Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.779
Filtrar
1.
J Toxicol Sci ; 45(9): 569-579, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879256

RESUMO

Indoxyl, a derivative of indole originating from tryptophan, may undergo phase-II sulfate-conjugation pathway, thereby forming indoxyl sulfate (IS) in vivo. We previously reported that IS, a well-known uremic toxin, can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model. Using the same cell model, the current study aimed to investigate whether indole and indoxyl (the metabolic precursors of indoxyl and IS, respectively) may cause macrophage immune dysfunction. Results obtained indicated that intracellular oxidation level and cytotoxicity markedly increased upon treatment with indole and indoxyl, in comparison with IS. Incubation of the cells with indole and indoxyl also resulted in attenuated phagocytic activity. Human serum albumin (HSA)-binding assay confirmed that tryptophan and IS, but not indole and indoxyl, could selectively bind to the site II in HSA. Collectively, the results indicated that indole and indoxyl may strongly down-regulate the phagocytic immune function of macrophages, whereas IS, formed upon sulfate conjugation of indoxyl, may exhibit enhanced HSA-binding capability, thereby reducing the adverse effects of indoxyl.


Assuntos
Indóis/efeitos adversos , Macrófagos/imunologia , Macrófagos/metabolismo , Oxirredução/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células HL-60 , Humanos , Indicã/metabolismo , Macrófagos/efeitos dos fármacos , Ligação Proteica , Albumina Sérica/metabolismo , Triptofano/metabolismo
2.
Int J Nanomedicine ; 15: 4919-4932, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764925

RESUMO

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. Diagnosing AD before symptoms arise will facilitate earlier intervention. The early diagnostic approaches are thus urgently needed. Methods: The multifunctional nanoparticles W20/XD4-SPIONs were constructed by the conjugation of oligomer-specific scFv antibody W20 and class A scavenger receptor (SR-A) activator XD4 onto superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs' stability and uniformity in size were measured by dynamic light scattering and transmission electron microscopy. The ability of W20/XD4-SPIONs for recognizing Aß oligomers (AßOs) and promoting AßOs phagocytosis was assessed by immunocytochemistry and flow cytometry analysis. The blood-brain barrier permeability of W20/XD4-SPIONs was determined by a co-culture transwell model. The in vivo probe distribution of W20/XD4-SPIONs in AD mouse brains was detected by magnetic resonance imaging (MRI). Results: W20/XD4-SPIONs, as an AßOs-targeted molecular MRI contrast probe, readily reached pathological AßOs regions in brains and distinguished AD transgenic mice from WT controls. W20/XD4-SPIONs retained the property of XD4 for SR-A activation and significantly promoted microglial phagocytosis of AßOs. Moreover, W20/XD4-SPIONs exhibited the properties of good biocompatibility, high stability and low cytotoxicity. Conclusion: Compared with W20-SPIONs or XD4-SPIONs, W20/XD4-SPIONs show the highest efficiency for AßOs-targeting and significantly enhance AßOs uptake by microglia. As a molecular probe, W20/XD4-SPIONs also specifically and sensitively bind to AßOs in AD brains to provide an MRI signal, demonstrating that W20/XD4-SPIONs are promising diagnostic agents for early-stage AD. Due to the beneficial effect of W20 and XD4 on neuropathology, W20/XD4-SPIONs may also have therapeutic potential for AD .


Assuntos
Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/imunologia , Imunoconjugados/química , Nanopartículas de Magnetita/química , Receptores Depuradores/metabolismo , Anticorpos de Cadeia Única/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Especificidade de Anticorpos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Diagnóstico Precoce , Imunoconjugados/farmacologia , Imagem por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/patologia , Nanopartículas Multifuncionais/química , Fagocitose/efeitos dos fármacos , Anticorpos de Cadeia Única/imunologia
3.
Nat Commun ; 11(1): 4027, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788676

RESUMO

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics.


