Proteomics of serum-derived extracellular vesicles are associated with the severity and different clinical profiles of patients with COVID-19: An exploratory secondary analysis.

BACKGROUND AIMS: Coronavirus disease 2019 (COVID-19) is characterized by a broad spectrum of clinical manifestations with the potential to progress to multiple organ dysfunction in severe cases. Extracellular vesicles (EVs) carry a range of biological cargoes, which may be used as biomarkers of disease state. METHODS: An exploratory secondary analysis of the SARITA-2 and SARITA-1 datasets (randomized clinical trials on patients with mild and moderate/severe COVID-19) was performed. Serum-derived EVs were used for proteomic analysis to identify enriched biological processes and key proteins, thus providing insights into differences in disease severity. Serum-derived EVs were separated from patients with COVID-19 by size exclusion chromatography and nanoparticle tracking analysis was used to determine particle concentration and diameter. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to identify and quantify protein signatures. Bioinformatics and multivariate statistical analysis were applied to distinguish candidate proteins associated with disease severity (mild versus moderate/severe COVID-19). RESULTS: No differences were observed in terms of the concentration and diameter of enriched EVs between mild (n = 14) and moderate/severe (n = 30) COVID-19. A total of 414 proteins were found to be present in EVs, of which 360 were shared while 48 were uniquely present in severe/moderate compared to mild COVID-19. The main biological signatures in moderate/severe COVID-19 were associated with platelet degranulation, exocytosis, complement activation, immune effector activation, and humoral immune response. Von Willebrand factor, serum amyloid A-2 protein, histone H4 and H2A type 2-C, and fibrinogen ß-chain were the most differentially expressed proteins between severity groups. CONCLUSION: Exploratory proteomic analysis of serum-derived EVs from patients with COVID-19 detected key proteins related to immune response and activation of coagulation and complement pathways, which are associated with disease severity. Our data suggest that EV proteins may be relevant biomarkers of disease state and prognosis.

Leishmania infection alters macrophage and dendritic cell migration in a three-dimensional environment.

Background: Leishmaniasis results in a wide spectrum of clinical manifestations, ranging from skin lesions at the site of infection to disseminated lesions in internal organs, such as the spleen and liver. While the ability of Leishmania-infected host cells to migrate may be important to lesion distribution and parasite dissemination, the underlying mechanisms and the accompanying role of host cells remain poorly understood. Previously published work has shown that Leishmania infection inhibits macrophage migration in a 2-dimensional (2D) environment by altering actin dynamics and impairing the expression of proteins involved in plasma membrane-extracellular matrix interactions. Although it was shown that L. infantum induces the 2D migration of dendritic cells, in vivo cell migration primarily occurs in 3-dimensional (3D) environments. The present study aimed to investigate the migration of macrophages and dendritic cells infected by Leishmania using a 3-dimensional environment, as well as shed light on the mechanisms involved in this process. Methods: Following the infection of murine bone marrow-derived macrophages (BMDM), human macrophages and human dendritic cells by L. amazonensis, L. braziliensis, or L. infantum, cellular migration, the formation of adhesion complexes and actin polymerization were evaluated. Results: Our results indicate that Leishmania infection inhibited 3D migration in both BMDM and human macrophages. Reduced expression of proteins involved in adhesion complex formation and alterations in actin dynamics were also observed in Leishmania-infected macrophages. By contrast, increased human dendritic cell migration in a 3D environment was found to be associated with enhanced adhesion complex formation and increased actin dynamics. Conclusion: Taken together, our results show that Leishmania infection inhibits macrophage 3D migration, while enhancing dendritic 3D migration by altering actin dynamics and the expression of proteins involved in plasma membrane extracellular matrix interactions, suggesting a potential association between dendritic cells and disease visceralization.

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].

Agathisflavone as a Single Therapy or in Association With Mesenchymal Stem Cells Improves Tissue Repair in a Spinal Cord Injury Model in Rats.

Agathisflavone is a flavonoid with anti-neuroinflammatory and myelinogenic properties, being also capable to induce neurogenesis. This study evaluated the therapeutic effects of agathisflavone-both as a pharmacological therapy administered in vivo and as an in vitro pre-treatment aiming to enhance rat mesenchymal stem cells (r)MSCs properties-in a rat model of acute spinal cord injury (SCI). Adult male Wistar rats (n = 6/group) underwent acute SCI with an F-2 Fogarty catheter and after 4 h were treated daily with agathisflavone (10 mg/kg ip, for 7 days), or administered with a single i.v. dose of 1 × 106 rMSCs either unstimulated cells (control) or pretreated with agathisflavone (1 µM, every 2 days, for 21 days in vitro). Control rats (n = 6/group) were treated with a single dose methylprednisolone (MP, 60 mg/kg ip). BBB scale was used to evaluate the motor functions of the animals; after 7 days of treatment, the SCI area was analyzed after H&E staining, and RT-qPCR was performed to analyze the expression of neurotrophins and arginase. Treatment with agathisflavone alone or with of 21-day agathisflavone-treated rMSCs was able to protect the injured spinal cord tissue, being associated with increased expression of NGF, GDNF and arginase, and reduced macrophage infiltrate. In addition, treatment of animals with agathisflavone alone was able to protect injured spinal cord tissue and to increase expression of neurotrophins, modulating the inflammatory response. These results support a pro-regenerative effect of agathisflavone that holds developmental potential for clinical applications in the future.

Leishmania-Induced Dendritic Cell Migration and Its Potential Contribution to Parasite Dissemination.

