reduced paw edema in bovine serum albumin-induced the delayed-type hypersensitivity response model. Our results demonstrate the potential of the ethanolic extract of P. angulata as an alternative for the treatment of immune-inflammatory diseases.

Genetic Engineering as a Strategy to Improve the Therapeutic Efficacy of Mesenchymal Stem/Stromal Cells in Regenerative Medicine.

Mesenchymal stem/stromal cells (MSCs) have been widely studied in the field of regenerative medicine for applications in the treatment of several disease settings. The therapeutic potential of MSCs has been evaluated in studies in vitro and in vivo, especially based on their anti-inflammatory and pro-regenerative action, through the secretion of soluble mediators. In many cases, however, insufficient engraftment and limited beneficial effects of MSCs indicate the need of approaches to enhance their survival, migration and therapeutic potential. Genetic engineering emerges as a means to induce the expression of different proteins and soluble factors with a wide range of applications, such as growth factors, cytokines, chemokines, transcription factors, enzymes and microRNAs. Distinct strategies have been applied to induce genetic modifications with the goal to enhance the potential of MCSs. This review aims to contribute to the update of the different genetically engineered tools employed for MSCs modification, as well as the factors investigated in different fields in which genetically engineered MSCs have been tested.

Leukemia Inhibitory Factor (LIF) Overexpression Increases the Angiogenic Potential of Bone Marrow Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) have the ability to secrete bioactive molecules, exerting multiple biological effects, such as tissue regeneration, reduction of inflammation, and neovascularization. The therapeutic potential of MSCs can be increased by genetic modification to overexpress cytokines and growth factors. Here we produced mouse MSCs overexpressing human leukemia inhibitory factor (LIF) to assess their proangiogenic potential in vitro and in vivo. Mouse bone marrow-derived MSCs were transduced by using a second-generation lentiviral system to express human LIF. Leukemia inhibitory factor expression was confirmed by RT-qPCR and by ELISA, allowing the quantification of the transcript and secreted protein, respectively. Flow cytometry analysis and trilineage differentiation assay showed that the MSC_LIF cell line maintained the immunophenotype and a multipotency characteristic of MSCs. The immunosuppressive activity of MSC_LIF was confirmed using a lymphoproliferation assay. Moreover, gene expression analysis demonstrated upregulation of genes coding for strategic factors in the neovascularization process, such as angiogenin, IL-8, MCP-1, and VEGF, and for the perivascular cell markers αSMA, Col4a1, SM22, and NG2. To evaluate the pro-angiogenic potential of MSC_LIF, we first tested its effects on endothelial cells obtained from umbilical vein in a scratch wound healing assay. Conditioned medium (CM) from MSC_LIF promoted a significant increase in cell migration compared to CM from control MSC. Additionally, in vitro tube formation of endothelial cells was increased by the presence of MSC_LIF, as shown in microvessel sprouting in aortic ring cultures. Finally, an in vivo Matrigel plug assay was performed, showing that MSC_LIF were more potent in promoting in vivo angiogenesis and tissue vascularization than control MSCs. In conclusion, LIF overexpression is a promising strategy to increase the proangiogenic potential of MSCs and sets precedents for future investigations of their potential applications for the treatment of ischemic diseases and tissue repair.

Mesenchymal Stem Cells and Atopic Dermatitis: A Review.

Mesenchymal stem/stromal cells (MSCs) are stromal-derived non-hematopoietic progenitor cells that reside in and can be expanded from various tissues sources of adult and neonatal origin, such as the bone marrow, umbilical cord, umbilical cord blood, adipose tissue, amniotic fluid, placenta, dental pulp and skin. The discovery of the immunosuppressing action of MSCs on T cells has opened new perspectives for their use as a therapeutic agent for immune-mediated disorders, including allergies. Atopic dermatitis (AD), a chronic and relapsing skin disorder that affects up to 20% of children and up to 3% of adults worldwide, is characterized by pruritic eczematous lesions, impaired cutaneous barrier function, Th2 type immune hyperactivation and, frequently, elevation of serum immunoglobulin E levels. Although, in the dermatology field, the application of MSCs as a therapeutic agent was initiated using the concept of cell replacement for skin defects and wound healing, accumulating evidence have shown that MSC-mediated immunomodulation can be applicable to the treatment of inflammatory/allergic skin disorders. Here we reviewed the pre-clinical and clinical studies and possible biological mechanisms of MSCs as a therapeutic tool for the treatment of atopic dermatitis.