Assuntos
Apoptose , Imageamento Tridimensional , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Fagocitose/efeitos dos fármacos , Fosfatidilserinas/metabolismo
4.
Anticancer Res ; 40(8): 4681-4685, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727792

RESUMO

BACKGROUND/AIM: The functions of macrophages change in response to environmental factors such as lipopolysaccharide (LPS). LPS derived from Pantoea agglomerans (LPSp) is involved in macrophage activation and tissue repair when administered dermally. LPSp-activated macrophages may be useful for restoring and maintaining homeostasis of the skin. MATERIALS AND METHODS: Phorbol myristate acetate-treated human monocytes (THP-1 cells) were activated with LPSp. The medium of LPSp-activated THP-1 cells was added to normal human dermal fibroblasts (NHDF cells). After 24 h, the expression of hyaluronan (HA) synthase (HAS)2, hyaluronidase (HYAL)1, and tropoelastin in NHDF cells was analyzed using quantitative real-time PCR. RESULTS: The expression of HAS2 and tropoelastin was significantly increased, but that of HYAL1 was significantly decreased. It was demonstrated that the abilities of HA and elastin synthesis in NHDF cells increased through LPSp-activated THP-1 cells. CONCLUSION: LPSp-activated macrophages may be useful for enhancing the abilities of HA and elastin synthesis in fibroblasts, subsequently improving dysfunction and reducing various age-related disorders.


Assuntos
Elastina/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ácido Hialurônico/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Linhagem Celular , Humanos , Ativação de Macrófagos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Pantoea/metabolismo , Fagocitose/efeitos dos fármacos , Células Th1
5.
Anticancer Res ; 40(8): 4707-4710, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727796

RESUMO

BACKGROUND/AIM: Serum-derived macrophage activating factor (serum-MAF) can rapidly activate macrophage phagocytic activity by inducing characteristic membrane ruffles designated as Frill-like structures. Serum-MAF contains γ-globulin, an activator of phagocytosis. This study examined whether serum-MAF and γ-globulin activate macrophages similarly. MATERIALS AND METHODS: Morphological changes in macrophages were observed by time-lapse imaging and the efficiency of engulfment was analysed quantitatively. Immunological staining of talin-1 and a calpain inhibitor were performed. RESULTS: The engulfment efficiency of serum-MAF- and γ-globulin-activated macrophages was significantly different. Talin-1 showed weak co-localisation with the Frill-like structures. Treatment with a calpain inhibitor similarly down-regulated phagocytosis irrespective of the activation factor. CONCLUSION: There was a difference between macrophage activation mechanisms by γ-globulin and serum-MAF. Talin may slightly contribute to serum-MAF activation. It is possible to distinguish between the calpain-dependent fundamental 'mechanism of phagocytosis' and the activating factor-dependent rapid 'activation mechanism'.


Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Fatores Ativadores de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , gama-Globulinas/farmacologia , Calpaína/farmacologia , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Humanos , Fagocitose/efeitos dos fármacos , Células THP-1
6.
Anticancer Res ; 40(8): 4711-4717, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727797

RESUMO

BACKGROUND: Continuous oral administration of lipopolysaccharide (LPS) enhances the phagocytic ability of macrophages, which is useful for preventing various diseases. Here, we attempted to create an in vitro model of continuous administration of LPS. MATERIALS AND METHODS: RAW264.7 cells were stimulated with LPS three times every 24 h (repeated stimulation), and phagocytic ability and inflammatory cytokine [interleukin-6 (IL6) and tumor necrosis factor-α (TNFα)] production were measured. RESULTS: The phagocytic ability was increased by a single stimulation with LPS and was maintained by repeated stimulation. IL6 production increased with a single stimulation with LPS; however, IL6 production by repeated stimulation with LPS was comparable to that of non-stimulation with LPS. On the other hand, the amount of TNFα was significantly increased by single and repeated stimulation with LPS. CONCLUSION: Repeated stimulation with LPS in RAW264.7 cells triggered a phenotype that was similar to that of macrophages after continuous oral administration of LPS. This suggests that this study model may reproduce the enhancement of macrophage phagocytosis, an effect afforded by continuous oral administration of LPS.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo
7.
Anticancer Res ; 40(8): 4755-4762, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727802

