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Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC. Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43S368 and Cx43S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1ß, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer. Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1ß, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43S368 and Cx43S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1ß, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells. Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease.
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Cardiomiopatia Chagásica , Conexina 43 , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Conexina 43/metabolismo , Conexina 43/genética , Animais , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/parasitologia , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/parasitologia , Miócitos Cardíacos/patologia , Inflamação/metabolismo , Fosforilação , Masculino , Doença Crônica , Trypanosoma cruzi , Modelos Animais de Doenças , Linhagem Celular , Citocinas/metabolismo , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/parasitologia , Arritmias Cardíacas/imunologia , FemininoRESUMO
BACKGROUND AIMS: Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model. METHODS: hUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model. RESULTS: hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury. CONCLUSIONS: hUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , Cordão Umbilical , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Vesículas Extracelulares/metabolismo , Humanos , Animais , Cordão Umbilical/citologia , Camundongos , Síndrome do Desconforto Respiratório/terapia , Meios de Cultivo Condicionados/farmacologia , Transplante de Células-Tronco Mesenquimais/métodos , Modelos Animais de Doenças , Células CultivadasRESUMO
Cutaneous leishmaniasis is an infectious disease that may lead to a single or multiple disseminated cutaneous lesions. The mechanisms involved in Leishmania dissemination to different areas of the skin and the internal organs remain poorly understood. Evidence shows that Very Late Antigen-4 (VLA-4)-dependent phagocyte adhesion is impaired by Leishmania infection, which may be related to the mechanisms of parasite dissemination. We investigated factors potentially associated with decreased VLA-4-mediated adhesion in Leishmania-infected macrophages, including lipid raft-mediated VLA-4 mobilization along the cellular membrane, integrin cluster formation at the cell base (adhesion site), and focal adhesion complex assembly. Phagocytes treated with Methyl-ß-Cyclodextrin (MßCD) demonstrated reduced adhesion, similarly to Leishmania amazonensis-infected J774 cells. Infected and MßCD-treated macrophages presented decreased VLA-4 mobilization to the adhesion plane, as well as reduced integrin clustering. Leishmania amazonensis-infected cells exhibited talin depletion, as well as a decreased mobilization of adhesion complex proteins, such as talin and viculin, which were associated with lower VLA-4 concentrations at the adhesion site and limited cell-spreading. Our results suggest that Leishmania infection may modulate the firm adhesion phase of the cell-spreading process, which could contribute to the bloodstream dissemination of infected cells.
Assuntos
Leishmania mexicana , Leishmania , Leishmaniose Cutânea , Humanos , Integrina alfa4beta1 , Talina , Leishmaniose Cutânea/parasitologia , Análise por ConglomeradosRESUMO
Biofilms are complex microecosystems with valuable ecological roles that can shelter a variety of microorganisms. Spirochetes from the genus Leptospira have been observed to form biofilms in vitro, in rural environments, and in the kidneys of reservoir rats. The genus Leptospira is composed of pathogenic and non-pathogenic species, and the description of new species is ongoing due to the advent of whole genome sequencing. Leptospires have increasingly been isolated from water and soil samples. To investigate the presence of Leptospira in environmental biofilms, we collected three distinct samples of biofilms formed in an urban setting with poor sanitation: Pau da Lima, in Salvador, Bahia, Brazil. All biofilm samples were negative for the presence of pathogenic leptospires via conventional PCR, but cultures containing saprophytic Leptospira were identified. Whole genomes were generated and analyzed for twenty isolates obtained from these biofilms. For species identification, we used digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analysis. The obtained isolates were classified into seven presumptive species from the saprophytic S1 clade. ANI and dDDH analysis suggest that three of those seven species were new. Classical phenotypic tests confirmed the novel isolated bacteria as saprophytic Leptospira. The isolates presented typical morphology and ultrastructure according to scanning electron microscopy and formed biofilms under in vitro conditions. Our data indicate that a diversity of saprophytic Leptospira species survive in the Brazilian poorly sanitized urban environment, in a biofilm lifestyle. We believe our results contribute to a better understanding of Leptospira biology and ecology, considering biofilms as natural environmental reservoirs for leptospires.
