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This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.
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Células Dendríticas , Pele , Animais , Humanos , Citometria de Fluxo , Células Mieloides , Rim , MamíferosRESUMO
Dendritic cells (DC), macrophages, and monocytes, collectively known as mononuclear phagocytes (MPs), critically control tissue homeostasis and immune defense. However, there is a paucity of models allowing to selectively manipulate subsets of these cells in specific tissues. The steady-state adult kidney contains four MP subsets with Clec9a-expression history that include the main conventional DC1 (cDC1) and cDC2 subtypes as well as two subsets marked by CD64 but varying levels of F4/80. How each of these MP subsets contributes to the different phases of acute kidney injury and repair is unknown. We created a mouse model with a Cre-inducible lox-STOP-lox-diphtheria toxin receptor cassette under control of the endogenous CD64 locus that allows for diphtheria toxin-mediated depletion of CD64-expressing MPs without affecting cDC1, cDC2, or other leukocytes in the kidney. Combined with specific depletion of cDC1 and cDC2, we revisited the role of MPs in cisplatin-induced kidney injury. We found that the intrinsic potency reported for CD11c+ cells to limit cisplatin toxicity is specifically attributed to CD64+ MPs, while cDC1 and cDC2 were dispensable. Thus, we report a mouse model allowing for selective depletion of a specific subset of renal MPs. Our findings in cisplatin-induced injury underscore the value of dissecting the functions of individual MP subsets in kidney disease, which may enable therapeutic targeting of specific immune components in the absence of general immunosuppression.
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Injúria Renal Aguda/prevenção & controle , Células Dendríticas/imunologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Fagócitos/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Antineoplásicos/toxicidade , Cisplatino/toxicidade , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Feminino , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Monócitos/patologia , Fagócitos/citologia , Receptores de IgGRESUMO
Cadmium (Cd) is a widespread toxic occupational and environmental pollutant, and its effect on lipid metabolism dysregulation has been observed. In this study, we utilized two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) technologies to explore changes in the blood plasma proteins of mice exposed to Cd. From the 2-DE, 8 protein spots were screened in response to Cd exposure, and the identities of these proteins were revealed by MALDI-TOF MS. Western blotting was applied to analyze the expression of the apolipoproteins in both plasma and liver, which were consistent with Cd-induced dyslipidemia of their composed lipid. Moreover, the Cd-induced apolipoprotein ApoE upregulation was due to inhibition of autopahgic flux in the Cd exposed mice. It was further observed from the mouse liver that Cd reduced the expression of the lipid uptake receptor low-density lipoprotein receptor (LDLR), which might be responsible for the coordinated elevation in blood triglycerides and abnormal apolipoproteins. This study may provide a new insight into the mechanism of Cd-induced dyslipidemia and the risk of cardiovascular diseases.
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Apolipoproteínas E/sangue , Cádmio/farmacologia , Triglicerídeos/sangue , Animais , Cádmio/administração & dosagem , Exposição Dietética , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
The prostate-specific antigen (PSA) test is considered an important way for preoperative diagnosis and accurate screening of prostate cancer. Current antigen detection methods, including radioimmunoassay, enzyme-linked immunosorbent assay and microfluidic electrochemical detection, feature expensive equipment, long testing time and poor stability. Here, we propose a portable biosensor composed of electrolyte-gated amorphous indium gallium zinc oxide (a-IGZO) transistors with an extended gate, which can achieve real-time, instant PSA detection at a low operating voltage (<2 V) owing to the liquid-free ionic conductive elastomer (ICE) serving as the gate dielectric. The electric double layer (EDL) capacitance in ICE enhances the accumulation of carriers in the IGZO channel, leading to strong gate modulation, which enables the IGZO transistor to have a small subthreshold swing (<0.5 V dec-1) and a high on-state current (â¼4 × 10-4 A). The separate, biodegradable, and pluggable sensing pad, serving as an extended gate connected to the IGZO transistor, prevents contamination and depletion arising from direct contact with biomolecular buffers, enabling the IGZO transistor to maintain superior electronic performance for at least six months. The threshold voltage and channel current of the transistor exhibit excellent linear response to PSA molecule concentrations across five orders of magnitude ranging from 1 fg mL-1 to 10 pg mL-1, with a detection limit of 400 ag mL-1 and a detection time of â¼5.1 s. The fabricated biosensors offer a point-of-care system for antigen detection, attesting the feasibility of the electrolyte-gated transistors in clinical screening, healthcare diagnostics and biological management.