Circulating miRNAs as Potential Biomarkers Associated with Cardiac Remodeling and Fibrosis in Chagas Disease Cardiomyopathy.

Chagas disease (CD) affects approximately 6-7 million people worldwide, from which 30% develop chronic Chagas cardiomyopathy (CCC), usually after being asymptomatic for years. Currently available diagnostic methods are capable of adequately identifying infected patients, but do not provide information regarding the individual risk of developing the most severe form of the disease. The identification of biomarkers that predict the progression from asymptomatic or indeterminate form to CCC, may guide early implementation of pharmacological therapy. Here, six circulating microRNAs (miR-19a-3p, miR-21-5p, miR-29b-3p, miR-30a-5p, miR-199b-5p and miR-208a-3p) were evaluated and compared among patients with CCC (n = 28), CD indeterminate form (n = 10) and healthy controls (n = 10). MiR-19a-3p, miR-21-5p, and miR-29b-3p were differentially expressed in CCC patients when compared to indeterminate form, showing a positive correlation with cardiac dysfunction, functional class, and fibrosis, and a negative correlation with ejection fraction and left ventricular strain. Cardiac tissue analysis confirmed increased expression of microRNAs in CCC patients. In vitro studies using human cells indicated the involvement of these microRNAs in the processes of cardiac hypertrophy and fibrosis. Our study suggests that miRNAs are involved in the process of cardiac fibrosis and remodeling presented in CD and indicate a group of miRNAs as potential biomarkers of disease progression in CCC.

IGF-1-Overexpressing Mesenchymal Stem/Stromal Cells Promote Immunomodulatory and Proregenerative Effects in Chronic Experimental Chagas Disease.

Mesenchymal stem/stromal cells (MSCs) have been investigated for the treatment of diseases that affect the cardiovascular system, including Chagas disease. MSCs are able to promote their beneficial actions through the secretion of proregenerative and immunomodulatory factors, including insulin-like growth factor-1 (IGF-1), which has proregenerative actions in the heart and skeletal muscle. Here, we evaluated the therapeutic potential of IGF-1-overexpressing MSCs (MSC_IGF-1) in a mouse model of chronic Chagas disease. C57BL/6 mice were infected with Colombian strain Trypanosoma cruzi and treated with MSCs, MSC_IGF-1, or vehicle (saline) six months after infection. RT-qPCR analysis confirmed the presence of transplanted cells in both the heart and skeletal muscle tissues. Transplantation of either MSCs or MSC_IGF-1 reduced the number of inflammatory cells in the heart when compared to saline controls. Moreover, treatment with MSCs or MSC_IGF-1 significantly reduced TNF-α, but only MSC treatment reduced IFN-γ production compared to the saline group. Skeletal muscle sections of both MSC- and MSC_IGF-1-treated mice showed a reduction in fibrosis compared to saline controls. Importantly, the myofiber area was reduced in T. cruzi-infected mice, and this was recovered after treatment with MSC_IGF-1. Gene expression analysis in the skeletal muscle showed a higher expression of pro- and anti-inflammatory molecules in MSC_IGF-1-treated mice compared to MSCs alone, which significantly reduced the expression of TNF-α and IL-1ß. In conclusion, our results indicate the therapeutic potential of MSC_IGF-1, with combined immunomodulatory and proregenerative actions to the cardiac and skeletal muscles.

Granulocyte-Colony Stimulating Factor-Overexpressing Mesenchymal Stem Cells Exhibit Enhanced Immunomodulatory Actions Through the Recruitment of Suppressor Cells in Experimental Chagas Disease Cardiomyopathy.

