RESUMO
Absent in melanoma-2 (AIM2) is an inflammasome-forming innate immune sensor for dsDNA but also exhibits inflammasome-independent functions such as restricting cellular proliferation. AIM2 is expressed in the kidney, but its localization and function are not fully characterized. In normal human glomeruli, AIM2 localized to podocytes. In patients with glomerulonephritis, AIM2 expression increased in CD44+-activated parietal epithelial cells within glomerular crescents. To explore AIM2 effects in glomerular disease, studies in Aim2 -/- mice were performed. Aim2-/- glomeruli showed reduced expression of Wilm tumor gene-1 (WT1), WT1-driven podocyte genes, and increased proliferation in outgrowth assays. In a nephrotoxic serum (NTS)-induced glomerulonephritis model, Aim2-/- (B6) mice exhibited more severe glomerular crescent formation, tubular injury, inflammation, and proteinuria compared with wild-type controls. Inflammasome activation markers were absent in both Aim2 -/- and wild-type kidneys, despite an increased inflammatory transcriptomic signature in Aim2 -/- mice. Aim2 -/- mice also demonstrated dysregulated cellular proliferation and an increase in CD44+ parietal epithelial cells during glomerulonephritis. The augmented inflammation and epithelial cell proliferation in Aim2 -/- (B6) mice was not due to genetic background, as Aim2 -/- (B6.129) mice demonstrated a similar phenotype during NTS glomerulonephritis. The AIM2-like receptor (ALR) locus was necessary for the inflammatory glomerulonephritis phenotype observed in Aim2 -/- mice, as NTS-treated ALR -/- mice displayed equal levels of injury as wild-type controls. Podocyte outgrowth from ALR -/- glomeruli was still increased, however, confirming that the ALR locus is dispensable for AIM2 effects on epithelial cell proliferation. These results identify a noncanonical role for AIM2 in suppressing inflammation and epithelial cell proliferation during glomerulonephritis.
Assuntos
Proteínas de Ligação a DNA/imunologia , Células Epiteliais/imunologia , Glomerulonefrite/imunologia , Inflamação/imunologia , Animais , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Feminino , Glomerulonefrite/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The NOD-like receptor (NLR) family of proteins is a group of pattern recognition receptors (PRRs) known to mediate the initial innate immune response to cellular injury and stress. The NLRP proteins represent a fourteen-member subset of the NLR family that contains an N-terminal pyrin domain. Some NLRs are known to form multi-protein complexes known as inflammasomes. Inflammasomes consist of an NLR, the adaptor protein ASC, and the effector molecule pro-caspase-1. Once activated, these inflammasomes facilitate the cleavage and activation of caspase-1, which in turn mediates the cleavage of the pro-inflammatory cytokines IL-1ß and IL-18 into their active and secreted forms. Activated caspase-1 also drives the cleavage of gasdermin D, which triggers an inflammatory form of cell death known as pyroptosis. Several NLRs are also known to possess non-canonical, inflammasome-independent functions, regulating a variety of signaling pathways. In this review, a thorough overview of both inflammasome-dependent and -independent NLR signaling will be presented, with highlights from the field as well as promising future directions and postulates based on the known science.
Assuntos
Inflamassomos/metabolismo , Proteínas NLR/metabolismo , Transdução de Sinais , Humanos , PiroptoseRESUMO
The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.
Assuntos
Trifosfato de Adenosina/metabolismo , Inflamassomos/metabolismo , Proteínas NLR/metabolismo , Células HEK293 , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , UbiquitinaçãoRESUMO
Benzodiazepine use and dependence are on the rise as well as the number of deaths attributable to the combination of opioids and benzodiazepines. Anxiety, the most frequent condition for which benzodiazepines are prescribed, occurs commonly, and is increasingly noted to coincide with pregnancy. Use of both benzodiazepine anxiolytics and anxiety in pregnancy is associated with preterm delivery and low birth weight. Short-term neonatal effects of hypotonia, depression, and withdrawal are described but long-term sequelae, if any, are poorly understood. Benzodiazepines are associated with physical dependence and withdrawal symptoms which can be serious. To avoid withdrawal, tapering off these medications is recommended. What is known about the pharmacology and pharmacokinetics, pregnancy implications, tapering schedules, and alternative strategies for anxiety are discussed.