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Técnicas Biossensoriais , Eletrólitos , Gálio , Antígeno Prostático Específico , Transistores Eletrônicos , Óxido de Zinco , Antígeno Prostático Específico/análise , Humanos , Eletrólitos/química , Óxido de Zinco/química , Técnicas Biossensoriais/instrumentação , Gálio/química , Masculino , Índio/química , Desenho de EquipamentoRESUMO
Urinary tract obstruction during renal development leads to inflammation, leukocyte infiltration, tubular cell death, and interstitial fibrosis. Interleukin-10 (IL-10) is an anti-inflammatory cytokine, produced mainly by monocytes/macrophages and regulatory T-cells. IL-10 inhibits innate and adaptive immune responses. IL-10 has a protective role in the adult model of obstructive uropathy. However, its role in neonatal obstructive uropathy is still unclear which led us to study the role of IL-10 in neonatal mice with unilateral ureteral obstruction (UUO). UUO serves as a model for congenital obstructive nephropathies, a leading cause of kidney failure in children. Newborn Il-10-/- and C57BL/6 wildtype-mice (WT) were subjected to complete UUO or sham-operation on the 2nd day of life. Neonatal kidneys were harvested at day 3, 7, and 14 of life and analyzed for different leukocyte subpopulations by FACS, for cytokines and chemokines by Luminex assay and ELISA, and for inflammation, programmed cell death, and fibrosis by immunohistochemistry and western blot. Compared to WT mice, Il-10-/- mice showed reduced infiltration of neutrophils, CD11bhi cells, conventional type 1 dendritic cells, and T-cells following UUO. Il-10-/- mice with UUO also showed a reduction in pro-inflammatory cytokine and chemokine release compared to WT with UUO, mainly of IP-10, IL-1α, MIP-2α and IL-17A. In addition, Il-10-/- mice showed less necroptosis after UUO while the rate of apoptosis was not different. Finally, α-SMA and collagen abundance as readout for fibrosis were similar in Il-10-/- and WT with UUO. Surprisingly and in contrast to adult Il-10-/- mice undergoing UUO, neonatal Il-10-/- mice with UUO showed a reduced inflammatory response compared to respective WT control mice with UUO. Notably, long term changes such as renal fibrosis were not different between neonatal Il-10-/- and neonatal WT mice with UUO suggesting that IL-10 signaling is different in neonates and adults with UUO.
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Nefropatias , Obstrução Ureteral , Adulto , Animais , Criança , Humanos , Camundongos , Animais Recém-Nascidos , Citocinas , Fibrose , Inflamação , Interleucina-10/genética , Camundongos Endogâmicos C57BLRESUMO
Inflammasome molecules make up a family of receptors that typically function to initiate a proinflammatory response upon infection by microbial pathogens. Dysregulation of inflammasome activity has been linked to unwanted chronic inflammation, which has also been implicated in certain autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus, and related animal models. Classical inflammasome activation-dependent events have intrinsic and extrinsic effects on both innate and adaptive immune effectors, as well as resident cells in the target tissue, which all can contribute to an autoimmune response. Recently, inflammasome molecules have also been found to regulate the differentiation and function of immune effector cells independent of classical inflammasome-activated inflammation. These alternative functions for inflammasome molecules shape the nature of the adaptive immune response, that in turn can either promote or suppress the progression of autoimmunity. In this review we will summarize the roles of inflammasome molecules in regulating self-tolerance and the development of autoimmunity.