Genetic modification of mesenchymal stem cells (MSCs) is a promising strategy to improve their therapeutic effects. Granulocyte-colony stimulating factor (G-CSF) is a growth factor widely used in the clinical practice with known regenerative and immunomodulatory actions, including the mobilization of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Here we evaluated the therapeutic potential of MSCs overexpressing G-CSF (MSC_G-CSF) in a model of inflammatory cardiomyopathy due to chronic Chagas disease. C57BL/6 mice were treated with wild-type MSCs, MSC_G-CSF, or vehicle (saline) 6 months after infection with Trypanosoma cruzi. Transplantation of MSC_G-CSF caused an increase in the number of circulating leukocytes compared to wild-type MSCs. Moreover, G-CSF overexpression caused an increase in migration capacity of MSCs to the hearts of infected mice. Transplantation of either MSCs or MSC_G-CSF improved exercise capacity, when compared to saline-treated chagasic mice. MSC_G-CSF mice, however, were more potent than MSCs in reducing the number of infiltrating leukocytes and fibrosis in the heart. Similarly, MSC_G-CSF-treated mice presented significantly lower levels of inflammatory mediators, such as IFNγ, TNFα, and Tbet, with increased IL-10 production. A marked increase in the percentage of Tregs and MDSCs in the hearts of infected mice was seen after administration of MSC_G-CSF, but not MSCs. Moreover, Tregs were positive for IL-10 in the hearts of T. cruzi-infected mice. In vitro analysis showed that recombinant hG-CSF and conditioned medium of MSC_G-CSF, but not wild-type MSCs, induce chemoattraction of MDSCs in a transwell assay. Finally, MDSCs purified from hearts of MSC_G-CSF transplanted mice inhibited the proliferation of activated splenocytes in a co-culture assay. Our results demonstrate that G-CSF overexpression by MSCs potentiates their immunomodulatory effects in our model of Chagas disease and suggest that mobilization of suppressor cell populations such as Tregs and MDSCs as a promising strategy for the treatment of chronic Chagas disease. Finally, our results reinforce the therapeutic potential of genetic modification of MSCs, aiming at increasing their paracrine actions.

The Presence and Extension of Myocardial Fibrosis in the Undetermined Form of Chagas' Disease: A Study Using Magnetic Resonance.

BACKGROUND: Previous data has shown that patients in the indeterminate form of Chagas disease may present myocardial fibrosis as shown on through magnetic resonance imaging (MRI). However, there is little information available regarding the degree of severity of myocardial fibrosis in these individuals. This variable has the potential to predict the evolution of Chagas' disease into its cardiac form. OBJECTIVES: To describe the frequency and extent of myocardial fibrosis evaluated using an MRI in patients in the indeterminate form, and to compare it with other forms of the disease. METHODS: Patients were admitted one after another. Their clinical history was collected and they were submitted to laboratory exams and an MRI. RESULTS: Sixty-one patients with Chagas' disease, with an average age of 58 ± 9 years old, 17 patients in the indeterminate form, 16 in the cardiac form without left ventricular (LV) dysfunction and 28 in the cardiac form with LV dysfunction were studied. P <0.05 was considered to be statistically significant. Late enhancement was detected in 37 patients (64%). Myocardial fibrosis was identified in 6 individuals in indeterminate form (41%; 95% CI 23-66) in a proportion similar to that observed in cardiac form without LV dysfunction (44%); p = 1.0. Among the individuals with fibrosis, the total area of the affected myocardium was 4.1% (IIQ: 2.1 - 10.7) in the indeterminate form versus 2.3% (IIQ: 1-5) in the cardiac form without LV (p = 0.18). The left ventricular fraction ejection in subjects in the indeterminate form was similar to that of the individuals in the cardiac form without ventricular dysfunction (p = 0.09). CONCLUSION: The presence of fibrosis in the indeterminate form of Chagas' disease has a frequency and extension similar to that of in the cardiac form without dysfunction, suggesting that the former is part of a subclinical disease spectrum, rather than lacking cardiac involvement.

