RESUMEN
Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of progesterone and GCs, and expression of steroidogenic enzymes and key HPA axis components (eg, corticotropin-releasing hormone [CRH], adrenocorticotropic hormone [ACTH]). We administered LPS (50 µg/kg intraperitoneally) or vehicle control to male and female C57BL/6J neonatal (postnatal day [PND] 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hours later. We measured progesterone, 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone via liquid chromatography-tandem mass spectrometry. We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via quantitative polymerase chain reaction. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominantly in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH.
Asunto(s)
Médula Ósea/metabolismo , Glucocorticoides/biosíntesis , Lipopolisacáridos/farmacología , Bazo/metabolismo , Timo/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Animales , Animales Recién Nacidos/metabolismo , Médula Ósea/efectos de los fármacos , Médula Ósea/enzimología , Corticosterona/análisis , Corticosterona/sangre , Femenino , Glucocorticoides/sangre , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , ARN Mensajero/análisis , Receptores de Hormona Liberadora de Corticotropina/genética , Bazo/efectos de los fármacos , Bazo/enzimología , Esteroide 11-beta-Hidroxilasa/genética , Timo/efectos de los fármacos , Timo/enzimologíaRESUMEN
BACKGROUND: Spinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury. METHODS: Contusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array. RESULTS: Syk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils. CONCLUSIONS: Syk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter sparing. These findings implicate Syk in pathogenic neutrophil activities that worsen long-term functional recovery after spinal cord injury.
Asunto(s)
Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/patología , Activación Neutrófila , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/patología , Bazo/enzimología , Quinasa Syk/genética , Animales , Apoptosis , Muerte Celular , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Femenino , Miembro Posterior/inervación , Masculino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Recuperación de la Función , Sustancia Blanca/patologíaRESUMEN
Heme oxygenase-1 (HO-1) is an evolutionarily conserved stress response enzyme and important in pregnancy maintenance, fetal and neonatal outcomes, and a variety of pathologic conditions. Here, we investigated the effects of an exposure to systemic inflammation late in gestation [embryonic day (E)15.5] on wild-type (Wt) and HO-1 heterozygous (Het, HO-1+/-) mothers, fetuses, and offspring. We show that alterations in fetal liver and spleen HO homeostasis during inflammation late in gestation can lead to a sustained dysregulation of innate immune cell populations and intracellular myeloid HO-1 expression in the spleen through young adolescence [postnatal day 25] in mice.
Asunto(s)
Feto/metabolismo , Hemo-Oxigenasa 1/metabolismo , Inmunidad Innata , Inflamación/patología , Animales , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Genotipo , Hemo-Oxigenasa 1/deficiencia , Hemo-Oxigenasa 1/genética , Inflamación/metabolismo , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Bazo/efectos de los fármacos , Bazo/enzimologíaRESUMEN
Perturbed expression of microRNAs (miRs) has been reported in different diseases including autoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR-25-3p and its targets in the central nervous system (CNS) tissue from mice with experimental autoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets in activated macrophages and splenocytes. EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyte glycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR-25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We next established primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR-25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and anti-CD3/anti-CD28 antibodies, respectively. MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression of phosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak of the disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR-25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significant upregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR-25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show any alterations in miR-25-3p expression compared with vehicle-treated cells. Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators during autoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.