RESUMO

BACKGROUND: Developmental disorders are associated with microglial dysfunction. Oral administration of lipopolysaccharide derived from Pantoea agglomerans bacteria (LPSp) leads to normalization of phagocytic activity of microglia and suppression of inflammation in mice. In this article, we report on a successful trial in which we achieved a significant improvement of symptoms in patients with developmental disorders. PATIENTS AND METHODS: Five pediatric patients diagnosed with autism spectrum disorders (ASD)/attention deficit hyperactivity disorder (ADHD) who visited our clinic received either 0.75 or 1 mg/day LPSp for 6 months or more, in addition to our usual therapy regimens (detoxification therapy, nutritional therapy, and vibration therapy). A survey questionnaire was completed by the patients' parents and evaluated using the Numerical Rating Scale. RESULTS: Behavior, verbal ability, and communication disabilities associated with ASD/ADHD improved in all patients. CONCLUSION: Oral administration of LPSp may represent a new treatment option in the area of developmental disorders where there is currently no treatment available.


Assuntos
Deficiências do Desenvolvimento/tratamento farmacológico , Lipopolissacarídeos/administração & dosagem , Pantoea/química , Administração Oral , Criança , Pré-Escolar , Citocinas/metabolismo , Deficiências do Desenvolvimento/metabolismo , Feminino , Humanos , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fagocitose/efeitos dos fármacos
8.
Anticancer Res ; 40(8): 4457-4464, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727775

RESUMO

BACKGROUND/AIM: Our previous studies suggested that oral administration of lipopolysaccharide (LPS) regulates the progression of various diseases via transformation of tissue-resident macrophages (MΦ). Recently, we characterized microglia transformed by repetitive low-dose LPS treatment (REPELL-microglia) in vitro, and this response was similar to that observed in response to oral administration of LPS in vivo. Here, we examined the characteristics of peritoneal tissue-resident MΦ (pMΦ) transformed by repetitive low-dose LPS treatment (REPELL-pMΦ). MATERIALS AND METHODS: Primary pMΦ were treated with low-dose LPS (1 ng/ml) three times; subsequently, phagocytic activity and gene expression were evaluated. RESULTS: REPELL-pMΦ exhibited high phagocytic activity and elevated expression of Arg1, Gipr, Gdnf, and Fpr2. The gene expression profiles observed in REPELL-pMΦ were distinct from those of REPELL-microglia. CONCLUSION: REPELL-pMΦ have the potential to promote clearance of xenobiotics and to suppress inflammation. The present study also demonstrates the diversity of tissue-resident MΦ transformation that reflect their tissue origin.


Assuntos
Arginase/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Lipopolissacarídeos/efeitos adversos , Macrófagos Peritoneais/fisiologia , Receptores de Formil Peptídeo/genética , Receptores dos Hormônios Gastrointestinais/genética , Administração Oral , Animais , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/administração & dosagem , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Especificidade de Órgãos , Fagocitose/efeitos dos fármacos , Fenótipo , Cultura Primária de Células , Regulação para Cima
9.
Exp Parasitol ; 216: 107939, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32535115

RESUMO

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.