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Leptospira , Leptospirose , Animais , Ratos , Leptospira/genética , Leptospirose/microbiologia , Brasil , Biofilmes , DNARESUMO
Leptospira interrogans is a biofilm-forming pathogen, however, there are few data involving Brazilian strains isolated from dogs and their antimicrobial sensitivity in planktonic and biofilm forms. The potential for biofilm formation and antimicrobial resistance in naturally infected dogs is a fundamental approach towards disease epidemiology and the establishment of consistent prophylaxis and control measures. The objective of this study was to evaluate in vitro biofilm formation of a reference strain (L. interrogans, sv. Copenhageni L1 130 - L20) and of L. interrogans isolated from dogs (C20, C29, C51, C82), with subsequent evaluation of antimicrobial susceptibility in planktonic and biofilm forms. The semi quantification of biofilm production revealed a dynamic process of development over time, with mature biofilm formation early on the seventh day of incubation. All strains were efficient for in vitro biofilm formation and, in this form, they were considerably more resistant compared to their planktonic form, with MIC90 of 1600 µg/mL for amoxicillin, 800 µg/mL for ampicillin, and >1600 µg/mL for doxycycline and ciprofloxacin. The strains studies were isolated on naturally infected dogs that might act as reservoirs and sentinels for human infections. The potential to antimicrobial resistance together with the close relation between dogs and humans indicates the need for greater actions on disease control and surveillance. Moreover, biofilm formation may contribute to the persistence of Leptospira interrogans in the host and these animals can act as chronic carriers, disseminating the agent in the environment.
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INTRODUCTION: Here we report estimates of glomerular basement membrane (GBM) thickness in the Brazilian population performed using direct (DM) and orthogonal interception methods (OIM), and comment on potential sources of variation among estimates made by different laboratories. METHODOLOGY: A total of 38 patients, ranging from 3 to 78 years of age, 26 (68%) males and 12 (32%) females, were submitted to kidney biopsy procedures for renal disease diagnosis. Glomeruli were diagnosed with minor histological changes by conventional, immunofluorescence and electron microscopy. GBM thickness was estimated using both DM and OIM methods. RESULTS: Estimates of GBM thickness obtained using DM were higher than those obtained by OIM. However, the application of a correction for non-perpendicular membrane sectioning to DM estimates yielded similar results to those obtained under OIM. The estimated GMB thickness using DM after correction was 289 + 44 nm, versus 287 + 48 nm by OIM. No statistically significant differences were detected in GMB thickness, nor with respect to patient age or sex. CONCLUSIONS: GBM thickness in the studied Brazilian population measured approximately 290 nm. The application of criteria for estimating the shortest distance between the endothelial and podocyte cell membranes with correction for non-perpendicular membrane sectioning can increase the accuracy of GBM thickness estimates using DM and OIM.
Assuntos
Membrana Basal Glomerular/patologia , Nefropatias/patologia , Adolescente , Adulto , Idoso , Biópsia , Brasil , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Adulto JovemAssuntos
COVID-19/complicações , Traço Falciforme/complicações , Doenças Vasculares/diagnóstico , COVID-19/virologia , Evolução Fatal , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Doenças Vasculares/etiologia , Doenças Vasculares/terapiaRESUMO
Rattus norvegicus (Norway rat) is the main reservoir host of pathogenic Leptospira, the causative agent of leptospirosis, in urban environments. Pathogenic Leptospira forms biofilms in the environment, possibly contributing for bacterial survival and maintenance. Nonetheless, biofilms have not yet been studied in natural animal reservoirs presenting leptospiral renal carriage. Here, we described biofilm formation by pathogenic Leptospira inside the renal tubules of R. norvegicus naturally infected and captured in an urban slum endemic for leptospirosis. From the 65 rats carrying Leptospira in their kidneys, 24 (37%) presented biofilms inside the renal tubules. The intensity of leptospiral colonization in the renal tubules (OR: 1.00; 95% CI 1.05-1.1) and the type of occlusion pattern of the colonized renal tubules (OR: 3.46; 95% CI 1.20-9.98) were independently associated with the presence of Leptospira biofilm. Our data showed that Leptospira interrogans produce biofilms during renal chronic colonization in rat reservoirs, suggesting a possible role for leptospiral biofilms in the pathogenesis of leptospirosis and bacterial carriage in host reservoirs.
Assuntos
Biofilmes , Reservatórios de Doenças/microbiologia , Leptospira interrogans/fisiologia , Leptospirose/veterinária , Doenças dos Roedores/microbiologia , Animais , Rim/microbiologia , Leptospira interrogans/genética , Leptospirose/microbiologia , Masculino , RatosRESUMO
Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen-/- ) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen-/- and wild-type promastigotes were similar, but axenic and intracellular LbCen-/- amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen-/- were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen-/- , differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen-/- can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.