Assuntos
Ansiolíticos/efeitos adversos , Benzodiazepinas/efeitos adversos , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Ansiolíticos/administração & dosagem , Ansiolíticos/farmacocinética , Ansiolíticos/farmacologia , Ansiedade/complicações , Ansiedade/terapia , Benzodiazepinas/administração & dosagem , Benzodiazepinas/farmacocinética , Benzodiazepinas/farmacologia , Feminino , Humanos , Gravidez , Nascimento Prematuro/etiologia , Efeitos Tardios da Exposição Pré-Natal/etiologia , Síndrome de Abstinência a Substâncias/fisiopatologiaRESUMO
Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1ß cleavage in Aim2-/- or WT mice that received WT bone marrow than in WT mice that received Aim2-/- bone marrow. Intravital microscopy of the kidney in LysM(gfp/gfp) mice 5-6 days after UUO demonstrated the significant recruitment of GFP+ proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CX3CR1+ renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1ß, as well as the formation of AIM2+ ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1ß levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.
Assuntos
Proteínas de Ligação a DNA/fisiologia , DNA/metabolismo , Inflamassomos/fisiologia , Macrófagos/fisiologia , Insuficiência Renal Crônica/metabolismo , Animais , Transplante de Medula Óssea , Caspase 1/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Nefropatias Diabéticas/metabolismo , Ativação Enzimática , Fibrose , Humanos , Interleucina-1beta/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais/metabolismo , Leucócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Nefroesclerose/metabolismo , Fagocitose , Fenótipo , Quimera por Radiação , Células THP-1 , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologiaRESUMO
The success of a home hemodialysis program depends largely on a patient safety framework and the risk tolerance of a home dialysis program. Dialysis treatments require operators to perform dozens of steps repeatedly and reliably in a complex procedure. For home hemodialysis, those operators are patients themselves or their care partners, so attention to safety and risk mitigation is front of mind. While newer, smaller, and more user-friendly dialysis machines designed explicitly for home use are slowly entering the marketplace, teaching patients to perform their own treatments in an unsupervised setting hundreds of times remains a foundational programmatic obligation regardless of machine. Just how safe is home hemodialysis? How does patient training affect this safety? There is a surprising lack of literature surrounding these questions. No consensus exists among home hemodialysis programs regarding optimized training schedules or methods, with each program adopting its own approach on the basis of local experience. Furthermore, there are little available data on the safety of home hemodialysis as compared with conventional in-center hemodialysis. This review will outline considerations for training patients on home hemodialysis, discuss the safety of home hemodialysis with an emphasis on the risk of serious and life-threatening adverse effects, and address the methods by which adverse events are monitored and prevented.
Assuntos
Hemodiálise no Domicílio , Educação de Pacientes como Assunto , Segurança do Paciente , Humanos , Hemodiálise no Domicílio/efeitos adversos , Hemodiálise no Domicílio/educação , Fatores de Risco , Medição de RiscoRESUMO
High-density lipoprotein (HDL) cholesterol levels are inversely associated with risk of atherosclerotic cardiovascular disease. At least 50% of the variation in HDL cholesterol levels is genetically determined, but the genes responsible for variation in HDL levels have not been fully elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two members of the triacylglyerol (TG) lipase family, both influence HDL metabolism and the HL (LIPC) locus has been associated with variation in HDL cholesterol levels in humans. We describe here the cloning and in vivo functional analysis of a new member of the TG lipase family. In contrast to other family members, this new lipase is synthesized by endothelial cells in vitro and thus has been termed endothelial lipase (encoded by the LIPG gene). EL is expressed in vivo in organs including liver, lung, kidney and placenta, but not in skeletal muscle. In contrast to LPL and HL, EL has a lid of only 19 residues. EL has substantial phospholipase activity, but less triglyceride lipase activity. Overexpression of EL in mice reduced plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I. The endothelial expression, enzymatic profile and in vivo effects of EL suggest that it may have a role in lipoprotein metabolism and vascular biology.