The Presence and Extension of Myocardial Fibrosis in the Undetermined Form of Chagas' Disease: A Study Using Magnetic Resonance / Presença e Extensão da Fibrose Miocárdica na Forma Indeterminada da Doença de Chagas: Estudo de Ressonância Magnética

Abstract Background: Previous data has shown that patients in the indeterminate form of Chagas disease may present myocardial fibrosis as shown on through magnetic resonance imaging (MRI). However, there is little information available regarding the degree of severity of myocardial fibrosis in these individuals. This variable has the potential to predict the evolution of Chagas' disease into its cardiac form. Objectives: To describe the frequency and extent of myocardial fibrosis evaluated using an MRI in patients in the indeterminate form, and to compare it with other forms of the disease. Methods: Patients were admitted one after another. Their clinical history was collected and they were submitted to laboratory exams and an MRI. Results: Sixty-one patients with Chagas' disease, with an average age of 58 ± 9 years old, 17 patients in the indeterminate form, 16 in the cardiac form without left ventricular (LV) dysfunction and 28 in the cardiac form with LV dysfunction were studied. P <0.05 was considered to be statistically significant. Late enhancement was detected in 37 patients (64%). Myocardial fibrosis was identified in 6 individuals in indeterminate form (41%; 95% CI 23-66) in a proportion similar to that observed in cardiac form without LV dysfunction (44%); p = 1.0. Among the individuals with fibrosis, the total area of the affected myocardium was 4.1% (IIQ: 2.1 - 10.7) in the indeterminate form versus 2.3% (IIQ: 1-5) in the cardiac form without LV (p = 0.18). The left ventricular fraction ejection in subjects in the indeterminate form was similar to that of the individuals in the cardiac form without ventricular dysfunction (p = 0.09). Conclusion: The presence of fibrosis in the indeterminate form of Chagas' disease has a frequency and extension similar to that of in the cardiac form without dysfunction, suggesting that the former is part of a subclinical disease spectrum, rather than lacking cardiac involvement.

Resumo Fundamento: Dados prévios têm demonstrado que pacientes na forma indeterminada podem apresentar fibrose miocárdica à ressonância magnética (RM). No entanto, são poucas as informações disponíveis quanto ao grau de fibrose miocárdica apresentada por esses indivíduos, o que guardaria relação com o potencial dessa variável na predição de evolução para a forma cardíaca da doença de Chagas. Objetivos: Descrever a frequência e extensão da fibrose miocárdica avaliada por RM em pacientes da forma indeterminada, comparando com as outras formas da doença. Métodos: Pacientes consecutivamente admitidos tiveram história clínica colhida e foram submetidos à realização de exames laboratoriais e RM. Resultados: Foram estudados 61 pacientes portadores da doença de Chagas, com média de idade de 58 ± 9 anos, sendo 17 pacientes na forma indeterminada, 16 na forma cardíaca sem disfunção do ventrículo esquerdo (VE) e 28 na forma com disfunção do VE. Foi considerado estatisticamente significante p < 0,05. Realce tardio foi detectado em 37 pacientes (64%). Foi identificada fibrose miocárdica em 6 indivíduos na forma indeterminada (41%; IC95% 23 - 66), proporção semelhante à observada na forma cardíaca sem disfunção do VE (44%); p = 1,0. Entre os indivíduos com fibrose, a área total do miocárdio acometida foi de 4,1% (IIQ: 2,1 - 10,7) na forma indeterminada versus 2,3% (IIQ: 1 - 5) na forma cardíaca sem disfunção do VE (p = 0,18). A fração de ejeção do ventrículo esquerdo nos indivíduos na forma indeterminada foi semelhante aos portadores da forma cardíaca sem disfunção ventricular (p = 0,09). Conclusão: A presença de fibrose na forma indeterminada da doença de Chagas tem frequência e extensão semelhante à forma cardíaca sem disfunção, o que sugere que a primeira faz parte de um espectro de doença subclínica, em vez da ausência de acometimento cardíaco.

Generation and characterization of transgenic mouse mesenchymal stem cell lines expressing hIGF-1 or hG-CSF.