Asunto(s)
Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/enzimología , Mediadores de Inflamación/metabolismo , Macrófagos/enzimología , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Bazo/enzimología , Linfocitos T/enzimología , Animales , Autoinmunidad , Células Cultivadas , Citocinas/genética , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Adyuvante de Freund , Regulación de la Expresión Génica , Factor 4 Similar a Kruppel/genética , Factor 4 Similar a Kruppel/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Ratones Endogámicos C57BL , MicroARNs/genética , Glicoproteína Mielina-Oligodendrócito , Fosfohidrolasa PTEN/genética , Fragmentos de Péptidos , Transducción de Señal , Bazo/inmunología , Linfocitos T/inmunologíaRESUMEN
The splenic microenvironment regulates hematopoietic stem and progenitor cell (HSPC) function, particularly during demand-adapted hematopoiesis; however, practical strategies to enhance splenic support of transplanted HSPCs have proved elusive. We have previously demonstrated that inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH), using the small molecule (+)SW033291 (PGDHi), increases BM prostaglandin E2 (PGE2) levels, expands HSPC numbers, and accelerates hematologic reconstitution after BM transplantation (BMT) in mice. Here we demonstrate that the splenic microenvironment, specifically 15-PGDH high-expressing macrophages, megakaryocytes (MKs), and mast cells (MCs), regulates steady-state hematopoiesis and potentiates recovery after BMT. Notably, PGDHi-induced neutrophil, platelet, and HSPC recovery were highly attenuated in splenectomized mice. PGDHi induced nonpathologic splenic extramedullary hematopoiesis at steady state, and pretransplant PGDHi enhanced the homing of transplanted cells to the spleen. 15-PGDH enzymatic activity localized specifically to macrophages, MK lineage cells, and MCs, identifying these cell types as likely coordinating the impact of PGDHi on splenic HSPCs. These findings suggest that 15-PGDH expression marks HSC niche cell types that regulate hematopoietic regeneration. Therefore, PGDHi provides a well-tolerated strategy to therapeutically target multiple HSC niches, promote hematopoietic regeneration, and improve clinical outcomes of BMT.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hematopoyesis Extramedular/efectos de los fármacos , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Regeneración , Bazo/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Femenino , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Bazo/enzimología , Bazo/metabolismoRESUMEN
Sialic acid and its associated metabolic enzymes have emerged as important components of the pathophysiology of type 2 diabetes (T2D). There is an elevation in the serum concentration of sialic acid in humans and animals with T2D. The present study investigated the modulation of mRNA expression level of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) and neuraminidase 1 (NEU1) genes in some organs of type 2 diabetic rats. T2D was induced using fructose-streptozotocin model and eight weeks after the induction of diabetes, sialic acid was assayed in the blood and organs (adipose tissue, brain, colon, kidney, liver, pancreas, skeletal muscle and spleen) followed by quantification of mRNA expression level of GNE and NEU1 genes by qPCR. The results showed a significant (P < 0.05) increase in sialic acid level in the serum and all the afore-mentioned organs investigated except in the adipose tissue and skeletal muscle of the diabetic rats compared the normal control. The expression GNE gene was only increased in the pancreas (1.8-fold) of the diabetic rats while there was a decrease in the expression of the gene in the colon. In contrast, the expression of NEU1 gene was increased in the spleen (3.5-fold), brain (2.2-fold), liver (1.9-fold), colon (1.5-fold) and kidney of the diabetic rats. It was concluded that the elevated level of sialic acid in the organs of diabetic rats, except the pancreas, might not be due to increased endogenous synthesis of sialic acid.