Assuntos
Glucosilceramidase/antagonistas & inibidores , Leishmania/fisiologia , Macrófagos/parasitologia , Fagocitose , Animais , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Doença de Gaucher/complicações , Doença de Gaucher/fisiopatologia , Glucosilceramidase/efeitos dos fármacos , Glucosilceramidase/genética , Inositol/análogos & derivados , Inositol/farmacologia , Leishmania infantum/fisiologia , Leishmania major/fisiologia , Leishmania mexicana/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fagocitose/efeitos dos fármacos
10.
Anticancer Res ; 40(6): 3139-3145, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487608

RESUMO

BACKGROUND/AIM: The aim of this study was to directly compare the anti-infectious and anti-cancer effects of five commercially available glucans. MATERIALS AND METHODS: We used five different glucans isolated from algae, yeast, bacteria, oat, and mushroom. We compared their effects on the stimulation of phagocytosis of blood cells, on the secretion of IL-2, and on the inhibition of melanoma and breast and lung cancers. In addition, we evaluated the effects of glucan supplementation on two experimental models of infection. RESULTS: Most of the tested glucans stimulated phagocytosis and IL-2 secretion, reduced cancer growth, and ameliorated some effects of experimental infections. CONCLUSION: Glucans can produce significant pleiotropic effects, but the activity varies among individual samples.


Assuntos
Anti-Infecciosos/uso terapêutico , Fagocitose/efeitos dos fármacos , beta-Glucanas/uso terapêutico , Animais , Anti-Infecciosos/farmacologia , Linhagem Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , beta-Glucanas/farmacologia
11.
Am J Respir Cell Mol Biol ; 63(3): 306-316, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32469619

RESUMO

Electronic nicotine delivery system (ENDS) use is outpacing our understanding of its potential harmful effects. Homeostasis of the lung is maintained through proper balance of cell death, efferocytic clearance, and phagocytosis of pathogens. To investigate whether ENDS use has the potential to alter this balance, we developed physiologically relevant ENDS exposure paradigms for lung epithelial cells and primary macrophages. In our studies, cells were exposed directly to aerosol made from carefully controlled components with and without nicotine. We found that ENDS aerosol exposure led to apoptosis, secondary necrosis, and necrosis in lung epithelial cell models. In contrast, macrophages died mostly by apoptosis and inflammatory caspase-mediated cell death when exposed to ENDS aerosol. The clearance of dead cells and pathogens by efferocytosis and phagocytosis, respectively, is an important process in maintaining a healthy lung. To investigate the impact of ENDS aerosol on macrophage function independent of general toxicity, we used an exposure time that did not induce cell death in primary macrophages. Exposure to ENDS aerosol containing nicotine inhibited nearly all phagocytic and greatly reduced the efferocytic abilities of primary macrophages. When challenged with a bacterial pathogen, there was decreased bacterial clearance. The presence of nicotine in the ENDS aerosol increased its toxicity and functional impact; however, nicotine exposure alone did not have any deleterious effects. These data demonstrate that ENDS aerosol exposure could lead to increased epithelial cell and macrophage death in the lung and impair important macrophage functions that are essential for maintenance of lung function.


Assuntos
Morte Celular/efeitos dos fármacos , Sistemas Eletrônicos de Liberação de Nicotina , Células Epiteliais/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Aerossóis/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Humanos , Pulmão/efeitos dos fármacos , Nicotina/farmacologia , Fagocitose/efeitos dos fármacos
12.
Nat Commun ; 11(1): 2358, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398649

RESUMO

Sphingosine kinase1 (SphK1) is an acetyl-CoA dependent acetyltransferase which acts on cyclooxygenase2 (COX2) in neurons in a model of Alzheimer's disease (AD). However, the mechanism underlying this activity was unexplored. Here we show that N-acetyl sphingosine (N-AS) is first generated by acetyl-CoA and sphingosine through SphK1. N-AS then acetylates serine 565 (S565) of COX2, and the N-AS-acetylated COX2 induces the production of specialized pro-resolving mediators (SPMs). In a mouse model of AD, microglia show a reduction in N-AS generation, leading to decreased acetyl-S565 COX2 and SPM production. Treatment with N-AS increases acetylated COX2 and N-AS-triggered SPMs in microglia of AD mice, leading to resolution of neuroinflammation, an increase in microglial phagocytosis, and improved memory. Taken together, these results identify a role of N-AS in the dysfunction of microglia in AD.


