ABSTRACT
BACKGROUND: Silicosis is an occupational disease that affects workers who inhale silica particles, leading to extensive lung fibrosis and ultimately causing respiratory failure. Mesenchymal stromal cells (MSCs) have been shown to exert therapeutic effects in lung diseases and represent an alternative treatment for silicosis. Recently, it has been suggested that similar effects can be achieved by the therapeutic use of extracellular vesicles (EVs) obtained from MSCs. The aim of this study was to investigate the effects of adipose-tissue-derived MSCs (AD-MSCs) or their EVs in a model of silicosis. METHODS: Silicosis was induced by intratracheal instillation of silica in C57BL/6 mice. After the onset of disease, animals received saline, AD-MSCs, or EVs, intratracheally. RESULTS: At day 30, AD-MSCs and EVs led to a reduction in collagen fiber content, size of granuloma, and in the number of macrophages inside granuloma and in the alveolar septa. In addition, the expression levels of interleukin 1ß and transforming growth factor beta in the lungs were decreased. Higher dose of EVs also reduced lung static elastance when compared with the untreated silicosis group. CONCLUSIONS: Both AD-MSCs and EVs, locally delivered, ameliorated fibrosis and inflammation, but dose-enhanced EVs yielded better therapeutic outcomes in this model of silicosis.
Subject(s)
Adipose Tissue/transplantation , Disease Models, Animal , Extracellular Vesicles/transplantation , Mesenchymal Stem Cell Transplantation/methods , Silicon Dioxide/toxicity , Silicosis/therapy , Adipose Tissue/cytology , Animals , Female , Mesenchymal Stem Cells , Mice , Mice, Inbred C57BL , Silicosis/pathology , Treatment OutcomeABSTRACT
We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1×105), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; 2) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased keratinocyte-derived chemokine (KC, a mouse analog of interleukin-8) and transforming growth factor-ß levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial growth factor. Only BM-MSCs (IV > IT) increased the number of M2 macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M2.
Subject(s)
Bone Marrow Cells/physiology , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/physiology , Pulmonary Emphysema/pathology , Pulmonary Emphysema/therapy , Animals , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Random Allocation , Treatment OutcomeABSTRACT
BACKGROUND: Influenza viruses cause highly contagious acute respiratory illnesses with significant mortality, especially among young children, elderly people, and individuals with serious medical conditions. This encourages the development of new treatments for human flu. Biotherapies are diluted solutions prepared from biological products compounded following homeopathic procedures. OBJECTIVES: To develop a biotherapy prepared from the infectious influenza A virus (A/Aichi/2/68 H3N2) and to verify its in vitro response. METHODS: The ultradiluted influenza virus solution was prepared in the homeopathic dilution 30dH, it was termed Influenzinum RC. The cellular alterations induced by this preparation were analyzed by optical and electron microscopy, MTT and neutral red assays. Glycolytic metabolism (PFK-1) was studied by spectrophotometric assay. Additionally, the production of tumor necrosis factor-α (TNF-α) by J774.G8 macrophage cells was quantified by ELISA before and after infection with H3N2 influenza virus and treatment. RESULTS: Influenzinum RC did not cause cytotoxic effects but induced morphological alterations in Madin-Darby canine kidney (MDCK) cells. After 30 days, a significant increase (p < 0.05) in mitosis rate was detected compared to control. MDCK mitochondrial activity was changed after treatment for 10 and 30 days. Treatment significantly diminished (p < 0.05) PFK-1 activity. TNF-α in biotherapy-stimulated J774.G8 macrophages indicated a significant (p < 0.05) increase in this cytokine when the cell supernatant was analyzed. CONCLUSION: Influenzinum RC altered cellular and biochemical features of MDCK and J774G8 cells.
Subject(s)
Homeopathy/methods , Influenza A Virus, H3N2 Subtype/physiology , Animals , Biological Therapy , Cell Line/virology , Dogs , Indicator Dilution Techniques , Macrophages/metabolism , Microscopy, Electron , Mitosis , Phosphofructokinase-1/metabolism , Solutions/analysis , Spectrophotometry , Tumor Necrosis Factor-alpha/metabolismABSTRACT
SCOPE: Perinatal maternal moderately high-fat diet (mHFD) is associated with obesity and fatty liver disease in offspring, and maternal fish oil (FO: n-3 PUFA source) supplementation may attenuate these disorders. This study evaluates the effects of FO given to pregnant rats fed a mHFD on the offspring's liver at weaning. METHODS AND RESULTS: Female Wistar rats receive an isoenergetic, control (CT: 10.9% from fat) or high-fat (HF: 28.7% from fat) diet before mating, and throughout pregnancy and lactation. FO supplementation (HFFO: 2.9% of FO in the HF diet) is given to one subgroup of HF dams during pregnancy. At weaning, male and female mHFD offspring display higher body mass, adiposity, and hepatic cellular damage, steatosis, and inflammation, accompanied by increased damaged mitochondria. FO does not protect pups from systemic metabolic alterations and partially mitigates hepatic histological damage induced by mHFD only in females. However, FO reduces mRNA expression of lipogenic genes, and mitochondrial damage, and modified mitochondrial morphology suggestive of early adaptations via mitochondrial dynamics. CONCLUSIONS: Gestational FO supplementation has limited beneficial effects on the damage caused by perinatal mHFD consumption in offspring's liver at weaning. However, FO imprinting effect on lipid metabolism and mitochondria may have beneficial long-term outcomes.
