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1.
BMC Biol ; 21(1): 36, 2023 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-36797789

RESUMO

BACKGROUND: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis. RESULTS: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis. CONCLUSIONS: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.


Assuntos
COVID-19 , Testículo , Tropismo Viral , Animais , Humanos , Masculino , Angiotensina II/metabolismo , Chlorocebus aethiops , COVID-19/patologia , SARS-CoV-2 , Testículo/imunologia , Testículo/virologia , Células Vero
2.
Immunology ; 160(1): 78-89, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32107769

RESUMO

Annexins are well-known Ca2+ phospholipid-binding proteins, which have a wide variety of cellular functions. The role of annexin A1 (AnxA1) in the innate immune system has focused mainly on the anti-inflammatory and proresolving properties through its binding to the formyl-peptide receptor 2 (FPR2)/ALX receptor. However, studies suggesting an intracellular role of AnxA1 are emerging. In this study, we aimed to understand the role of AnxA1 for interleukin (IL)-1ß release in response to activators of the nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3 (NLRP3) inflammasome. Using AnxA1 knockout mice, we observed that AnxA1 is required for IL-1ß release in vivo and in vitro. These effects were due to reduction of transcriptional levels of IL-1ß, NLRP3 and caspase-1, a step called NLRP3 priming. Moreover, we demonstrate that AnxA1 co-localize and directly bind to NLRP3, suggesting the role of AnxA1 in inflammasome activation is independent of its anti-inflammatory role via FPR2. Therefore, AnxA1 regulates NLRP3 inflammasome priming and activation in a FPR2-independent manner.


Assuntos
Anexina A1/metabolismo , Inflamassomos/imunologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Administração Intranasal , Animais , Cartilagem Articular , Caspase 1/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Gota/induzido quimicamente , Gota/imunologia , Gota/patologia , Humanos , Inflamassomos/metabolismo , Injeções Intra-Articulares , Pulmão/imunologia , Pulmão/patologia , Macrófagos , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , Ligação Proteica/imunologia , Dióxido de Silício/administração & dosagem , Dióxido de Silício/toxicidade , Silicose/imunologia , Silicose/patologia , Transcrição Gênica/imunologia , Ácido Úrico/administração & dosagem , Ácido Úrico/toxicidade
3.
Eur J Immunol ; 47(4): 646-657, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28294319

RESUMO

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF), where hepatocyte necrotic products trigger liver inflammation, release of CXC chemokine receptor 2 (CXCR2) ligands (IL-8) and other neutrophil chemotactic molecules. Liver infiltration by neutrophils is a major cause of the life-threatening tissue damage that ensues. A GRPR (gastrin-releasing peptide receptor) antagonist impairs IL-8-induced neutrophil chemotaxis in vitro. We investigated its potential to reduce acetaminophen-induced ALF, neutrophil migration, and mechanisms underlying this phenomenon. We found that acetaminophen-overdosed mice treated with GRPR antagonist had reduced DILI and neutrophil infiltration in the liver. Intravital imaging and cell tracking analysis revealed reduced neutrophil mobility within the liver. Surprisingly, GRPR antagonist inhibited CXCL2-induced migration in vivo, decreasing neutrophil activation through CD11b and CD62L modulation. Additionally, this compound decreased CXCL8-driven neutrophil chemotaxis in vitro independently of CXCR2 internalization, induced activation of MAPKs (p38 and ERK1/2) and downregulation of neutrophil adhesion molecules CD11b and CD66b. In silico analysis revealed direct binding of GRPR antagonist and CXCL8 to the same binding spot in CXCR2. These findings indicate a new potential use for GRPR antagonist for treatment of DILI through a mechanism involving adhesion molecule modulation and possible direct binding to CXCR2.


Assuntos
Bombesina/análogos & derivados , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Neutrófilos/imunologia , Fragmentos de Peptídeos/farmacologia , Receptores da Bombesina/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Animais , Bombesina/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Quimiotaxia/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Camundongos , Camundongos Endogâmicos , Ativação de Neutrófilo/efeitos dos fármacos , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos
4.
Inflamm Res ; 66(7): 591-602, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28386622