Assuntos
Doença de Alzheimer/imunologia , Anti-Inflamatórios/farmacologia , Encéfalo/imunologia , Microglia/imunologia , Esfingosina/análogos & derivados , Acetilcoenzima A/metabolismo , Acetilação , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Anti-Inflamatórios/uso terapêutico , Encéfalo/patologia , Linhagem Celular , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/patologia , Mutagênese , Neurônios , Fagocitose/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Presenilina-1/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Esfingosina/metabolismo
13.
Chem Biol Interact ; 325: 109126, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32430275

RESUMO

Alzheimer's disease (AD) is a common neurodegenerative disease, and its pathogenesis is closely related to ß-amyloid (Aß) peptide. The deposition of Aß in the brain due to impaired Aß clearance is considered as an important cause of AD. The decrease in Aß clearance is closely related to the autophagy dysfunction in brains of AD patients. It is feasible to treat AD by increasing the autophagy level of cells such as microglia and neurons to accelerate Aß clearance. In this article we explored the ability of graphene oxide (GO) to clear Aß through activating autophagy. Our work demonstrated that GO could inhibit the mTOR signaling pathway by activating AMPK to induce the autophagy of microglial and neurons. As expected, with the improvement of autophagy ability of microglia, GO promoted microglia-mediated Aß phagocytosis. Under the conditions of co-culture of microglia and neurons, GO induced the autophagy of microglia and neurons, especially the autophagy of microglia, thereby promoting the clearance of Aß, and ultimately achieved the effect of protecting neurons. Moreover, GO was not only non-cytotoxic to microglia and neurons but also able to reduce the toxicity of Aß to neurons through its clearance. These results have shown the potential of GO in treating Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Autofagia/efeitos dos fármacos , Grafite/farmacologia , Microglia/citologia , Microglia/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Microglia/metabolismo , Neurônios/metabolismo , Fagocitose/efeitos dos fármacos
14.
Ecotoxicol Environ Saf ; 200: 110713, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32464436

RESUMO

Calcutta Leather Complex of the state of West Bengal, India has been designated as an industrially active zone with around 400 active tannery units. This area spanning 4.5 km2 is surrounded by human habitation. The soil of this region is contaminated with metal pollutants and exhibited an alteration in selected physicochemical parameters, namely cation exchange capacity, moisture content, pH, total nitrogen, total organic carbon and water holding capacity. Metaphire posthuma, a common variety of endogeic earthworm inhabiting this region is thus continuously exposed to these toxic metals. Coelomocytes, the chief immune effector cells of earthworm presented a shift in phagocytosis, lysosomal membrane stability, lysozyme and phosphatase activity, physiological apoptosis and cell cycle profile of M. posthuma sampled from the soil of tannery industry. Presence of high concentration of toxic metals and change in the physicochemical characteristics of soil led to a state of cellular stress and immunocompromisation in M. posthuma, a common inhabitant of soil of this region. Experimental endpoints bear ecotoxicological significance as biomarkers of physiological stress in earthworm for monitoring the health of soil around this tannery industrial zone.


Assuntos
Metais/toxicidade , Oligoquetos/efeitos dos fármacos , Poluentes do Solo/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Biomarcadores Ambientais , Humanos , Índia , Indústrias , Lisossomos/efeitos dos fármacos , Muramidase/metabolismo , Oligoquetos/enzimologia , Oligoquetos/imunologia , Oligoquetos/metabolismo , Fagocitose/efeitos dos fármacos , Solo/química
15.
PLoS One ; 15(4): e0226661, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240171