Mesenchymal stem cells (MSC) are promising tools in the fields of cell therapy and regenerative medicine. In addition to their differentiation potential, MSC have the ability to secrete bioactive molecules that stimulate tissue regeneration. Thus, the overexpression of cytokines and growth factors may enhance the therapeutic effects of MSC. Here we generated and characterized mouse bone marrow MSC lines overexpressing hG-CSF or hIGF-1. MSC lines overexpressing hG-CSF or hIGF-1 were generated through lentiviral vector mediated gene transfer. The expression of hG-CSF or hIGF-1 genes in the clones produced was quantified by qRT-PCR, and the proteins were detected in the cell supernatants by ELISA. The cell lines displayed cell surface markers and differentiation potential into adipocytes, osteocytes and chondrocytes similar to the control MSC cell lines, indicating the conservation of their phenotype even after genetic modification. IGF-1 and G-CSF transgenic cells maintained immunosuppressive activity. Finally, we performed a comparative gene expression analysis by qRT-PCR array in the cell lines expressing hIGF-1 and hG-CSF when compared to the control cells. Our results demonstrate that the cell lines generated may be useful tools for cell therapy and are suitable for testing in disease models.

Lack of association between serum syndecan-4, myocardial fibrosis and ventricular dysfunction in subjects with chronic Chagas disease.

BACKGROUND: Syndecan-4 is a transmembrane glycoprotein associated with inflammation and fibrosis. Increased syndecan-4 levels were previously detected after acute myocardial infarction and in subjects with heart failure. However, the levels of syndecan-4 in subjects with Chagas disease have not so far been investigated. The aim of this study was to investigate the potential role of serum sydencan-4 as a novel biomarker for myocardial fibrosis and cardiac dysfunction in subjects with Chagas disease. METHODS: This study comprised subjects with Chagas disease (n = 56), being 14 (25%) with the indeterminate form, 16 (29%) with the cardiac form without ventricular dysfunction, and 26 (46%) with the cardiac form with ventricular dysfunction. RESULTS: Syndecan-4 serum concentrations did not correlate with presence or absence of myocardial fibrosis (P = 0.386) nor disease severity in subjects with Chagas disease (P = 0.918). Additionally, no correlation was found either between the degree of myocardial fibrosis and serum syndecan-4 [r = 0.08; P = 0.567] or between left ventricular ejection fraction and syndecan-4 [r = 0.02; P = 0.864]. In contrast, NT-proBNP levels correlated with ejection fraction and myocardial fibrosis. CONCLUSIONS: Our results demonstrate the lack of correlations between serum syndecan-4, myocardial fibrosis and cardiac dysfunction in subjects with Chagas disease. Further studies are required to show if syndecan-4 concentrations can be marker for prognosis assessment or disease progression.

Therapeutic effects of sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) in experimental chronic Chagas disease cardiomyopathy.

Chagas disease cardiomyopathy is a parasite-driven inflammatory disease to which there are no effective treatments. Here we evaluated the therapeutic potential of N,N-dimethylsphingosine(DMS), which blocks the production of sphingosine-1-phosphate(S1P), a mediator of cellular events during inflammatory responses, in a model of chronic Chagas disease cardiomyopathy. DMS-treated, Trypanosoma cruzi-infected mice had a marked reduction of cardiac inflammation, fibrosis and galectin-3 expression when compared to controls. Serum concentrations of galectin-3, IFNγ and TNFα, as well as cardiac gene expression of inflammatory mediators were reduced after DMS treatment. The gene expression of M1 marker, iNOS, was decreased, while the M2 marker, arginase1, was increased. DMS-treated mice showed an improvement in exercise capacity. Moreover, DMS caused a reduction in parasite load in vivo. DMS inhibited the activation of lymphocytes, and reduced cytokines and NO production in activated macrophage cultures in vitro, while increasing IL-1ß production. Analysis by qRT-PCR array showed that DMS treatment modulated inflammasome activation induced by T. cruzi on macrophages. Altogether, our results demonstrate that DMS, through anti-parasitic and immunomodulatory actions, can be beneficial in the treatment of chronic phase of T. cruzi infection and suggest that S1P-activated processes as possible therapeutic targets for the treatment of Chagas disease cardiomyopathy.