RESUMO

OBJECTIVE AND DESIGN: This study aimed to evaluate the effect of probucol in inflammatory hyperalgesia and leukocyte recruitment in mice. TREATMENT: Probucol at 0.3-3 mg/kg was administrated per oral 1 h before inflammatory stimulus.Author: Kindly check and confirm the affiliation 1 have been correctly processed or not and amend if necessary.Thank you. We have corrected affiliation 1. We added the information to the appropriate boxes. However the state and the postal code are in a different order when compared to the other affiliations. METHODS: Overt pain-like behaviors were determined by the number of abdominal writhings induced by phenyl-p-benzoquinone and acetic acid. Mechanical and thermal hyperalgesia induced by carrageenan were determined using an electronic anesthesiometer and hot plate apparatus, respectively. Leukocyte recruitment was evaluated by direct count or by determination of myeloperoxidase and N-acetylglucosaminidase activities. Antioxidant ability was determined by measurement of GSH levels, ABTS and FRAP assays. Cytokine production and NF-кB activation were evaluated by ELISA. Data were analyzed by ANOVA followed by Tukey's post-hoc. p < 0.05 was considered significant. RESULTS: Probucol reduced overt pain-like behavior, and carrageenan-induced mechanical and thermal hyperalgesia. These effects were accompanied by reduced leukocyte influx in both paw skin and peritoneum exudate. Probucol did not alter carrageenan-induced tissue antioxidant capacity at anti-inflammatory/analgesic dose. On the other hand, probucol inhibited carrageenan-induced IL-1ß, TNF-α and CXCL1 production as well as NF-кB activation. CONCLUSION: Probucol presents analgesic and anti-inflammatory activities by employing mechanisms other than its antioxidant properties. These mechanisms involve targeting of pro-inflammatory cytokines and NF-кB activation.


Assuntos
Analgésicos/farmacologia , Analgésicos/uso terapêutico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Probucol/farmacologia , Probucol/uso terapêutico , Ácido Acético , Animais , Comportamento Animal/efeitos dos fármacos , Benzoquinonas , Carragenina , Citocinas/imunologia , Edema/induzido quimicamente , Edema/tratamento farmacológico , Edema/imunologia , Temperatura Alta , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Hiperalgesia/imunologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Masculino , Camundongos , NF-kappa B/imunologia , Dor/induzido quimicamente , Dor/tratamento farmacológico , Dor/imunologia , Cavidade Peritoneal , Estimulação Física , Pele/efeitos dos fármacos , Pele/imunologia
5.
Nutr Cancer ; 67(3): 486-93, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25803482

RESUMO

Beneficial effects of L-arginine on immune responses and bowel function have been reported. Mucositis is a side effect of chemotherapy treatment that affects approximately 40% of patients. This complication is characterized by inflammation that affects the gastrointestinal tract, increasing permeability and causing abdominal pain, nausea, vomiting, and diarrhea, which worsen the patient's nutritional status and increases morbimortality. The aim of this study was to evaluate the effect of pretreating with 2% L-arginine supplementation in water on mucositis as induced by 5-fluorouracil (5-FU; a single dose of 200 mg/kg body weight) in Swiss male mice. The effect of L-arginine on weight, intestinal permeability, morphology, and the histopathological score of the small intestine (from 0 to 12), oxidative stress, myeloperoxidase (MPO), and N-acetylglucosaminidase (NAG) activities were evaluated. Intestinal length improvement was observed, in addition to the partial recovery of the mucosal architecture. L-arginine attenuated the histopathological score and MPO activity. There was also an improvement in intestinal permeability, despite weight loss after 5-FU administration. In conclusion, L-arginine can positively impact intestinal mucositis by promoting partial mucosal recovery, reducing inflammation and improving intestinal permeability.


Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Arginina/farmacologia , Fluoruracila/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Mucosite/prevenção & controle , Animais , Masculino , Camundongos , Mucosite/induzido quimicamente , Estresse Oxidativo , Peroxidase/metabolismo
6.
JHEP Rep ; 6(2): 100984, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38293685

RESUMO

Background & Aims: Lipid droplet (LD) accumulation in cells and tissues is understood to be an evolutionarily conserved tissue tolerance mechanism to prevent lipotoxicity caused by excess lipids; however, the presence of excess LDs has been associated with numerous diseases. Sepsis triggers the reprogramming of lipid metabolism and LD accumulation in cells and tissues, including the liver. The functions and consequences of sepsis-triggered liver LD accumulation are not well known. Methods: Experimental sepsis was induced by CLP (caecal ligation and puncture) in mice. Markers of hepatic steatosis, liver injury, hepatic oxidative stress, and inflammation were analysed using a combination of functional, imaging, lipidomic, protein expression and immune-enzymatic assays. To prevent LD formation, mice were treated orally with A922500, a pharmacological inhibitor of DGAT1. Results: We identified that liver LD overload correlates with liver injury and sepsis severity. Moreover, the progression of steatosis from 24 h to 48 h post-CLP occurs in parallel with increased cytokine expression, inflammatory cell recruitment and oxidative stress. Lipidomic analysis of purified LDs demonstrated that sepsis leads LDs to harbour increased amounts of unsaturated fatty acids, mostly 18:1 and 18:2. An increased content of lipoperoxides within LDs was also observed. Conversely, the impairment of LD formation by inhibition of the DGAT1 enzyme reduces levels of hepatic inflammation and lipid peroxidation markers and ameliorates sepsis-induced liver injury. Conclusions: Our results indicate that sepsis triggers lipid metabolism alterations that culminate in increased liver LD accumulation. Increased LDs are associated with disease severity and liver injury. Moreover, inhibition of LD accumulation decreased the production of inflammatory mediators and lipid peroxidation while improving tissue function, suggesting that LDs contribute to the pathogenesis of liver injury triggered by sepsis. Impact and Implications: Sepsis is a complex life-threatening syndrome caused by dysregulated inflammatory and metabolic host responses to infection. The observation that lipid droplets may contribute to sepsis-associated organ injury by amplifying lipid peroxidation and inflammation provides a rationale for therapeutically targeting lipid droplets and lipid metabolism in sepsis.