RESUMO

CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRPα receptors, that block the SIRPα-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRPα and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRPα variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRPα binding. The SIRPα-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRPα-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Antígenos de Diferenciação/genética , Antígeno CD47/genética , Neoplasias/tratamento farmacológico , Receptores Imunológicos/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Imunidade Adaptativa/genética , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/química , Antígenos de Diferenciação/química , Antígeno CD47/química , Desenvolvimento de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoterapia/métodos , Células Jurkat , Citometria de Varredura a Laser , Ligantes , Oncologia/tendências , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Fagocitose/efeitos dos fármacos , Mapas de Interação de Proteínas/genética , Receptores Imunológicos/química , Bibliotecas de Moléculas Pequenas/química
16.
Toxicology ; 436: 152437, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32169474

RESUMO

Mild cognitive impairment in Parkinson's disease (PD-MCI) is considered as a nonmotor clinical symptom in Parkinson's disease (PD). Microglia-mediated inflammation contributes to cognitive function impairment. Poloxamer 188 (P188) is an amphipathic polymer which has cytoprotective effect in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced dopaminergic (DA) neurons degeneration in PD. But whether P188 could ameliorate cognitive impairment in PD is still illusive. In the present study, we showed in a mouse model that paraquat (10 mg/kg) and maneb (30 mg/kg) (P + M) treatment intraperitoneally twice a week for 6 consecutive weeks resulted in cognitive deficits and synapse loss in hippocampus, together with DA neuron damage in the substantia nigra pars compacta (SNpc). P188 (0.8 g/kg) injection via tail vein 30 min after P + M administration significantly restored DA neuron numbers in SNpc and synapse density in hippocampus, and alleviated P + M-mediated cognitive function impairment in novel object recognition task and morris water maze task (MWM). Pathological synapse loss might be attributed to increased microglial phagocytic activity and cell density, and P188 prevented P + M-induced phagocytic state changes of microglia, such as increase in cell body size and decrease in process length, and upregulated microglia abundance in hippocampus. Consistently, P188 attenuated P + M-mediated increased mRNA levels of microglia proliferation related CSF1r and CSF2ra, microglial engulfment associated CD68, ICAM1, and ICAM2, and pro-inflammatory IL-6, IL-1ß, CD11b, and TNF-α in hippocampus. Together, these findings suggest that the biocompatible polymer P188 blunts microglia activation which may promote synaptic loss and exacerbate cognitive function in a mouse model of PD-MCI.


Assuntos
Anti-Inflamatórios/farmacologia , Comportamento Animal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Disfunção Cognitiva/prevenção & controle , Hipocampo/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Transtornos Parkinsonianos/tratamento farmacológico , Parte Compacta da Substância Negra/efeitos dos fármacos , Poloxâmero/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/psicologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Maneb , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Degeneração Neural , Paraquat , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/psicologia , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Fagocitose/efeitos dos fármacos , Poloxâmero/farmacocinética , Reconhecimento Psicológico/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/patologia
17.
Proc Natl Acad Sci U S A ; 117(14): 7971-7980, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32205444