Subject(s)
Fish Oils , Non-alcoholic Fatty Liver Disease , Pregnancy , Humans , Rats , Male , Female , Animals , Fish Oils/pharmacology , Diet, High-Fat/adverse effects , Rats, Wistar , Obesity/metabolism , Liver/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Mitochondria , Maternal Nutritional Physiological Phenomena , Dietary SupplementsABSTRACT
Acute ischemic stroke is associated with pulmonary complications, and often dexmedetomidine and propofol are used to decrease cerebral metabolic rate. However, it is unknown the immunomodulatory actions of dexmedetomidine and propofol on brain and lungs during acute ischemic stroke. The effects of dexmedetomidine and propofol were compared on perilesional brain tissue and lung damage after acute ischemic stroke in rats. Further, the mean amount of both sedatives was directly evaluated on alveolar macrophages and lung endothelial cells primarily extracted 24-h after acute ischemic stroke. In twenty-five Wistar rats, ischemic stroke was induced and after 24-h treated with sodium thiopental (STROKE), dexmedetomidine and propofol. Dexmedetomidine, compared to STROKE, reduced diffuse alveolar damage score [median(interquartile range); 12(7.8-15.3) vs. 19.5(18-24), p = 0.007)], bronchoconstriction index [2.28(2.08-2.36) vs. 2.64(2.53-2.77), p = 0.006], and TNF-α expression (p = 0.0003), while propofol increased VCAM-1 expression compared to STROKE (p = 0.0004). In perilesional brain tissue, dexmedetomidine, compared to STROKE, decreased TNF-α (p = 0.010), while propofol increased VCAM-1 compared to STROKE (p = 0.024). In alveolar macrophages and endothelial cells, dexmedetomidine decreased IL-6 and IL-1ß compared to STROKE (p = 0.002, and p = 0.040, respectively), and reduced IL-1ß compared to propofol (p = 0.014). Dexmedetomidine, but not propofol, induced brain and lung protection in experimental acute ischemic stroke.
Subject(s)
Brain/drug effects , Dexmedetomidine/administration & dosage , Hypnotics and Sedatives/administration & dosage , Ischemic Stroke/drug therapy , Lung/drug effects , Propofol/administration & dosage , Animals , Brain Ischemia/prevention & control , Dexmedetomidine/adverse effects , Disease Models, Animal , Endothelial Cells/drug effects , Hypnotics and Sedatives/adverse effects , Interleukin-1beta/biosynthesis , Interleukin-6/biosynthesis , Macrophages, Alveolar/drug effects , Male , Propofol/adverse effects , Rats , Rats, Wistar , Thiopental , Tumor Necrosis Factor-alpha/biosynthesis , Vascular Cell Adhesion Molecule-1/biosynthesisABSTRACT
In the present study, we analyzed the role of purinergic P2X7 receptor in Mycobacterium tuberculosis infection and host interaction mechanisms in vitro and in vivo. For experimental procedures, a macrophage murine cell line RAW 264.7, and male Swiss, wild-type C57BL/6 and P2X7 receptor knockout (P2X7R−/−) mice were used throughout this study. We have demonstrated that treatment of RAW 264.7 cells with ATP (3 and 5 mM) resulted in a statistically significant reduction of M. tuberculosis-colony-forming units. The purinergic P2X7 receptor expression was found significantly augmented in the lungs of mice infected with M. tuberculosis H37Rv. Infected wild-type mice showed a marked increase in the spleen weight, in comparison to non-infected animals. Furthermore, M. tuberculosis-infected P2X7R−/− mice showed an increase of M. tuberculosis burden in lung tissue, when compared to infected wild-type mice. In P2X7R−/− spleens, we observed a significant decrease in the populations of Treg (CD4+Foxp3+), T cells (CD4+, CD8+CD25+ and CD4+CD25+), dendritic cells (CD11c+) and B220+ cells. However, a significant increase in CD11b+ cells was observed in P2X7R−/− mice, when compared to wild-type animals. In the lungs, P2X7R−/− M. tuberculosisinfected mice exhibited pulmonary infiltrates containing an increase of Treg cells (CD4+Foxp3+), T cells (CD4+ and CD8+) and a decrease in the B220+ cells, when compared with wild-type M. tuberculosis-infected mice. The findings observed in the present study provide novel evidence on the role of P2X7 receptors in the pathogenesis of tuberculosis.