7.
Curr Dev Nutr ; 5(12): nzab138, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34993389

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is intricately linked to metabolic disease (including obesity, glucose intolerance, and insulin resistance) and encompasses a spectrum of disorders including steatosis, nonalcoholic steatohepatitis (NASH), and fibrosis. Rodents consuming high-fat (HF; ∼40 kcal% fat including fats containing higher concentrations of saturated and trans fats), high-fructose (HFr), and high-cholesterol (HC) diets display many clinically relevant characteristics of NASH, along with other metabolic disorders. C57BL/6 mice are the most commonly used animal model because they can develop significant metabolic disorders including severe NASH with fibrosis after months of feeding, but other models also are susceptible. The significant number of diets that contain these different factors (i.e., HF, HFr, and HC), either alone or in combination, makes the choice of diet difficult. This methodology review describes the efficacy of these nutrient manipulations on the NAFLD phenotype in mice, rats, guinea pigs, hamsters, and nonhuman primates.

8.
Cells ; 11(1)2021 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-35011636

RESUMO

The early detection of bacterial pathogens through immune sensors is an essential step in innate immunity. STING (Stimulator of Interferon Genes) has emerged as a key mediator of inflammation in the setting of infection by connecting pathogen cytosolic recognition with immune responses. STING detects bacteria by directly recognizing cyclic dinucleotides or indirectly by bacterial genomic DNA sensing through the cyclic GMP-AMP synthase (cGAS). Upon activation, STING triggers a plethora of powerful signaling pathways, including the production of type I interferons and proinflammatory cytokines. STING activation has also been associated with the induction of endoplasmic reticulum (ER) stress and the associated inflammatory responses. Recent reports indicate that STING-dependent pathways participate in the metabolic reprogramming of macrophages and contribute to the establishment and maintenance of a robust inflammatory profile. The induction of this inflammatory state is typically antimicrobial and related to pathogen clearance. However, depending on the infection, STING-mediated immune responses can be detrimental to the host, facilitating bacterial survival, indicating an intricate balance between immune signaling and inflammation during bacterial infections. In this paper, we review recent insights regarding the role of STING in inducing an inflammatory profile upon intracellular bacterial entry in host cells and discuss the impact of STING signaling on the outcome of infection. Unraveling the STING-mediated inflammatory responses can enable a better understanding of the pathogenesis of certain bacterial diseases and reveal the potential of new antimicrobial therapy.


Assuntos
Infecções Bacterianas/metabolismo , Inflamação/metabolismo , Espaço Intracelular/microbiologia , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Estresse do Retículo Endoplasmático , Humanos
9.
Nat Protoc ; 10(2): 258-68, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25569332

RESUMO

Imaging of live animals using intravital microscopy (IVM) has provided a substantial advance in our understanding of cell biology. Here we describe how to adapt a conventional, relatively low-cost laser-scanning microscope to operate as a versatile imaging station. We present the surgical procedures needed to perform liver confocal IVM in mice, thereby allowing one to image different cells in their native environment, including hepatocytes, endothelial cells and leukocytes, as well as to analyze their morphology and function under physiological or pathological conditions. In addition, we propose a plethora of working doses of antibodies and probes to stain multiple cells and molecules simultaneously in vivo. Considering the central role of the liver in metabolism and immunity and the growing interest in the relationship between immune and parenchymal cells, this protocol, in which 20 min of preparation yields up to 4 h of imaging, provides useful insights for various research fields. In addition, the protocol can be easily adapted to investigate adipose tissue, mesentery, intestines, spleen and virtually any abdominal organ.


Assuntos
Fígado/citologia , Microscopia Confocal/métodos , Animais , Anticorpos , Diagnóstico por Imagem/métodos , Desenho de Equipamento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Indóis/química , Fígado/cirurgia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal/instrumentação , Software , Coloração e Rotulagem/métodos
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