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.

In vitro and in vivo antiparasitic activity of Physalis angulata L. concentrated ethanolic extract against Trypanosoma cruzi.

BACKGROUND: The current treatment of Chagas disease, endemic in Latin America and emerging in several countries, is limited by the frequent side effects and variable efficacy of benznidazole. Natural products are an important source for the search for new drugs. AIM/HYPOTHESIS: Considering the great potential of natural products as antiparasitic agents, we investigated the anti-Trypanosoma cruzi activity of a concentrated ethanolic extract of Physalis angulata (EEPA). METHODS: Cytotoxicity to mammalian cells was determined using mouse peritoneal macrophages. The antiparasitic activity was evaluated against axenic epimastigote and bloodstream trypomastigote forms of T. cruzi, and against amastigote forms using T. cruzi-infected macrophages. Cell death mechanism was determined in trypomastigotes by flow cytometry analysis after annexin V and propidium iodide staining. The efficacy of EEPA was examined in vivo in an acute model of infection by monitoring blood parasitaemia and survival rate 30 days after treatment. The effect against trypomastigotes of EEPA and benznidazole acting in combination was evaluated. RESULTS: EEPA effectively inhibits the epimastigote growth (IC50 2.9 ± 0.1 µM) and reduces bloodstream trypomastigote viability (EC50 1.7 ± 0.5 µM). It causes parasite cell death by necrosis. EEPA impairs parasite infectivity as well as amastigote development in concentrations noncytotoxic to mammalian cells. In mice acutely-infected with T. cruzi, EEPA reduced the blood parasitaemia in 72.7%. When combined with benznidazole, EEPA showed a synergistic anti-T. cruzi activity, displaying CI values of 0.8 ± 0.07 at EC50 and 0.83 ± 0.1 at EC90. CONCLUSION: EEPA has antiparasitic activity against T. cruzi, causing cell death by necrosis and showing synergistic activity with benznidazole. These findings were reinforced by the observed efficacy of EEPA in reducing parasite load in T. cruzi-mice. Therefore, this represents an important source of antiparasitic natural products.

Bone marrow-derived cells migrate to the liver and contribute to the generation of different cell types in chronic Schistosoma mansoni infection.

The main pathogenic event caused by Schistosoma mansoni infection is characterized by a granulomatous inflammatory reaction around parasite eggs and fibrosis in the liver. We have previously shown that transplantation of bone marrow cells (BMC) promotes a reduction in liver fibrosis in chronically S. mansoni-infected mice. Here we investigated the presence and phenotype of bone marrow-derived cells in livers of S. mansoni-infected mice. During the chronic phase of infection, C57BL/6 mice had an increased number of circulating mesenchymal stem cells and endothelial progenitor cells in the peripheral blood when compared to uninfected controls. In order to investigate the fate of BMC in the liver, we generated bone marrow chimeric mice by transplanting BMC from transgenic green fluorescent protein (GFP) mice into lethally irradiated wild-type C57BL/6 mice. S. mansoni-infected chimeric mice did not demonstrate increased mortality and developed similar liver histopathological features, when compared to wild-type S. mansoni-infected mice. GFP(+) bone marrow-derived cells were found in the liver parenchyma, particularly in periportal regions. CD45(+)GFP(+) cells were found in the granulomas. Flow cytometry analysis of digested liver tissue characterized GFP(+) cells as lymphocytes, myeloid cells and stem cells. GFP(+) cells were also found in areas of collagen deposition, although rare GFP(+) cells expressed the myofibroblast cell marker α-SMA. Additionally GFP(+) endothelial cells (co-stained with von Willebrand factor) were frequently observed, while BMC-derived hepatocytes (GFP(+) albumin(+) cells) were sparsely found in the liver of chimeric mice chronically infected with S. mansoni. In conclusion, BMC are recruited to the liver during chronic experimental infection with S. mansoni and contribute to the generation of different cell types involved, not only in disease pathogenesis, but possibly in liver regeneration and repair.