Assuntos
Leishmania braziliensis , Leishmania , Leishmaniose Cutânea , Animais , Sistemas CRISPR-Cas , Leishmania braziliensis/genética , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Combinação Trimetoprima e SulfametoxazolRESUMO
BACKGROUND: Survival and therapeutic actions of bone marrow-derived mesenchymal stem cells (BMMSCs) can be limited by the hostile microenvironment present during acute spinal cord injury (SCI). Here, we investigated whether BMMSCs overexpressing insulin-like growth factor 1 (IGF-1), a cytokine involved in neural development and injury repair, improved the therapeutic effects of BMMSCs in SCI. METHODS: Using a SCI contusion model in C57Bl/6 mice, we transplanted IGF-1 overexpressing or wild-type BMMSCs into the lesion site following SCI and evaluated cell survival, proliferation, immunomodulation, oxidative stress, myelination, and functional outcomes. RESULTS: BMMSC-IGF1 transplantation was associated with increased cell survival and recruitment of endogenous neural progenitor cells compared to BMMSC- or saline-treated controls. Modulation of gene expression of pro- and anti-inflammatory mediators was observed after BMMSC-IGF1 and compared to saline- and BMMSC-treated mice. Treatment with BMMSC-IGF1 restored spinal cord redox homeostasis by upregulating antioxidant defense genes. BMMSC-IGF1 protected against SCI-induced myelin loss, showing more compact myelin 28 days after SCI. Functional analyses demonstrated significant gains in BMS score and gait analysis in BMMSC-IGF1, compared to BMMSC or saline treatment. CONCLUSIONS: Overexpression of IGF-1 in BMMSC resulted in increased cell survival, immunomodulation, myelination, and functional improvements, suggesting that IGF-1 facilitates the regenerative actions of BMMSC in acute SCI.
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Fator de Crescimento Insulin-Like I/genética , Transplante de Células-Tronco Mesenquimais , Células-Tronco Neurais/transplante , Traumatismos da Medula Espinal/terapia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Modelos Animais de Doenças , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Bainha de Mielina/genética , Bainha de Mielina/patologia , Células-Tronco Neurais/citologia , Recuperação de Função Fisiológica , Regeneração/genética , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologiaRESUMO
Lipophosphoglycan (LPG) is the major surface glycoconjugate of metacyclic Leishmania promastigotes and is associated with virulence in various species of this parasite. Here, we generated a LPG-deficient mutant of Leishmania infantum, the foremost etiologic agent of visceral leishmaniasis in Brazil. The L. infantum LPG-deficient mutant (Δlpg1) was obtained by homologous recombination and complemented via episomal expression of LPG1 (Δlpg1 + LPG1). Deletion of LPG1 had no observable effect on parasite morphology or on the presence of subcellular organelles, such as lipid droplets. While both wild-type and add-back parasites reached late phase in axenic cultures, the growth of Δlpg1 parasites was delayed. Additionally, the deletion of LPG1 impaired the outcome of infection in murine bone marrow-derived macrophages. Although no significant differences were observed in parasite load after 4 h of infection, survival of Δlpg1 parasites was significantly reduced at 72 h post-infection. Interestingly, L. infantum LPG-deficient mutants induced a strong NF-κB-dependent activation of the inducible nitric oxide synthase (iNOS) promoter compared to wild type and Δlpg1 + LPG1 parasites. In conclusion, the L. infantum Δlpg1 mutant constitutes a powerful tool to investigate the role(s) played by LPG in host cell-parasite interactions.
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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.
Assuntos
Apoptose , Mitose , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/virologia , Infecção por Zika virus/metabolismo , Zika virus/metabolismo , Células Cultivadas , Humanos , Células-Tronco Neurais/patologia , Infecção por Zika virus/patologiaRESUMO
Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum.
Assuntos
Antígenos de Bactérias/metabolismo , Fator I do Complemento/metabolismo , Evasão da Resposta Imune , Leptospira/fisiologia , Leptospirose/imunologia , Antígenos de Bactérias/imunologia , Sobrevivência Celular , Complemento C3b/metabolismo , Complemento C4b/metabolismo , Fator H do Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Cadeia Alimentar , Humanos , Leptospira/patogenicidade , Ligação ProteicaRESUMO
We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.
Assuntos
Variação Genética , Genótipo , Leptospira interrogans/classificação , Leptospira interrogans/isolamento & purificação , Leptospirose/veterinária , Doenças dos Roedores/microbiologia , Animais , Cidades , Feminino , Imuno-Histoquímica , Rim/microbiologia , Rim/patologia , Leptospira interrogans/genética , Leptospirose/microbiologia , Leptospirose/patologia , Masculino , Áreas de Pobreza , RatosRESUMO
Abstract We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.(AU)
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Animais , Cobaias , Leptospira interrogans , Ratos , LeptospiroseRESUMO
Abstract We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.
Assuntos
Animais , Feminino , Masculino , Ratos , Variação Genética , Genótipo , Leptospira interrogans/classificação , Leptospira interrogans/isolamento & purificação , Leptospirose/veterinária , Doenças dos Roedores/microbiologia , Cidades , Imuno-Histoquímica , Rim/microbiologia , Rim/patologia , Leptospira interrogans/genética , Leptospirose/microbiologia , Leptospirose/patologia , Áreas de PobrezaRESUMO
Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] µm(2) for uninfected and 41 [34-51] µm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 µm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 µm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.