RESUMO

Timely resolution of bacterial infections critically depends on phagocytosis of invading pathogens by polymorphonuclear neutrophil granulocytes (PMNs), followed by PMN apoptosis and efferocytosis. Here we report that bacterial DNA (CpG DNA) and mitochondrial DNA impair phagocytosis and attenuate phagocytosis-induced apoptosis in human PMNs through Toll-like receptor 9 (TLR9)-mediated release of neutrophil elastase and proteinase 3 and subsequent down-regulation of the complement receptor C5aR. Consistently, CpG DNA delays pulmonary clearance of Escherichia coli in mice and suppresses PMN apoptosis, efferocytosis, and generation of proresolving lipid mediators, thereby prolonging lung inflammation evoked by E. coli Genetic deletion of TLR9 renders mice unresponsive to CpG DNA. We also show that aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 (17-epi-RvD1) through the receptor ALX/FPR2 antagonize cues from CpG DNA, preserve C5aR expression, restore impaired phagocytosis, and redirect human PMNs to apoptosis. Treatment of mice with 15-epi-LXA4 or 17-epi-RvD1 at the peak of inflammation accelerates clearance of bacteria, blunts PMN accumulation, and promotes PMN apoptosis and efferocytosis, thereby facilitating resolution of E. coli-evoked lung injury. Collectively, these results uncover a TLR9-mediated endogenous mechanism that impairs PMN phagocytosis and prolongs inflammation, and demonstrate both endogenous and therapeutic potential for 15-epi-LXA4 and 17-epi-RvD1 to restore impaired bacterial clearance and facilitate resolution of acute lung inflammation.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Infecções por Escherichia coli/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Pneumonia/imunologia , Receptor Toll-Like 9/metabolismo , Adulto , Idoso , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Células Cultivadas , Ilhas de CpG/imunologia , DNA Bacteriano/imunologia , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Escherichia coli/genética , Escherichia coli/imunologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Voluntários Saudáveis , Humanos , Lipoxinas/farmacologia , Lipoxinas/uso terapêutico , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Pneumonia/patologia , Cultura Primária de Células , Receptores de Formil Peptídeo/imunologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/imunologia , Receptores de Lipoxinas/metabolismo
18.
J Neurosci ; 40(17): 3320-3331, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32060170

RESUMO

Microglial cells are considered as sensors of brain pathology by detecting any sign of brain lesions, infections, or dysfunction and can influence the onset and progression of neurological diseases. They are capable of sensing their neuronal environment via many different signaling molecules, such as neurotransmitters, neurohormones and neuropeptides. The neuropeptide VGF has been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide and has been shown to signal via C3aR1 and C1qBP receptors. The effect of TLQP21 on microglial functions in health or disease is not known. Studying microglial cells in acute brain slices, we found that TLQP21 impaired metabotropic purinergic signaling. Specifically, it attenuated the ATP-induced activation of a K+ conductance, the UDP-stimulated phagocytic activity, and the ATP-dependent laser lesion-induced process outgrowth. These impairments were reversed by blocking C1qBP, but not C3aR1 receptors. While microglia in brain slices from male mice lack C3aR1 receptors, both receptors are expressed in primary cultured microglia. In addition to the negative impact on purinergic signaling, we found stimulating effects of TLQP21 in cultured microglia, which were mediated by C3aR1 receptors: it directly evoked membrane currents, stimulated basal phagocytic activity, evoked intracellular Ca2+ transient elevations, and served as a chemotactic signal. We conclude that TLQP21 has differential effects on microglia depending on C3aR1 activation or C1qBP-dependent attenuation of purinergic signaling. Thus, TLQP21 can modulate the functional phenotype of microglia, which may have an impact on their function in health and disease.SIGNIFICANCE STATEMENT The neuropeptide VGF and its peptides have been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide that activates C1qBP receptors, which are expressed by microglia. We show here, for the first time, that TLQP21 impairs P2Y-mediated purinergic signaling and related functions. These include modulation of phagocytic activity and responses to injury. As purinergic signaling is central for microglial actions in the brain, this TLQP21-mediated mechanism might regulate microglial activity in health and disease. We furthermore show that, in addition to C1qBP, functional C3aR1 responses contribute to TLQP21 action on microglia. However, C3aR1 responses were only present in primary cultures but not in situ, suggesting that the expression of these receptors might vary between different microglial activation states.


Assuntos
Quimiotaxia/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fagocitose/efeitos dos fármacos , Receptores Purinérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cálcio/metabolismo , Células Cultivadas , Quimiotaxia/fisiologia , Feminino , Masculino , Camundongos , Microglia/metabolismo , Fagocitose/fisiologia , Transdução de Sinais/fisiologia
19.
Biochem Biophys Res Commun ; 524(2): 371-377, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32005517