Assuntos
Endotélio Vascular/enzimologia , Lipase/genética , Lipase/metabolismo , Lipoproteínas HDL/metabolismo , Sequência de Aminoácidos , Animais , Anticolesterolemiantes/farmacologia , Apolipoproteína A-I/genética , Northern Blotting , Células COS/enzimologia , Células Cultivadas , HDL-Colesterol/sangue , HDL-Colesterol/efeitos dos fármacos , Clonagem Molecular , Endotélio Vascular/citologia , Feminino , Humanos , Lipoproteínas HDL/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Placenta , Gravidez , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Jackson-Weiss syndrome is an autosomal dominant condition characterized by craniosynostosis, foot anomalies and great phenotypic variability. Recently mutations in fibroblast growth factor receptor 2 (FGFR2) have been found in patients with another craniosynostotic syndrome, Crouzon syndrome. FGFR2 is a member of the tyrosine kinase receptor superfamily, having a high affinity for peptides that signal the transduction pathways for mitogenesis, cellular differentiation and embryogenesis. We now report an FGFR2 mutation in the conserved region of the immunoglobulin IIIc domain in the Jackson-Weiss syndrome family in which the syndrome was originally described. In addition, in four of 12 Crouzon syndrome cases, we identified two new mutations and found two previously described mutations in the same region.
Assuntos
Alelos , Disostose Craniofacial/genética , Craniossinostoses/genética , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/genética , Mutação , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 10 , Sequência Consenso , Análise Mutacional de DNA , Feminino , Genes , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Receptores Proteína Tirosina Quinases/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , SíndromeRESUMO
OBJECTIVE: Breast cancer fatality rates are high in low- and middle-income countries because of the late stage at diagnosis. We investigated patient-mediated determinants for late-stage presentation of breast cancer in Egypt. METHODS: A case-case comparison was performed for 343 women with breast cancer, comparing those who had been initially diagnosed at Stage I or II with those diagnosed at Stage III or IV. Patients were recruited from the National Cancer Institute of Cairo University and Tanta Cancer Center in the Nile delta. Patients were either newly diagnosed or diagnosed within the year preceding the study. Interviews elicited information on disease history and diagnosis, beliefs and attitudes toward screening practices, distance to treatment facility, education, income, and reproductive history. RESULTS: Forty-six per cent of the patients had presented at late stage. Women seen in Cairo were more likely to present at late stages than patients in Tanta (OR=5.05; 95% CI=1.30, 19.70). Women without any pain were more likely to present at later stage (OR=2.68; 95% CI=1.18, 6.08). Knowledge of breast self-examination increased the likelihood of women to present in early stages significantly (OR=0.24; 95% CI=0.06, 0.94). CONCLUSIONS: Despite increasing numbers of cancer centers in Egypt during the past 20 years, additional regional facilities are needed for cancer management. In addition, increasing awareness about breast cancer will have significant long-term impact on breast cancer prevention.
Assuntos
Neoplasias da Mama/diagnóstico , Diagnóstico Tardio , Adulto , Idoso , Idoso de 80 Anos ou mais , Atitude Frente a Saúde , Neoplasias da Mama/psicologia , Estudos de Casos e Controles , Diagnóstico Precoce , Escolaridade , Egito/epidemiologia , Feminino , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Análise Multivariada , Dor/diagnóstico , Dor/psicologia , Fatores de Tempo , Adulto JovemRESUMO
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-alpha and PPAR-gamma are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-alpha may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR-activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-alpha and PPAR-gamma activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-alpha and PPAR-gamma activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor alpha, an oxysterol-activated nuclear receptor which induces ABCA1-promoter transcription. Moreover, PPAR-alpha and PPAR-gamma activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-alpha or PPAR-gamma activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-alpha and PPAR-gamma in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , Células Espumosas/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Transportador 1 de Cassete de Ligação de ATP , Sequência de Bases , Transporte Biológico , Células Cultivadas , Primers do DNA , HumanosRESUMO
The pryin domain (PYD) domain is involved in protein interactions that lead to assembly of immune-sensing complexes such as inflammasomes. The repertoire of PYD-containing genes expressed by a cell type arms tissues with responses against a range of stimuli. The transcriptional regulation of the PYD gene family however is incompletely understood. Alternative promoter utilization was identified as a mechanism regulating the tissue distribution of human PYD gene family members, including NLRP6 that is translationally silenced outside of intestinal tissue. Results show that alternative transcriptional promoters mediate NLRP6 silencing in mice and humans, despite no upstream genomic synteny. Human NLRP6 contains an internal alternative promoter within exon 2 of the PYD, resulting in a truncated mRNA in nonintestinal tissue. In mice, a proximal promoter was used that expanded the 5' leader sequence restricting nuclear export and abolishing translational efficiency. Nlrp6 was dispensable in disease models targeting the kidney, which expresses noncanonical isoforms. Thus, alternative promoter use is a critical mechanism not just for isoform modulation but for determining expression profile and function of PYD family members.