Assuntos
Fibronectinas/metabolismo , Integrina beta1/metabolismo , Leishmania/metabolismo , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Monócitos/metabolismo , Monócitos/parasitologia , Adesão Celular/fisiologia , Humanos , Integrina alfa4beta1/metabolismo , Cinética , Leucócitos/metabolismo , Leucócitos/parasitologiaRESUMO
BACKGROUND: Extracellular vesicles (EVs) are structures with phospholipid bilayer membranes and 100-1000 nm diameters. These vesicles are released from cells upon activation of surface receptors and/or apoptosis. The production of EVs by dendritic cells, mast cells, macrophages, and B and T lymphocytes has been extensively reported in the literature. EVs may express MHC class II and other membrane surface molecules and carry antigens. The aim of this study was to investigate the role of EVs from Leishmania-infected macrophages as immune modulatory particles. METHODOLOGY/PRINCIPAL FINDINGS: In this work it was shown that BALB/c mouse bone marrow-derived macrophages, either infected in vitro with Leishmania amazonensis or left uninfected, release comparable amounts of 50-300 nm-diameter extracellular vesicles (EVs). The EVs were characterized by flow cytometry and electron microscopy. The incubation of naïve macrophages with these EVs for 48 hours led to a statistically significant increase in the production of the cytokines IL-12, IL-1ß, and TNF-α. CONCLUSIONS/SIGNIFICANCE: EVs derived from macrophages infected with L. amazonensis induce other macrophages, which in vivo could be bystander cells, to produce the proinflammatory cytokines IL-12, IL-1ß and TNF-α. This could contribute both to modulate the immune system in favor of a Th1 immune response and to the elimination of the Leishmania, leading, therefore, to the control the infection.
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Vesículas Extracelulares/imunologia , Leishmania/imunologia , Leishmania/parasitologia , Leishmaniose/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Inhibition of Trypanosoma brucei and Leishmania spp. sirtuins has shown promising antiparasitic activity, indicating that these enzymes may be used as targets for drug discovery against trypanosomatid infections. In the present work we carried out a virtual screening focused on the C pocket of Sir2 from Trypanosoma cruzi. Using this approach, the best ligand found was nicotinamide. In vitro tests confirmed the anti-T. cruzi activity of nicotinamide on epimastigote and trypomastigote forms. Moreover, treatment of T. cruzi-infected macrophages with nicotinamide caused a significant reduction in the number of amastigotes. In addition, alterations in the mitochondria and an increase in the vacuolization in the cytoplasm were observed in epimastigotes treated with nicotinamide. Analysis of the complex of Sir2 and nicotinamide revealed the details of the possible ligand-target interaction. Our data reveal a potential use of TcSir2 as a target for anti-T. cruzi drug discovery.
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Doença de Chagas/parasitologia , Niacinamida/farmacologia , Sirtuínas/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Sobrevivência Celular , Doença de Chagas/tratamento farmacológico , Descoberta de Drogas , Humanos , Ligantes , Macrófagos/parasitologia , Modelos Moleculares , Proteínas de Protozoários , Alinhamento de Sequência , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/fisiologiaRESUMO
The genus Botryococcus comprises a group of cosmopolitan species of freshwater colonial green algae, some of which synthesize and accumulate an unusually high level (15-76%) of liquid hydrocarbons. This characteristic suggests the possibility of exploiting species from this group as renewable sources for jet fuel. An oil-rich strain of Botryococcus (Trebouxiophyceae) was isolated from a freshwater pond in the state of Bahia, Brazil, and is presently maintained under standard conditions at the Culture Collection of the Institute of Biology, Federal University of Bahia. The taxonomic classification of the species was based on light microscopy (LM); and TEM and SEM were used to better characterize its features, which have never before been described at this level. The LM characterization included the size of the colonies (35.7-157 µm) and cells (8-10 × 5-9 µm) and their connection in sub-colonies by mucilaginous strands, as well as the presence of mucilaginous processes on the periphery of some of the colonies, with most of the cells included inside the colony. Reproduction occurred through divisions into two to four autospores. These features characterized the species as Botryococcus terribilis Komárek and Marvan. The TEM study showed, in addition to the presence of starch grains, pyrenoids that are penetrated by thick thylakoids. The pyrenoid bodies appear as electron-dense protein inclusions located in the chloroplast and surrounded by a starch sheath. These structures, which contain most if not all of the Ribulose-1,5-bisphosphate carboxylase oxygenase in several algal species that have been studied closely, are newly discovered for this species.