Subject(s)
Macrophages/immunology , Mycobacterium tuberculosis/immunology , Receptors, Purinergic P2X7/immunology , Tuberculosis/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line , Dendritic Cells/immunology , Host-Pathogen Interactions/immunology , Lung/metabolism , Lung/microbiology , Lymphocyte Count , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Purinergic P2X7/genetics , Tuberculosis/microbiologyABSTRACT
BACKGROUND: The Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens characterized by decreased IL-2 synthesis. Trypanosoma cruzi mucin (Tc Muc) has been implicated in this phenomenom. These molecules contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated the ability of Tc Muc to modulate the activation of CD4(+) T cells. Our data show that cross-linking of CD3 on naïve CD4(+) T cells in the presence of Tc Muc resulted in the inhibition of both cytokine secretion and proliferation. We further show that the sialylated O-Linked Glycan residues from tc mucin potentiate the suppression of T cell response by inducing G1-phase cell cycle arrest associated with upregulation of mitogen inhibitor p27(kip1). These inhibitory effects cannot be reversed by the addition of exogenous IL-2, rendering CD4(+) T cells anergic when activated by TCR triggering. Additionally, in vivo administration of Tc Muc during T. cruzi infection enhanced parasitemia and aggravated heart damage. Analysis of recall responses during infection showed lower frequencies of IFN-γ producing CD4(+) T cells in the spleen of Tc Muc treated mice, compared to untreated controls. CONCLUSIONS/SIGNIFICANCE: Our results indicate that Tc Muc mediates inhibitory efects on CD4(+) T expansion and cytokine production, by blocking cell cycle progression in the G1 phase. We propose that the sialyl motif of Tc Muc is able to interact with sialic acid-binding Ig-like lectins (Siglecs) on CD4(+) T cells, which may allow the parasite to modulate the immune system.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Chagas Disease/immunology , Disease Susceptibility/immunology , Sialoglycoproteins/immunology , Trypanosoma cruzi/immunology , Animals , CD3 Complex/immunology , Cell Cycle Checkpoints/immunology , Cell Proliferation , G1 Phase/immunology , Interferon-gamma/immunology , Interleukin-2/immunology , Male , Mice , Mice, Inbred BALB C , Mucins/immunologyABSTRACT
Introduction: Influenza viruses have been responsible for highly contagious acute respiratory illnesses with high mortality, mainly in the elderly, which encourages the development of new drugs for the treatment of human flu. The biotherapics are medicines prepared from biological products, which are not chemically defined. They are compounded following the homeopathic procedures indicated for infectious diseases with known etiology [1]. Aim: The purpose of the present study is to verify cellular alterations induced by a biotherapic prepared from the infectious influenza A virus. Methodology: This biotherapic was prepared for this study in the homeopathic potency of 30X according to the Brazilian Homeopathic Pharmacopeia [2]. The concentration of 10% was not cytotoxic to cells, as verified by neutral red assay. The cellular alterations observed in MDCK cells were analyzed by optical microscopy for the quantification of mitosis, nucleoli and lipid bodies. The mitochondrial activity was assessed by MTT assay and the phosphosfructokinase-1 (PFK-1) enzyme activity was analyzed on the MDCK cells treated for 5, 10 and 30 days. Macrophages J778.G8 were treated with this biotherapic to evaluate the immunostimulatory cytokine release. Results: The cellular alterations observed in MDCK cells were verified by optical microscopy. The number of lipid bodies present in MDCK cells stimulated for 10 days was significantly lower (p <0.05) when compared to controls. The biotherapic significantly increased (p <0.05) the number of mitosis and the mitochondrial activity of MDCK cells stimulated for 10 and 30 days. These changes were confirmed by a significant reduction (p <0.05) on the PFK-1 activity. These results suggest that the biotherapic was able to activate the Krebs cycle and pentosephosphate metabolism to the generation of amino acids and nucleotides, situations common to cells whose rate of mitosis is increased. The quantification of immunostimulatory cytokines by macrophages J774.G8 indicated that the tumor necrosis factor (TNF-?) production was higher (p <0.05) in the supernatant of the macrophages pre-treated with this biotherapic and infected with influenza virus, suggesting an activation of the macrophages by this biotherapic. Conclusion: This biotherapic is able to induce some cellular alterations, which show strong evidence that it might be a promising option for the human flu. New experiments are being developed to understand the mechanisms of action of this biotherapic.