Assuntos
Processamento Alternativo/genética , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Córtex Renal/metabolismo , Regiões Promotoras Genéticas/genética , Domínio Pirina/genética , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Animais , Células Cultivadas , Éxons , Expressão Gênica , Regulação da Expressão Gênica , Genes Reguladores , Humanos , Inflamassomos/metabolismo , Mucosa Intestinal/patologia , Córtex Renal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismoRESUMO
The fibroblast or heparin-binding growth factors (HBGFs) are thought to be modulators of cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. A better understanding of the structural basis for the different activities of these proteins should facilitate the development of agonists and antagonists of specific HBGF activities and identification of the signal transduction pathways involved in the mechanisms of action of these growth factors. Chemical modification studies of Harper and Lobb (Harper, J. W., and R. R. Lobb. 1988. Biochemistry. 27:671-678) implicated lysine 132 in HBGF-1 (acidic fibroblast growth factor) as being important to the heparin-binding, receptor-binding, and mitogenic activities of the protein. We changed lysine 132 to a glutamic acid residue by site-directed mutagenesis of the human cDNA and expressed the mutant protein in Escherichia coli to obtain sufficient quantities for functional studies. Replacement of this lysine with glutamic acid reduces the apparent affinity of HBGF-1 for immobilized heparin (elutes at 0.45 M NaCl vs. 1.1 M NaCl for wild-type). Mitogenic assays established two points: (a) human recombinant HBGF-1 is highly dependent on the presence of heparin for optimal mitogenic activity, and (b) the change of lysine 132 to glutamic acid drastically reduces the specific mitogenic activity of HBGF-1. The poor mitogenic activity of the mutant protein does not appear to be due to a reduced affinity for the HBGF receptor. Similarly, the mutant HBGF-1 can stimulate tyrosine kinase activity and induce protooncogene expression. Differences in the biological properties of the wild-type and mutant proteins were observed in transfection studies. Mutant HBGF-1 expression in transfected NIH 3T3 cells did not induce the same transformed phenotype characteristic of cells expressing wild-type HBGF-1. Together these data indicate that different functional properties of HBGF-1 may be dissociated at the structural level.
Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Heparina/metabolismo , Mitose/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Fator 1 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica , Glutamatos , Ácido Glutâmico , Lisina , Mutagênese Sítio-Dirigida , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Receptores de Fatores de Crescimento de Fibroblastos , TransfecçãoRESUMO
Endothelial cells, which line the interior walls of blood vessels, proliferate at the site of blood vessel injury. Knowledge of the factors that control the proliferation of these cells would help elucidate the role of endothelial cells in wound healing, tumor growth, and arteriosclerosis. In vitro, endothelial cells organize into viable, three-dimensional tubular structures in environments that limit cell proliferation. The process of endothelial cell organization was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNA transcript for fibronectin. This situation was reversed on transition from the organized structure to a proliferative monolayer. These results suggest a reciprocity for two biological response modifiers involved in the regulation of endothelial cell proliferation and differentiation in vitro.
Assuntos
Endotélio/citologia , Regulação da Expressão Gênica , Fator de Crescimento Derivado de Plaquetas/genética , RNA Mensageiro/genética , Transcrição Gênica , Diferenciação Celular , Divisão Celular , Células Cultivadas , Meios de Cultura , Fatores de Crescimento Endotelial , Endotélio/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/biossíntese , Fibronectinas/genética , Substâncias de Crescimento/farmacologia , HumanosRESUMO
Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.