Leishmania, an intracellular parasite species, causes lesions on the skin and in the mucosa and internal organs. The dissemination of infected host cells containing Leishmania is crucial to parasite survival and the establishment of infection. Migratory phenomena and the mechanisms underlying the dissemination of Leishmania-infected human dendritic cells (hDCs) remain poorly understood. The present study aimed to investigate differences among factors involved in hDC migration by comparing infection with visceral leishmaniasis (VL) induced by Leishmaniainfantum with diverse clinical forms of tegumentary leishmaniasis (TL) induced by Leishmaniabraziliensis or Leishmania amazonensis. Following the infection of hDCs by isolates obtained from patients with different clinical forms of Leishmania, the formation of adhesion complexes, actin polymerization, and CCR7 expression were evaluated. We observed increased hDC migration following infection with isolates of L. infantum (VL), as well as disseminated (DL) and diffuse (DCL) forms of cutaneous leishmaniasis (CL) caused by L. braziliensis and L. amazonensis, respectively. Increased expression of proteins involved in adhesion complex formation and actin polymerization, as well as higher CCR7 expression, were seen in hDCs infected with L. infantum, DL and DCL isolates. Together, our results suggest that hDCs play an important role in the dissemination of Leishmania parasites in the vertebrate host.

Betulinic Acid Exerts Cytoprotective Activity on Zika Virus-Infected Neural Progenitor Cells.

Zika virus (ZIKV), a member of the Flaviviridae family, was brought into the spotlight due to its widespread and increased pathogenicity, including Guillain-Barré syndrome and microcephaly. Neural progenitor cells (NPCs), which are multipotent cells capable of differentiating into the major neural phenotypes, are very susceptible to ZIKV infection. Given the complications of ZIKV infection and potential harm to public health, effective treatment options are urgently needed. Betulinic acid (BA), an abundant terpenoid of the lupane group, displays several biological activities, including neuroprotective effects. Here we demonstrate that Sox2+ NPCs, which are highly susceptible to ZIKV when compared to their neuronal counterparts, are protected against ZIKV-induced cell death when treated with BA. Similarly, the population of Sox2+ and Casp3+ NPCs found in ZIKV-infected cerebral organoids was significantly higher in the presence of BA than in untreated controls. Moreover, well-preserved structures were found in BA-treated organoids in contrast to ZIKV-infected controls. Bioinformatics analysis indicated Akt pathway activation by BA treatment. This was confirmed by phosphorylated Akt analysis, both in BA-treated NPCs and brain organoids, as shown by immunoblotting and immunofluorescence analyses, respectively. Taken together, these data suggest a neuroprotective role of BA in ZIKV-infected NPCs.

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.

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.

Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells.

Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection, we used human neural progenitor cells (NPC), derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation, ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2+ cells was demonstrated by activation of caspases 3/7, 8 and 9, and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2+ cells was prevented by incubation with the pan-caspase inhibitor, Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities, including multipolar spindle, chromosome laggards, micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy, such as monosomy, trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain, including microcephaly.

Administration of granulocyte colony-stimulating factor induces immunomodulation, recruitment of T regulatory cells, reduction of myocarditis and decrease of parasite load in a mouse model of chronic Chagas disease cardiomyopathy.

Chagas disease, caused by Trypanosoma cruzi infection, is a leading cause of heart failure in Latin American countries. In a previous study, we showed beneficial effects of granulocyte colony-stimulating factor (G-CSF) administration in the heart function of mice with chronic T. cruzi infection. Presently, we investigated the mechanisms by which this cytokine exerts its beneficial effects. Mice chronically infected with T. cruzi were treated with human recombinant G-CSF (3 courses of 200 µg/kg/d for 5 d). Inflammation and fibrosis were reduced in the hearts of G-CSF-treated mice, compared with the hearts of vehicle-treated mice, which correlated with decreased syndecan-4, intercellular adhesion molecule-1, and galectin-3 expressions. Marked reductions in interferon-γ and tumor necrosis factor-α and increased interleukin-10 and transforming growth factor-ß were found after G-CSF administration. Because the therapy did not induce a Th1 to Th2 immune response deviation, we investigated the role of regulatory T (Treg) cells. A significant increase in CD3(+)Foxp3(+) cells was observed in the hearts of G-CSF-treated mice. In addition, a reduction of parasitism was observed after G-CSF treatment. Our results indicate a role of Treg cells in the immunosuppression induced by G-CSF treatment and reinforces its potential therapeutic use for patients with Chagas disease.

Transplantation of bone marrow mononuclear cells decreases seizure incidence, mitigates neuronal loss and modulates pro-inflammatory cytokine production in epileptic rats.

Approximately 30% of patients with mesial temporal lobe epilepsy do not respond to treatment with antiepileptic drugs. We have previously shown that transplantation of mononuclear bone marrow cells (BMC) has an anticonvulsant effect in acute epilepsy. Here, we used pilocarpine to induce epilepsy in rats and studied the effects of BMC injected intravenously either at the onset of seizures or after 10 months of recurrent seizures. BMC effectively decreased seizure frequency and duration. In addition, decreased levels of proinflammatory cytokines (TNF-α, IL-1ß and IL-6) and increased levels of anti-inflammatory cytokine (IL-10) were observed in the brain and serum of BMC-treated rats. Transplants performed at seizure-onset protected against pilocarpine-induced neuronal loss and gliosis and stimulated the proliferation of new neurons in epileptic rats. Our data demonstrate that BMC transplantation has potent therapeutic effects and could be a potential therapy for clinically intractable epilepsies.

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.