Asunto(s)
Diabetes Mellitus Tipo 2/enzimología , Complejos Multienzimáticos/genética , Animales , Encéfalo/enzimología , Colon/enzimología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Regulación Enzimológica de la Expresión Génica , Hígado/enzimología , Ácido N-Acetilneuramínico/sangre , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/genética , Páncreas/enzimología , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Bazo/enzimologíaRESUMEN
Systemic inflammatory response syndrome and sepsis are considered to contribute to hypercytokinemia in both patients with severe infection and immunocompromised condition. Past research has demonstrated that antibiotics and antifungals not only have antimicrobial efficacy but also affect the immune system. We previously examined whether immune cells were modulated by antibiotics such as tetracyclines or macrolides. The modulation of lipopolysaccharide-stimulated cells by those agents was elucidated. However, few reports about the modulation of the immune system by antifungal agents were found. In this study, the production of pro-inflammatory cytokines and chemokines and signaling pathways involved were investigated in zymosan-activated THP-1 cells. The effects were examined using antifungal agents such as echinocandin including caspofungin (CAS) and micafungin. Pro-inflammatory cytokine and chemokine levels were determined using enzyme-linked immunosorbent assay. Protein phosphorylation was evaluated by western blot analysis. CAS significantly decreased zymosan-induced pro-inflammatory cytokine and chemokine release in THP-1 cells. CAS (30 µg/mL) also downregulated tumor necrosis factor alpha levels, as shown by enzyme-linked immunosorbent assay. In western blot analysis, inhibitor of nuclear factor-kappa-B alpha, p38, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and nuclear factor of activated T-cells phosphorylation and activation of caspase-1 and spleen tyrosine kinase (Syk) were downregulated. The major underlying mechanism of pro-inflammatory cytokine and chemokine suppression by CAS is to inhibit activation of Syk and its downstream signaling molecules. Based on the results, it can be concluded that CAS activity possibly involves Syk signaling pathways and has potential to prevent hypercytokinemia in fungal sepsis.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antifúngicos/farmacología , Caspofungina/farmacología , Quimiocinas/metabolismo , Citocinas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Bazo/enzimología , Zimosan/farmacología , Humanos , Transducción de Señal , Células THP-1RESUMEN
The enzymatic family of heme oxygenase (HO) is accountable for heme breakdown. Among the two isoforms characterized to date, HO-2 is poorly investigated due to the lack of potent HO-2 chemical modulators and the greater attentiveness towards HO-1 isoform. In the present paper, we report the rational design and synthesis of HO-2 inhibitors achieved by modulating the volume of known HO-1 inhibitors. The inhibition preference has been moved from HO-1 to HO-2 by merely increasing the volume of the substituent in the western region of the inhibitors. Docking studies demonstrated that new derivatives soak differently in the two binding pockets, probably due to the presence of a Tyr187 residue in HO-2. These findings could be useful for the design of new selective HO-2 compounds.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Nitrilos/farmacología , Algoritmos , Animales , Encéfalo/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Hemo Oxigenasa (Desciclizante)/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Estructura Molecular , Nitrilos/síntesis química , Nitrilos/química , Ratas , Bazo/enzimología , Relación Estructura-ActividadRESUMEN
Obesity is a major risk factor for the development of insulin resistance and type 2 diabetes. However, the mechanisms that trigger the underlying adipose tissues inflammation are not completely understood. Here, we show that the E3 ubiquitin ligase March1 controls the phenotypic and functional properties of CD8+ T cells in mice white adipose tissue. In a diet-induced obesity model, mice lacking March1 [March1 knockout (KO)] show increased insulin resistance compared to their WT counterparts. Also, in obese March1 KO mice, the proportions of effector/memory (Tem) and resident/memory (Trm) CD8+ T cells were higher in the visceral adipose tissue, but not in the spleen. The effect of March1 on insulin resistance and on the phenotype of adipose tissue CD8+ T cells was independent of major histocompatibility complex class II ubiquitination. Interestingly, we adoptively transferred either WT or March1 KO splenic CD8+ T cells into obese WT chimeras that had been reconstituted with Rag1-deficient bone marrow. We observed an enrichment of Tem and Trm cells and exacerbated insulin resistance in mice that received March1 KO CD8 T cells. Mechanistically, we found that March1 deficiency alters the metabolic activity of CD8+ T cells. Our results provide additional evidence of the involvement of CD8+ T cells in adipose tissue inflammation and suggest that March1 controls the metabolic reprogramming of these cells.
Asunto(s)
Tejido Adiposo Blanco/enzimología , Linfocitos T CD8-positivos/metabolismo , Memoria Inmunológica , Resistencia a la Insulina , Obesidad/enzimología , Ubiquitina-Proteína Ligasas/deficiencia , Tejido Adiposo Blanco/inmunología , Traslado Adoptivo , Animales , Glucemia/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/trasplante , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Metabolismo Energético , Activación de Linfocitos , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Obesidad/inmunología , Fenotipo , Bazo/enzimología , Bazo/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Acid sphingomyelinase deficiency (ASMD) also known as Niemann-Pick disease, is a rare lysosomal storage disorder with a diverse disease spectrum that includes slowly progressive, chronic visceral (type B) and neurovisceral forms (intermediate type A/B), in addition to infantile, rapidly progressive fatal neurovisceral disease (type A). PURPOSE AND METHODS: We review the published evidence on the relevance of splenomegaly and reduced lung diffusion capacity to the clinical burden of chronic forms of ASMD. Targeted literature searches were conducted to identify relevant ASMD and non-ASMD studies for associations between diffusing capacity of the lungs for carbon monoxide (DLCO) and splenomegaly, with clinical parameters and outcome measures. RESULTS: Respiratory disease and organomegaly are primary and independent contributors to mortality, disease burden, and morbidity for patients with chronic ASMD. The degree of splenomegaly correlates with short stature, atherogenic lipid profile, and degree of abnormality of hematologic parameters, and thus may be considered a surrogate marker for bleeding risk, abnormal lipid profiles and possibly, liver fibrosis. Progressive lung disease is a prevalent clinical feature of chronic ASMD, contributing to a decreased quality of life (QoL) and an increased disease burden. In addition, respiratory-related complications are a major cause of mortality in ASMD. CONCLUSIONS: The reviewed evidence from ASMD natural history and observational studies supports the use of lung function and spleen volume as clinically meaningful endpoints in ASMD trials that translate into important measures of disease burden for patients.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades de Niemann-Pick/genética , Esfingomielina Fosfodiesterasa/genética , Esplenomegalia/genética , Monóxido de Carbono/metabolismo , Terapia de Reemplazo Enzimático , Humanos , Pulmón/metabolismo , Pulmón/patología , Enfermedades por Almacenamiento Lisosomal/epidemiología , Enfermedades por Almacenamiento Lisosomal/patología , Enfermedades por Almacenamiento Lisosomal/terapia , Mutación/genética , Enfermedades de Niemann-Pick/epidemiología , Enfermedades de Niemann-Pick/patología , Enfermedades de Niemann-Pick/terapia , Bazo/enzimología , Bazo/patología , Esplenomegalia/epidemiología , Esplenomegalia/patología , Esplenomegalia/terapiaRESUMEN
The expression and localization of different isoforms of creatine kinase in Pelodiscus sinensis (PSCK) were studied to reveal the role of PSCK isozymes (PSCK-B, PSCK-M, PSCK-S) under bacterial infection-induced immunologic stress. The computational molecular dynamics simulations predicted that PSCK-S would mostly possess a kinase function in a structural aspect when compared to PSCK-B and PSCK-M. The assay of biochemical parameters such as total superoxide dismutase (T-SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), and the content of ATP were measured along with total PSCK activity in different tissue samples under bacterial infection. The expression detections of PSCK isozymes in vitro and in vivo were overall well-matched where PSCK isozymes were expressed differently in P. sinensis tissues. The results showed that PSCK-B mostly contributes to the spleen, followed by the liver and myocardium; PSCK-M mostly contributes to the liver, followed by the myocardium and skeletal muscle, while PSCK-S contributes to the spleen and is uniquely expressed in skeletal muscle. Our study suggests that the various alterations of PSCK isozymes in tissues of P. sinensis are prone to defense the bacterial infection and blocking energetic imbalance before severe pathogenesis turned on in P. sinensis.
Asunto(s)
Infecciones Bacterianas/enzimología , Creatina Quinasa/química , Isoformas de Proteínas/química , Estrés Fisiológico/inmunología , Tortugas/metabolismo , Adenosina Trifosfato/metabolismo , Aeromonas hydrophila/inmunología , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Catalasa/metabolismo , Creatina Quinasa/genética , Creatina Quinasa/metabolismo , Regulación de la Expresión Génica/inmunología , Inmunohistoquímica , L-Lactato Deshidrogenasa/metabolismo , Hígado/química , Hígado/enzimología , Malondialdehído/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Miocardio/química , Miocardio/enzimología , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análisis de Secuencia de Proteína , Bazo/química , Bazo/enzimología , Superóxido Dismutasa/metabolismo , Tortugas/genética , Tortugas/inmunología , Tortugas/microbiologíaRESUMEN
Copper (Cu) is a necessary trace mineral due to its biological activity. Excessive Cu can induce inflammatory response in humans and animals, but the underlying mechanism is still unknown. Here, 240 broilers were used to study the effects of excessive Cu on oxidative stress and NF-κB-mediated inflammatory responses in immune organs. Chickens were fed with diet containing different concentrations of Cu (11, 110, 220, and 330 mg of Cu/kg dry matter). The experiment lasted for 49 days. Spleen, thymus, and bursa of Fabricius (BF) on day 49 were collected for histopathological observation and assessment of oxidative stress status. Additionally, the mRNA and protein levels of NF-κB and inflammatory cytokines were also analyzed. The results indicated that excess Cu could increase the number and area of splenic corpuscle as well as the ratio of cortex and medulla in thymus and BF. Furthermore, excessive Cu intake could decrease activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); but increase contents of malondialdehyde (MDA), TNF-α, IL-1, IL-1ß; up-regulate mRNA levels of TNF-α, IFN-γ, IL-1, IL-1ß, IL-2, iNOS, COX-2, NF-κB and protein levels of TNF-α, IFN-γ, NF-κB, p-NF-κB in immune organs. In conclusion, excessive Cu could cause pathologic changes and induce oxidative stress with triggered NF-κB pathway, and might further regulate the inflammatory response in immune organs of chicken.
Asunto(s)
Pollos/inmunología , Cobre/toxicidad , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Bolsa de Fabricio/enzimología , Bolsa de Fabricio/inmunología , Bolsa de Fabricio/metabolismo , Bolsa de Fabricio/patología , Catalasa/metabolismo , Pollos/genética , Pollos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Glutatión Peroxidasa/metabolismo , Inflamación/genética , Inflamación/metabolismo , Malondialdehído/metabolismo , FN-kappa B/genética , Bazo/enzimología , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Superóxido Dismutasa/metabolismo , Timo/enzimología , Timo/inmunología , Timo/metabolismo , Timo/patologíaRESUMEN
Heme oxygenase-1 (HO-1) has been recognized as extensively involved in the development and aggravation of cancer, cell propagation and at in the mechanism of chemoresistance development. Low micromolar HO-1 inhibitors selective towards HO-2 has been recently reported, wherein the azole core and the hydrophobic residues are linked through a phenylethanolic spacer bearing a chiral center. Since less information are known about the stereoselective requirements for HO-1 inhibition, here we report the enantiomeric resolution of 1-(biphenyl-3-yl)-2-(1H-imidazol-1-yl)ethanol (1) and 1-[4-[(4-bromobenzyl)oxy]phenyl]-2-(1H-imidazol-1-yl)ethanol (2), two among the most potent and selective HO-1 inhibitors known thus far when tested as racemates. The absolute configuration was established for 1 by a combination of experimental and in silico derived electronic circular dichroism spectra, while docking approaches were useful in the case of compound 2. Biological evaluation of pure enantiomers highlighted higher HO-1 inhibitory activity of (R)-enantiomers. Docking studies demonstrated the importance of hydrogen bond interaction, more pronounced for the (R)-enantiomers, with a consensus water molecule within the binding pocket. The present study demonstrates that differences in three-dimensional structure amongst compounds 1 and 2 enantiomers affect significantly the selectivity of these HO-1 inhibitors.
Asunto(s)
Azoles/farmacología , Inhibidores Enzimáticos/farmacología , Alcohol Feniletílico/farmacología , Animales , Azoles/química , Teoría Funcional de la Densidad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/metabolismo , Masculino , Simulación del Acoplamiento Molecular , Estructura Molecular , Alcohol Feniletílico/química , Ratas , Ratas Sprague-Dawley , Bazo/enzimología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Peste des petits ruminant (PPR), a highly contagious viral disease of small ruminants, is characterized by erosive stomatitis and pneumo-enteritis. However, its neurovirulence potential as observed with other morbilliviruses has not been fully investigated. The present study describes the neuropathological alterations induced by PPR virus through apoptotic pathway. A total number of 12 carcasses of local breed goat kids of either sex were received for postmortem examination. The clinical history was described as symptoms of mucopurulent nasal discharge, high to low grade fever, erosive stomatitis, dyspnoea and profuse watery diarrhoea followed by mortality of 35 goat kids within a week. The pathoanatomical lesions and immunohistochemical demonstration of PPRV antigen in lungs, intestine, spleen and lymph nodes confirmed PPR disease in goats. Grossly, five brain specimens showed moderate to severe leptomeningeal congestion during necropsy. Microscopically, brain sections showed leptomeningitis and nonsuppurative encephalitis characterized by vascular congestion, haemorrhages in the parenchyma, perivascular cuffing with mild to moderate mononuclear cells (mainly lymphocytes and few macrophages), focal to diffuse microgliosis, neuronal degeneration, satellitosis and neuronophagia. Immunolabelling of viral antigen was observed in the cytoplasm of neurons and glial cells. The RT-PCR amplification of N gene fragment also confirmed the presence of PPRV in the brain. The strong immunoreactivity of Caspase-3, Caspase-8 and comparatively lower expression of caspase-9 along with the absence of any reactivity for Apaf-1 antigen in the brain sections indicated the role of caspase dependent extrinsic pathway in inducing neuropathological changes. The presence of apoptotic neurons in the brain by TUNEL assay further confirmed the apoptosis and strong immunoreactivity of iNOS in neurons which suggested the generation of oxidative stress, that might have induced the apoptosis. The overall findings confirm the neurovirulence potential of PPR virus, via the extrinsic pathway of apoptosis, in natural cases of PPR disease in goat kids.
Asunto(s)
Caspasas/metabolismo , Enfermedades de las Cabras/enzimología , Peste de los Pequeños Rumiantes/enzimología , Animales , Apoptosis , Encéfalo/enzimología , Encéfalo/patología , Encéfalo/virología , Caspasas/genética , Femenino , Enfermedades de las Cabras/patología , Enfermedades de las Cabras/fisiopatología , Enfermedades de las Cabras/virología , Cabras , Pulmón/enzimología , Pulmón/patología , Pulmón/virología , Masculino , Neuropatología , Peste de los Pequeños Rumiantes/patología , Peste de los Pequeños Rumiantes/fisiopatología , Peste de los Pequeños Rumiantes/virología , Virus de la Peste de los Pequeños Rumiantes/fisiología , Bazo/enzimología , Bazo/patología , Bazo/virologíaRESUMEN
The tree shrew (Tupaia belangeri) is a rat-sized mammal, which is more closely related to humans than mice and rats. However, the use of tree shrew to explore the patho-mechanisms of human inflammatory disorders has been limited since nothing is known about eicosanoid metabolism in this mammalian species. Eicosanoids are important lipid mediators exhibiting pro- and anti-inflammatory activities, which are biosynthesized via lipoxygenase and cyclooxygenase pathways. When we searched the tree shrew genome for the presence of cyclooxygenase and lipoxygenase isoforms we found copies of functional COX1, COX2 and LOX genes. Interestingly, we identified four copies of ALOX15 genes, which encode for four structurally distinct ALOX15 orthologs (tupALOX15a-d). To explore the catalytic properties of these enzymes we expressed tupALOX15a and tupALOX15c as catalytically active proteins and characterized their enzymatic properties. As predicted by the Evolutionary Hypothesis of ALOX15 specificity we found that the two enzymes converted arachidonic acid predominantly to 12S-HETE and they also exhibited membrane oxygenase activities. However, their reaction kinetic properties (KM for arachidonic acid and oxygen, T- and pH-dependence) and their substrate specificities were remarkably different. In contrast to mice and humans, tree shrew ALOX15 isoforms are highly expressed in the brain suggesting a role of these enzymes in cerebral function. The genomic multiplicity and the tissue expression patterns of tree shrew ALOX15 isoforms need to be considered when the results of in vivo inflammation studies obtained in this animal are translated into the human situation.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Evolución Molecular , Tupaia/metabolismo , Animales , Araquidonato 15-Lipooxigenasa/genética , Ácido Araquidónico/metabolismo , Encéfalo/enzimología , Isoenzimas/genética , Isoenzimas/metabolismo , Pulmón/enzimología , Modelos Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Bazo/enzimología , Tupaia/genéticaRESUMEN
Neospora caninum is a protozoan that has tropism for the central nervous system. The aim of this study was to determine whether experimental infection of gerbils would interfere with activity of enzymes associated with energy metabolism. We randomized 20 gerbils into two groups (ten animals per group): the control group (healthy animals; uninfected) and the infected group (experimentally infected with dose 7.8â¯×â¯102 tachyzoites of N. caninum per gerbil). On day six and twelve post-infection (PI), brain and spleen tissues were collected for biochemical and histopathological analyses. No histopathological lesions were observed in the brains of infected animals; however, inflammatory infiltrates were found in the spleen. Significantly greater levels of reactive oxygen species (ROS) were observed in the brain and spleen of infected gerbils than in the control group at 12 days PI. Cytosolic creatine kinase (CK-CYT), mitochondrial creatine kinase (CK-MIT), and pyruvate kinase (PK) activities were lower in the brains of infected gerbils than in those of the control group on day 12 PI. There was significantly less CK-CYT activity in the spleens of infected gerbils on day 6 and 12 PI. Finally, there was significantly less sodium-potassium ion pump (Na+/K+ ATPase) activity in the brains and spleens of infected gerbils on day 12 PI. These data suggest that experimental infection with N. caninum interfered with energy metabolism associated with ATP homeostasis in the brain and spleen, directly or indirectly, apparently mediated by ROS overproduction, contributing to inhibition of Na+/K+ ATPase activity.
Asunto(s)
Encéfalo/enzimología , Coccidiosis/enzimología , Metabolismo Energético , Neospora , Bazo/enzimología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Coccidiosis/metabolismo , Creatina Quinasa/metabolismo , Citosol/enzimología , Gerbillinae , Masculino , Mitocondrias/enzimología , Piruvato Quinasa/metabolismo , Distribución Aleatoria , Especies Reactivas de Oxígeno/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Bazo/química , Bazo/patologíaRESUMEN
Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5'-4,4'-dimethyltryptamine-thymidine-succinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL-4B were 40 mg/mL trypsin solution, pH 7 at 4 °C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) by the immobilized trypsin were 55 °C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 µM of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.
Asunto(s)
Enzimas Inmovilizadas/química , Proteínas de Peces/química , Bazo/enzimología , Tripsina/química , Atún , Animales , Estabilidad de Enzimas , Calor , Concentración de Iones de HidrógenoRESUMEN
The immunoproteasome (iP) is a variant of the constitutive proteasome (cP) that is abundantly expressed in immune cells which can also be induced in somatic cells by cytokines such as TNF-α or IFN-γ. Accumulating evidence support that the iP is closely linked to multiple facets of inflammatory response, eventually leading to the development of several iP inhibitors as potential therapeutic agents for autoimmune diseases. Recent studies also found that the iP is upregulated in reactive glial cells surrounding amyloid ß (Aß) deposits in brains of Alzheimer's disease (AD) patients, but the role it plays in the pathogenesis of AD remains unclear. In this study, we investigated the effects of several proteasome inhibitors on cognitive function in AD mouse models and found that YU102, a dual inhibitor of the iP catalytic subunit LMP2 and the cP catalytic subunit Y, ameliorates cognitive impairments in AD mouse models without affecting Aß deposition. The data obtained from our investigation revealed that YU102 suppresses the secretion of inflammatory cytokines from microglial cells. Overall, this study indicates that there may exist a potential link between LMP2/Y and microglia-mediated neuroinflammation and that inhibition of these subunits may offer a new therapeutic strategy for AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Cisteína Endopeptidasas/genética , Neuroglía/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/patología , Línea Celular , Disfunción Cognitiva/enzimología , Disfunción Cognitiva/genética , Disfunción Cognitiva/patología , Cisteína Endopeptidasas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Proteínas Quimioatrayentes de Monocitos/genética , Proteínas Quimioatrayentes de Monocitos/metabolismo , Neuroglía/enzimología , Neuroglía/patología , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Bazo/efectos de los fármacos , Bazo/enzimología , Bazo/patologíaRESUMEN
Circadian clocks, biological timekeepers that are present in almost every cell of our body, are complex systems whose disruption is connected to various diseases. Controlling cellular clock function with high temporal resolution in an inducible manner would yield an innovative approach for the circadian rhythm regulation. In the present study, we present structure-guided incorporation of photoremovable protecting groups into a circadian clock modifier, longdaysin, which inhibits casein kinase I (CKI). Using photodeprotection by UV or visible light (400 nm) as the external stimulus, we have achieved quantitative and light-inducible control over the CKI activity accompanied by an accurate regulation of circadian period in cultured human cells and mouse tissues, as well as in living zebrafish. This research paves the way for the application of photodosing in achieving precise temporal control over the biological timing and opens the door for chronophotopharmacology to deeper understand the circadian clock system.
Asunto(s)
Adenina/análogos & derivados , Quinasa de la Caseína I/antagonistas & inhibidores , Relojes Circadianos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Rayos Ultravioleta , Pez Cebra/metabolismo , Adenina/química , Adenina/farmacología , Animales , Línea Celular , Relojes Circadianos/efectos de la radiación , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/efectos de la radiación , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Larva/efectos de los fármacos , Larva/enzimología , Larva/efectos de la radiación , Fototransducción , Ratones , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Bazo/efectos de los fármacos , Bazo/enzimología , Bazo/efectos de la radiación , Factores de TiempoRESUMEN
Polychlorinated biphenyls (PCBs) would do serious damage to multiple systems, while coplanar polychlorinated biphenyls, the most toxic member of the family, has been widely taken into consideration. In this study, ICR mice were fed with different doses of PCB126 to explore the underlying molecular mechanisms on immunotoxicity. The results showed that PCB126 caused immunosuppression as evidenced by inhibiting the ratios of thymus and spleen weights, changing the organizational structure and decreasing levels and mRNA expression of TNF-α, IFN-γ and IL-2. PCB126 inhibited the SOD activity and spurred the accumulation of MDA in spleen and thymus. Meanwhile, it also disturbed the Nrf2 signaling pathway as evidenced by up-regulating the mRNA expression of Nrf2 and Keap1. Additionally, a remarkable reduction in the mRNA expression of AhR and enhancement in the mRNA expression of Cyp1 enzymes (Cyp1a1, Cyp1a2 and Cyp1b1) were observed, which increased the ROS levels. PCB126 could increase protein expression of Bax, Caspase-3, Caspase-8 and Caspase-9, while the protein expression of Bcl-2 was decreased. In summary, the results indicated that PCB126 modulated the AhR signaling pathway, which interacted with apoptosis and oxidative stress to induce immunotoxicity, enrich the immunotoxicological mechanisms of PCB126.