Assuntos
Mapeamento Cromossômico , Substâncias de Crescimento/genética , Sequência de Bases , Tronco Encefálico/metabolismo , Clonagem Molecular , DNA/genética , Fatores de Crescimento Endotelial , Humanos , Interleucina-1/genética , Fígado/metabolismo , Hibridização de Ácido Nucleico , RNA Mensageiro/genéticaRESUMO
Analysis of gene expression is often used to evaluate the effects of experimental manipulations in laboratory animals. Blood is a rich source of potential biomarkers, including gene expression information, which may be obtained from whole blood. When compared with the end of a study, when whole blood samples can be easily obtained for gene expression measurements, the limiting volumes of whole blood obtainable from animals during the course of an experiment requires a method for RNA isolation from a minimal volume of whole blood. The PAXgene Blood RNA Extraction System originally designed for isolation of total RNA from 2.5 mL of human whole blood, was modified and successfully used to isolate high-integrity total RNA from as little as 50 microL of mouse whole blood. Fifty microlitres of mouse whole blood yielded an average of 2.3 microg highly intact total RNA, of sufficient quality and quantity allowing for multiple gene expression determinations. The utility of this method was demonstrated by confirming the time- and dose-dependent upregulation of haem oxygenase-1 (Hmox1) mRNA in response to a single injection of cobalt protoporphyrin. The successful isolation of total RNA from small volumes of mouse whole blood can allow for serial sampling on the same animals, thereby reducing the number of animals required for experimentation.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Regulação da Expressão Gênica/fisiologia , RNA/sangue , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/sangue , Heme Oxigenase-1/genética , Ciência dos Animais de Laboratório/métodos , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Camundongos , Protoporfirinas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacosRESUMO
The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1ß secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1ß production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.
Assuntos
Caspases Iniciadoras/metabolismo , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Toxina Shiga/farmacologia , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Proteínas de Ligação a Fosfato , Piroptose/efeitos dos fármacosRESUMO
Radiographic contrast agents cause acute kidney injury (AKI), yet the underlying pathogenesis is poorly understood. Nod-like receptor pyrin containing 3-deficient (Nlrp3-deficient) mice displayed reduced epithelial cell injury and inflammation in the kidney in a model of contrast-induced AKI (CI-AKI). Unexpectedly, contrast agents directly induced tubular epithelial cell death in vitro that was not dependent on Nlrp3. Rather, contrast agents activated the canonical Nlrp3 inflammasome in macrophages. Intravital microscopy revealed diatrizoate (DTA) uptake within minutes in perivascular CX3CR1+ resident phagocytes in the kidney. Following rapid filtration into the tubular luminal space, DTA was reabsorbed and concentrated in tubular epithelial cells via the brush border enzyme dipeptidase-1 in volume-depleted but not euvolemic mice. LysM-GFP+ macrophages recruited to the kidney interstitial space ingested contrast material transported from the urine via direct interactions with tubules. CI-AKI was dependent on resident renal phagocytes, IL-1, leukocyte recruitment, and dipeptidase-1. Levels of the inflammasome-related urinary biomarkers IL-18 and caspase-1 were increased immediately following contrast administration in patients undergoing coronary angiography, consistent with the acute renal effects observed in mice. Taken together, these data show that CI-AKI is a multistep process that involves immune surveillance by resident and infiltrating renal phagocytes, Nlrp3-dependent inflammation, and the tubular reabsorption of contrast via dipeptidase-1.
Assuntos
Injúria Renal Aguda/etiologia , Meios de Contraste/efeitos adversos , Dipeptidases/metabolismo , Vigilância Imunológica , Rim/enzimologia , Rim/imunologia , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/metabolismo , Animais , Meios de Contraste/farmacocinética , Modelos Animais de Doenças , Proteínas Ligadas por GPI/metabolismo , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Rim/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Fagócitos/imunologia , Fagócitos/metabolismoRESUMO
The cloned complementary DNA for coagulation Factor IX (FIX) detects a frequent restriction fragment length polymorphism (RFLP) in human genomic DNAs digested with the restriction endonuclease Taq I. This genetic marker was used, in parallel with coagulation and immunological assays, to follow the segregation of an abnormal FIX gene in a large Hemophilia B family. Among the six potential female carriers, functional assays showed that four had a high probability, and two a low probability of being carriers. Analysis at the DNA level with the cDNA probe was informative in five of the six cases, and in all these five the diagnosis of carrier state was definitively confirmed. This demonstrates the feasibility of using linkage analysis at the DNA level for the genetic screening of Hemophilia B. This method has the advantages over conventional assays of giving a diagnosis of certainty, and of being applicable to early prenatal diagnosis using biopsies of trophoblast villi. At present, the single known polymorphism associated with the FIX gene restricts the application of linkage analysis to informative cases (40%), but findings of additional RFLPs in this region should improve this figure.
Assuntos
Enzimas de Restrição do DNA/genética , Fator IX , Triagem de Portadores Genéticos/métodos , Hemofilia B/genética , Adolescente , Adulto , Idoso , Pré-Escolar , Feminino , Marcadores Genéticos , Hemofilia B/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo GenéticoRESUMO
Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identification of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation.
Assuntos
Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Análise de SequênciaRESUMO
Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.