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Doenças Autoimunes , Inflamassomos , Animais , Autoimunidade , Inflamação , Tolerância a Antígenos PrópriosRESUMO
BACKGROUND: In the enzymatic conversion of biomass, it becomes an important issue to efficiently and cost-effectively degrade cellulose into fermentable glucose. ß-Glucosidase (Bgluc), an essential member of cellulases, plays a critical role in cellulosic biomass degradation. The difficulty in improving the stability of Bgluc has been a bottleneck in the enzyme-dependent cellulose degradation. The traditional method of protein purification, however, leads to higher production cost and a decrease in activity. To simplify and efficiently purify Bgluc with modified special properties, Bgluc-tagged ELP and His with defined phase transitions was designed to facilitate the process. RESULTS: Here, a novel binary ELP and His tag was fused with Bgluc from termite Coptotermes formosanus to construct a Bgluc-linker-ELP-His recombinant fusion protein (BglucLEH). The recombinant plasmid Bgluc expressing a His tag (BglucH) was also constructed. The BglucLEH and BglucH were expressed in E. coli BL21 and purified using inverse transition cycling (ITC) or Ni-NTA resin. The optimum salt concentration for the ITC purification of BglucLEH was 0.5 M (NH4)2SO4 and the specific activity of BglucLEH purified by ITC was 75.5 U/mg for substrate p-NPG, which was slightly higher than that of BglucLEH purified by Ni-NTA (68.2 U/mg). The recovery rate and purification fold of BglucLEH purified by ITC and Ni-NTA were 77.8%, 79.1% and 12.60, 11.60, respectively. The results indicated that purification with ITC was superior to the traditional Ni-NTA. The K m of BglucLEH and BglucH for p-NPG was 5.27 and 5.73 mM, respectively. The K ca t/K m (14.79 S-1 mM-1) of BglucLEH was higher than that of BglucH (12.10 S-1 mM-1). The effects of ELP tag on the enzyme activity, secondary structure and protein stability were also studied. The results showed that ELP tag did not affect the secondary structure or enzyme activity of Bgluc. More importantly, ELP improved the protein stability in harsh conditions such as heating and exposure to denaturant. CONCLUSION: The Bgluc-linker-ELP-His system shows wide application prospect in maintaining the activity, efficient purification and improving the stability of Bgluc. These properties of BglucLEH make it an interesting tool to reduce cost, to improve the efficiency of biocatalyst and potentially to enhance the degradation of lignocellulosic biomass.
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Human exposure to cadmium (Cd) could lead to alterations in lipid metabolism. However, the underlying mechanism is still unclear. In the present study, the data revealed that Cd exposure induced cholesterol redistribution both in vivo from mouse liver tissue into the serum, and in vitro from the HepG2 cells to the cultured medium, which were associated with modulating the expressions of cholesterol efflux proteins, including upregulating cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) and downregulating oxysterol-binding protein (OSBP). Further investigation in HepG2 cells revealed that Cd upregulated ABCA1 expression with increased stability by inhibiting lysosomal pathway, and downregulated OSBP expression by increasing ubiquitination. Cd-induced cholesterol redistribution was completely inhibited by knockdown of ABCA1 expression using siRNA, and was significantly reduced by overexpression of OSBP. Taken together, these results suggested that Cd induced cholesterol redistribution by upregulating ABCA1 and downregulating OSBP.
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Transportador 1 de Cassete de Ligação de ATP/metabolismo , Cádmio/toxicidade , Colesterol/metabolismo , Receptores de Esteroides/metabolismo , Regulação para Baixo , Células Hep G2 , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Cadmium (Cd), as an extremely toxic metal could accumulate in kidney and induce renal injury. Previous studies have proved that Cd impact on renal cell proliferation, autophagy and apoptosis, but the detoxification drugs and the functional mechanism are still in study. In this study, we used mouse renal tubular epithelial cells (mRTECs) to clarify Cd-induced toxicity and signaling pathways. Moreover, we proposed to elucidate the prevent effect of activation of Ca2+ sensing receptor (CaSR) by Calcimimetic (R-467) on Cd-induced cytotoxicity and underlying mechanisms. Cd induced intracellular Ca2+ elevation through phospholipase C-inositol 1, 4, 5-trisphosphate (PLC) followed stimulating p38 mitogen-activated protein kinases (MAPK) activation and suppressing extracellular signal-regulated kinase (ERK) activation, which leaded to increase apoptotic cell death and inhibit cell proliferation. Cd induced p38 activation also contribute to autophagic flux inhibition that aggravated Cd induced apoptosis. R-467 reinstated Cd-induced elevation of intracellular Ca2+ and apoptosis, and it also increased cell proliferation and restored autophagic flux by switching p38 to ERK pathway. The identification of the activation of CaSR-mediated protective pathway in renal cells sheds light on a possible cellular protective mechanism against Cd-induced kidney injury.