Subject(s)
Biotherapics , Influenza, Human/immunologyABSTRACT
A progressive destruction of the myocardium occurs in approximately 30% of Trypanosoma cruzi-infected individuals, causing chronic chagasic cardiomyopathy, a disease so far without effective treatment. Syngeneic bone marrow cell transplantation has been shown to cause repair and improvement of heart function in a number of studies in patients and animal models of ischemic cardiopathy. The effects of bone marrow transplant in a mouse model of chronic chagasic cardiomyopathy, in the presence of the disease causal agent, ie, the T. cruzi, are described herein. Bone marrow cells injected intravenously into chronic chagasic mice migrated to the heart and caused a significant reduction in the inflammatory infiltrates and in the interstitial fibrosis characteristics of chronic chagasic cardiomyopathy. The beneficial effects were observed up to 6 months after bone marrow cell transplantation. A massive apoptosis of myocardial inflammatory cells was observed after the therapy with bone marrow cells. Transplanted bone marrow cells obtained from chagasic mice and from normal mice had similar effects in terms of mediating chagasic heart repair. These results show that bone marrow cell transplantation is effective for treatment of chronic chagasic myocarditis and indicate that autologous bone marrow transplant may be used as an efficient therapy for patients with chronic chagasic cardiomyopathy.
Subject(s)
Bone Marrow Transplantation , Chagas Cardiomyopathy/surgery , Myocardium/pathology , Animals , Apoptosis/physiology , Cell Movement , Chagas Cardiomyopathy/etiology , Chronic Disease , Disease Models, Animal , Female , Fibrosis/pathology , Fluorescent Antibody Technique , In Situ Nick-End Labeling , Male , Trypanosoma cruziABSTRACT
A biocompatibilidade de uma membrana de pericárdio bovino foi avaliada em tecido subcutâneo de camundongos 3,7, 15, 30 e 60 dias após a implantação. Os componentes celulares da resposta inflamatória, a degradação da membranae as características do colágeno foram analisadas em cortes histológicos corados pela hematoxilina-eosina, tricrômico de Masson e Picro-Sírius, respectivamente. Para verificar seu potencial como carreador celular, osteoblastos humanos(hFOB1.19, ATCC) foram semeados sobre a membrana e mantidos em DMEM/F12 por 7 dias. Os resultados in vitro mostraram que os osteoblastos proliferaram em monocamada na superfície da membrana, mas sem penetrar em seu interior. A análise dos cortes histológicos demonstrou 3 dias após a implantação apenas a formação da rede de fibrina. Aos 7 dias, o material implantado estava circundado por células inflamatórias mononucleares, com pouca penetração celular no seu interior. Após 15 dias foi observado um intenso infiltrado inflamatório em contato e dentro do material,bem como sinais de degradação interna e externa. No período de 30 dias, o material, em processo bastante avançado de absorção, estava totalmente tomado por fibroblastos e macrófagos. Aos 60 dias pós-implantação, o material não foi maisdetectado em quaisquer dos animais e a tecido subcutâneo apresentava-se normal. Os cortes corados com Picro-Sírius e observados sob luz polarizada mostraram o remodelamento tecidual. Em conclusão, a membrana de pericárdio é bioabsorvívele biocompatível, porém, in vitro, não proporciona uma adequada matriz tridimensional para osteoblastos.
The biocompatibility of a pericardium membrane was evaluated in the subcutaneous tissue of mouse killed 3, 7, 15, 30 and 60 days post implantation. The cellular components of inflammatory infiltrate, the membrane degradation, and the collagen characteristic were analyzed in histological sections stained with hematoxilyn and eosin, Tricromic of Masson and Sirius Red, respectively. The potential features as a tissue engineering scaffold was tested in vitro using human osteoblasts (h.Fob 1.19, ATCC) seeded over the membrane and maintained for 7 days in DMEM/F12. We observed in vitro the monolayer proliferation of osteoblasts, but without penetrating in the membrane. The histological sections showed after 3 days of implantation only the presence of a fibrin net. At the 7-day period, mononuclear inflammatory cells were observed around the implant, but a few one were observed inside the membrane. After 15 days the inflammatory infiltrate was more intense than in the previous period and the cells were inside and in close contact to the material showing evident signs of internal and external degradation. The implant degradation was intense after 30 days and theresidual material was fulfilled of fibroblasts and macrophages. No signs of membrane were observed after 60 days in any animals and the subcutaneous tissue presented normal aspect. Sirius Red staining at polarized light had evidenced the tissue remodeling throughout the experimental periods. In conclusion, the pericardium membrane is bioabsorbable and biocompatible, but, in vitro, do not fulfill the requirements as a tridimensional scaffold to osteoblast.