RESUMO

Osteopontin (OPN) is a phosphorylated glycoprotein expressed in various tissues, including brain, and mediates a wide range of cellular activities. In our previous studies, we reported recombinant OPN and RGD and SLAY-containing OPN-peptide icosamer (OPNpt20) exhibited robust neuroprotective activities in an animal model of transient focal ischemia, and attributed these effects to the anti-inflammatory, pro-angiogenic, and phagocytic functions of OPNpt20. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motif (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S) and their cell motility and migration inducing activities were examined in BV2 cells (a microglia cell line). All four peptides significantly enhanced BV2 cell motility and migration, but OPNpt7R, an RGD-containing 7-amino-acid OPN peptide (VPNGRGD), was found to be most potent and its potency was comparable to OPNpt20. Phagocytic activity and F-actin polymerization were also significantly enhanced in OPNpt7R-treated BV2 cells. Importantly, studies using two mutant peptides (OPNpt7R-RAA and OPNpt7R-RAD, wherein RGD in OPNpt7R was replaced with RAA or RAD, respectively) revealed that all these effects of OPNpt7R, motility, migration, F-actin polymerization, and phagocytosis induction, were RGD-dependent. Furthermore, the Erk, Fak, and Akt signaling pathways appeared to be involved in the induction of phagocytic activity by OPNpt7R. Co-treating cells with OPNpt7R and D98059 or wortmannin (pharmacological inhibitors of Erk and Akt, respectively) significantly suppressed OPNpt7R-mediated phagocytosis induction. These results indicate the RGD-containing OPN heptamer OPNpt7R triggers microglial motility, migration, and phagocytic activity and that the RGD motif plays a critical role in these activities.


Assuntos
Microglia/efeitos dos fármacos , Oligopeptídeos/farmacologia , Osteopontina/farmacologia , Fagocitose/efeitos dos fármacos , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Camundongos , Microglia/imunologia , Oligopeptídeos/química , Osteopontina/química
20.
J Mycol Med ; 30(2): 100924, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32037102

RESUMO

Cryptococcus neoformans, an encapsulated fungal pathogen is evolving as a major threat to immune-compromised patients and rarely to healthy individuals also. The cell wall bound capsular polysaccharide, melanin pigment and biofilm formation are major virulence factors that are known to contribute to cryptococcal meningitis. In the present study, a furanone derivative, (E)-5-benzylidenedihydrofuran-2(3H)-one (compound-6) was evaluated against biofilm of seven different strains of C. neoformans in melanized and non-melanized condition. In addition, the efficacy of compound-6 in activation of TLR-2, opsonophagocytosis, and modulation of cytokine expression during phagocytosis were studied. During the biofilm study, we found that moderate capsule size favored biofilm formation. Interestingly, the minimum biofilm eradication concentration (MBEC0.5) of melanized biofilm was found to be achieved at 1- to 1.7-fold higher MBEC0.5 of non-melanized cells. The maximum eradication of 77% and 69% of non-melanized and melanized biofilm were observed. The capsule size was reduced to half of its size with marked changes in morphology. Furthermore, expression of TLR2, iNOS and pro-inflammatory cytokines such as TNF-α, IL-12, and IFN-γ were also facilitated by compound-6. The correlation analysis showed a positive correlation between phagocytosis and the expression of TLR-2, iNOS, IL-6, IL-12. Collectively, the significant effect of compound-6, anti-melanization activity, antibiofilmand effective immunomodulant could be an interesting dual strategy drug agonist against cryptococcal meningitis.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Cryptococcus/efeitos dos fármacos , Proteínas Opsonizantes/fisiologia , Fagocitose/efeitos dos fármacos , Animais , Antifúngicos/síntese química , Antifúngicos/química , Cápsulas Bacterianas/efeitos dos fármacos , Cápsulas Bacterianas/fisiologia , Células Cultivadas , Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus/fisiologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/fisiologia , Furanos/síntese química , Furanos/química , Furanos/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Melaninas/metabolismo , Meningite Criptocócica/imunologia , Meningite Criptocócica/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Proteínas Opsonizantes/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA