RESUMEN
Glutamate functions as the major excitatory neurotransmitter for primary sensory neurons and has a crucial role in sensitizing peripheral nociceptor terminals producing sensitization. Glutaminase (GLS) is the synthetic enzyme that converts glutamine to glutamate. GLS-immunoreactivity (-ir) and enzyme activity are elevated in dorsal root ganglion (DRG) neuronal cell bodies during chronic peripheral inflammation, but the mechanism for this GLS elevation is yet to be fully characterized. It has been well established that, after nerve growth factor (NGF) binds to its high-affinity receptor tropomyosin receptor kinase A (TrkA), a retrograde signaling endosome is formed. This endosome contains the late endosomal marker Rab7GTPase and is retrogradely transported via axons to the cell soma located in the DRG. This complex is responsible for regulating the transcription of several critical nociceptive genes. Here, we show that this retrograde NGF signaling mediates the expression of GLS in DRG neurons during the process of peripheral inflammation. We disrupted the normal NGF/TrkA signaling in adjuvant-induced arthritic (AIA) Sprague Dawley rats by the pharmacological inhibition of TrkA or blockade of Rab7GTPase, which significantly attenuated the expression of GLS in DRG cell bodies. The results indicate that NGF/TrkA signaling is crucial for the production of glutamate and has a vital role in the development of neurogenic inflammation. In addition, our pain behavioral data suggest that Rab7GTPase can be a potential target for attenuating peripheral inflammatory pain.
Asunto(s)
Ganglios Espinales , Glutaminasa , Inflamación , Factor de Crecimiento Nervioso , Ratas Sprague-Dawley , Receptor trkA , Transducción de Señal , Animales , Ganglios Espinales/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Glutaminasa/metabolismo , Ratas , Receptor trkA/metabolismo , Inflamación/metabolismo , Inflamación/patología , Masculino , Neuronas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Chronic inflammatory diseases are considered the most significant cause of death worldwide. Current treatments for inflammatory diseases are limited due to the lack of understanding of the biological factors involved in early-stage disease progression. Nerve growth factor (NGF) is a neurotrophic factor directly associated with inflammatory and autoimmune diseases like osteoarthritis, multiple sclerosis, and rheumatoid arthritis. It has been shown that NGF levels are significantly upregulated at the site of inflammation and play a crucial role in developing a robust inflammatory response. However, little is known about NGF's temporal expression profile during the initial progressive phase of inflammation. This study aimed to determine the temporal expression patterns of NGF in rat skin (epidermis) during adjuvant-induced arthritis (AIA). Sprague Dawley rats were randomly divided into control and complete Freund's adjuvant (CFA)-treated groups. Levels of NGF were evaluated following unilateral AIA at different time points, and it was found that peripheral inflammation due to AIA significantly upregulated the expression of NGF mRNA and protein in a biphasic pattern. These results suggest that NGF signaling is crucial for initiating and maintaining peripheral neurogenic inflammation in rats during AIA.
Asunto(s)
Factor de Crecimiento Nervioso , Inflamación Neurogénica , Animales , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Nervioso/genética , Adyuvantes Inmunológicos , Adyuvantes Farmacéuticos , InflamaciónRESUMEN
Coastal ecosystems are vital for maintaining the biodiversity and human livelihoods, but they are increasingly subjected to anthropogenic pressures, including pollution from various sources. Present work intends to assess the possible threats in coastal ecosystem as well as coastal fish species, in particular, through haematological parameters caused due to exposure of environmental contaminants like polycyclic aromatic hydrocarbons (PAHs), potentially toxic metals (PTMs), etc. This study analysed the haematological parameters and probable toxicity levels in two important coastal fish species, viz., Mystus sp. and Mugil sp. widely available in Digha coastal belt. Different haematological parameters, such as WBCs (White Blood Cells), Lym (Lymphocytes), Gran (Granulocytes), Mid (Monocytes), RBCs (Red Blood Cells), HCT (Haematocrit) value, MCV (Mean Corpuscular Volume), MCH (Mean Corpuscular Haemoglobin), MCHC (Mean Corpuscular Haemoglobin Concentration), RDW- CV (Red Cells Distribution Width-Co-efficient of Variation), RDW- SD (Red Cells Distribution Width-Standard Deviation), PLT (Total Platelet Count), MPV (Mean Platelet Volume), PDW- SD (Platelet Distribution Width-Standard Deviation), PDW- CV (Platelet Distribution Width-Co-efficient of Variation), PCT (Plateletcrit), PLCR (Platelet Large Cell Ratio), PLCC (Platelet Large Cell Count) and many others were measured directly through Erba H360 Haematology Analyser, simultaneously air dried blood smear was stained by Haematoxylin-Eosin(H-E) and Giemsa stain for assessing morphometric alterations of RBCs, WBCs, platelets as well as to determine the differential counts of WBCs by observing through Leica DM2000 microscope. Evidence of several abnormalities in the erythrocyte's nucleus (ENAs) and the abundance of abnormal celled erythrocytes (ECAs), carcinoma (lymphoproliferative disorder, polycythaemia vera, Hodgkin lymphoma and non-Hodgkin lymphoma), elevation of WBCs content, Lym %(Lymphocyte percentage), Eo(Eosinophils), monocytes, HCT and gross depletion of Ne(Neutrophils), basophils, and PLCR levels indicated a sign of major impact of contamination to two intoxicated fishes which may also affect the human being through food chain and may result into leukaemia in mammalian species, finally. However, comprehensive evaluation of the long-term impacts of the contaminants like PAHs and/or PTMs, etc., on fish populations, human health risk and coastal ecosystem is required to be addressed.
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Contaminantes Químicos del Agua , Animales , India , Contaminantes Químicos del Agua/toxicidad , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/sangre , Peces , Monitoreo del Ambiente/métodos , Pruebas Hematológicas , Hidrocarburos Policíclicos Aromáticos/toxicidad , Hidrocarburos Policíclicos Aromáticos/análisisRESUMEN
Background and aims: Alcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p. Materials and methods: Bio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required. Results: The combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1ß/IL8/MCP1/IL6/TGFß) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1ß were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1ß was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1ß-31CT+TT than TNF-α-238GG/IL1ß-31CC. The TNF-α/IL1ß promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1ß over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFß and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1ß were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFßR2/MCP1, and ICAM1 respectively. Conclusion: Thus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1ß gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1ß and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients.
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Interleucina-1beta , Hepatopatías Alcohólicas , MicroARNs , Factor de Necrosis Tumoral alfa , Humanos , Quimiocinas/genética , Colágeno/genética , Cirrosis Hepática/genética , Hepatopatías Alcohólicas/genética , Luciferasas/genética , MicroARNs/genética , Polimorfismo de Nucleótido Simple , Factor de Crecimiento Transformador beta/genética , Factor de Necrosis Tumoral alfa/genética , Interleucina-1beta/genéticaRESUMEN
The present study was intended to determine the possible influences of direct field application of choline chloride into pond water in addition to farm-made-aqua-feed under a semi-intensive culture system on the growth and biochemical parameters of two Indian major carps (IMCs), Catla catla (Catla) and Labeo rohita (Rahu), and two air-breathing species, Clarias batrachus (Magur) and Anabas testudineus (Koi), cultured in a ratio of 2:5:1:1 (Catla/Rahu/Magur/Koi) in three experimental ponds for a period of 90 days during the breeding season (June to August). Results were compared with control (C: fed only with farm-made-aqua-feed) and treatment (T: P1 and P2: farm-made-aqua-feed plus choline chloride into pond water directly at the rate of 350 g bigha-1 fortnightly or 350 g per 1600 square meter fortnightly). A significant increasing trend was observed in the growth parameters including total length-final (TLF), standard length-final (SLF), mean weight-final (MWF), % gain of mean total length (MTL), % gain of mean standard length (MSL), % weight gain (WG), specific growth rate (SGR) % per day, and survivability %. However, a reverse pattern was noticed in the food conversion ratio (FCR) both in IMCs and air-breathing fish species under choline supplementation. Serum biochemical responses, e.g., total protein (PRO), lactate dehydrogenase (LDH), glucose (GLU), and calcium (Ca) showed significant enhancement, and alkaline phosphatase (ALP), alanine amino transaminase (ALT), aspartate amino transaminase (AST), cholesterol (CHOLES), and triglycerides (Trig) showed gradual significant reduction during the breeding season under choline exposure. Treated fishes showed prevention from liver dysfunction and fatty liver formation, and increased body crude protein content. Results indicated favorable growth and yield, which may benefit fish farmers during their culture practices, and the output fish species under choline supplementation resulted in quality food-fish for human consumption.
RESUMEN
Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin injury, inflammation, and/or sensitization. The goal of this study is to describe an epidermal-dermal separation protocol using thermolysin, a proteinase that is active at 4 °C. To illustrate this procedure, Sprague Dawley rats are anesthetized, and right hind paws are injected with carrageenan. Six and twelve hours after injection, rats with inflammation and naïve rats are euthanized, and a piece of hind paw, glabrous skin is placed in cold Dulbecco's Modified Eagle Medium. The epidermis is then separated at the basement membrane from the dermis by thermolysin in PBS with calcium chloride. Next, the dermis is secured by microdissection forceps, and the epidermis is gently teased away. Toluidine blue staining of tissue sections show that the epidermis is separated cleanly from the dermis at the basement membrane. All keratinocyte cell layers remain intact, and the epidermal rete ridges along with indentations from dermal papillae are clearly observed. Qualitative and real-time RT-PCR is used to determine nerve growth factor and interleukin-6 expression levels. Western blotting and immunohistochemistry are finally performed to detect amounts of nerve growth factor. This report illustrates that cold thermolysin digestion is an effective method to separate epidermis from dermis for evaluation of mRNA and protein alterations during inflammation.
Asunto(s)
Dermis , Epidermis , Animales , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , TermolisinaRESUMEN
Pseudo-unipolar cell bodies of somatosensory primary neurons are located in the dorsal root ganglia (DRG). The somatic and peripheral domains of DRG neurons are often studied in sensory pain research to understand molecular mechanisms involved in the activation of pain and maintenance of inflammation. Adjuvant-induced arthritis (AIA) is an inflammatory model that elicits a robust and rapid onset immune response with a maximal swelling period of 24-48 h and persisting for several weeks. The AIA model in the hind paw of the rat elicits a potent inflammatory response of the dermis and epidermis, leading to protein expression changes for sensitization of many DRG neurons; however, it is unknown if the AIA model in the hind paw of the rat induces DRG neuronal injury, necrosis, or apoptosis at the somatic level. Neuronal nuclei (NeuN) antigen is a biomarker for post-mitotic neurons, neuronal identification, protein alterations, injury, and loss. Calcitonin gene-related peptide (CGRP) is expressed in C and Aδ DRG neurons, a subset of DRG neurons known to play a role in peripheral sensitization. The focus of this research was to evaluate the expression pattern of NeuN immunoreactivity, in size (soma) and CGRP subpopulations of DRG neurons in naïve and inflamed groups. Confirmed by both immunofluorescence and immunoprecipitation, DRG neuronal expression of NeuN was localized to nuclear and cytoplasmic subcellular compartments. NeuN increased within the nucleus of small CGRP positive DRG neurons during inflammation, indicating a potential role for NeuN in a subset of nociceptive neurons.
Asunto(s)
Antígenos Nucleares/metabolismo , Artralgia/inmunología , Artritis Experimental/complicaciones , Péptido Relacionado con Gen de Calcitonina/metabolismo , Ganglios Espinales/inmunología , Proteínas del Tejido Nervioso/metabolismo , Adyuvantes Inmunológicos/administración & dosificación , Animales , Artritis Experimental/inmunología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Femenino , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Humanos , Inmunohistoquímica , Masculino , Neuronas Aferentes/inmunología , Neuronas Aferentes/metabolismo , Nocicepción/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Polyaromatic compounds are the major, widespread contaminants in the aquatic environment. However, the adverse impacts of these compounds on blood pathophysiology (hematological profiling and serum biochemical responses) are poorly understood. As a consequence, this study was intended to evaluate the toxic effects of naphthalene, one of the polycyclic aromatic hydrocarbons, on the blood pathophysiology of Anabas testudineus using multiple end-point biomarker approach. A. testudineus was exposed to short-term (1 and 5 d) and long-term (10, 15, and 21 d) naphthalene concentrations, that is, T1 (0.71 mg/L indicates 25% of LC50) and T2 (1.42 mg/L indicates 50% of LC50 value). The results disclosed significant decrease in red blood cells, hemoglobin (Hb), packed cell volume, and platelet levels, while other blood parameters, namely, white blood cells, percent lymphocyte, mean cell volume, mean corpuscular Hb, and mean corpuscular Hb concentration showed enhanced levels under naphthalene intoxication. Results were more detrimental under T2 concentration. Cholesterol, glucose, calcium, high-density lipoprotein, and low-density lipoprotein levels gradually increased throughout the different exposure periods under T1 and T2 concentrations, while the triglyceride level gradually decreased during exposure periods. Finally, integrated biomarker responses (IBR) analysis indicated that serum biochemical parameters are more powerful than hematological parameters for determining the naphthalene-induced fish health status. Additionally, the IBR study clearly identified that long-term (>5 d) exposure was more harmful than short-term (<5 d) naphthalene exposure. So, these responses may be derived as biomarkers for monitoring naphthalene pollution in an aquatic ecosystem.
RESUMEN
Anabas testudineus (Bloch) was exposed to 0.71â¯mg/L and 1.42â¯mg/L (25 and 50% of LC50 value respectively) naphthalene, a polycyclic aromatic hydrocarbon (PAH), for 21 days. Blood biochemical parameters and erythrocytic morphological alterations were assessed to describe the naphthalene toxicity. Biochemical analysis showed a significant increase in glutamic pyruvic transaminase, GPT (576.7 ± 11.79 and 608.9 ± 12.08 U/L, respectively) and alkaline phosphatase, ALP (12.9 ± 0.69 and 13.4⯱â¯0.64 U/L, respectively) activities under two doses compared with control. Protein and albumin (ALB) content in blood decreased significantly, in comparison with control value in the tune of 22.67⯱â¯1.04 and 23.97⯱â¯1.24â¯g/dl, respectively and 10.7⯱â¯0.79 and 11.1⯱â¯0.67â¯g/dl, respectively. Erythrocytes showed varied symptomatic morphological changes under naphthalene exposure, which included severe denaturation, swelling in cells, appearance of sickle and tear drop cells, and cellular vacuolation. In particularly, the changes were more prominent under higher naphthalene exposure. Following the results, it has been able to establish that GPT, ALP, protein and ALB, and the morphological manifestations of erythrocytes would be good tools of biomarker in monitoring toxicological paradigm, especially to naphthalene exposure in aquatic bodies.
Asunto(s)
Eritrocitos/efectos de los fármacos , Peces/sangre , Naftalenos/toxicidad , Contaminantes Químicos del Agua/toxicidad , Alanina Transaminasa/sangre , Fosfatasa Alcalina/sangre , Animales , Eritrocitos/patología , Proteínas de Peces/sangre , Albúmina Sérica/análisisRESUMEN
Present study highlighted the ultramicroscopic (SEM) alterations of the skin, eye, barbel, and fins of spawn of an air-breathing teleost (Clarias batrachus, Linn. 1758) induced by UV-B radiation (280-320 nm) at a dose (@4.07 × 10-20J/photon/m2) under the time-frame of 5, 10 and 15 min/d in the laboratory condition for the periods of 5 and 10 days. Limnological parameters revealed no significant changes throughout the period of experimentation which were measured by PCS Testr 35 Multi-Parameter. Morphometric analysis revealed that during the extended exposure period of 10 days the spawn size and weight were reduced as analysed through Specific Growth Rate (SGR). SGR values in terms of weight for 5 and 10 days under 3 time-frames were 17.12%, 12.52%, 11.46% and 9.09%, 6.43%, 6.09% respectively, which revealed a declined trend along with the exposure days. In the skin of C. batrachus, the compact regular orientation of the stratified epithelial cells and mucous cells became distorted and the microridges and double-ridged structures showed destruction and fragmentations. The body striations and microfolds became shrinked and swollen and finally degenerated to form a mass. The distribution of mucous cells throughout the epidermis was disorganised and releasing secretory contents on the surface through small pores. Appearance of huge quantity of biogenic semi-hexagonal plate like crystals (guanine platelets) on the skin surface of the body was the most significant observations during UV-B radiation. In the developmental phases the eyeball showed shrinkage loosing normal regular concave structure and to become a dome-shaped one. The supportive connective infoldings became loosened. The choroid coat displayed deformities and the iris deformed the pupil. The fibroblast on the epithelium and melanocytes depicted dispersed arrangement. The pairs of ventral barbels near the mouth depicted the presence of taste buds that became severely damaged exposing the sensory as well as neuroepithelial cells. Compact regular arrangement of the SECs was completely destroyed leaving long and deep channels inbetween them; the disintegrated concentric MRs also showed a mass.
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Aletas de Animales/efectos de la radiación , Bagres , Ojo/efectos de la radiación , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Aletas de Animales/ultraestructura , Animales , Relación Dosis-Respuesta en la Radiación , Epidermis/efectos de la radiación , Epidermis/ultraestructura , Epitelio/efectos de la radiación , Epitelio/ultraestructura , Ojo/ultraestructura , Piel/ultraestructuraRESUMEN
Opioid-immune crosstalk occurs when opioid drugs alter the activity of the immune system. In this study, the opioid antagonist ß-funaltrexamine (ß-FNA) decreases the expression and release of an inflammatory chemokine, interferon-γ inducible protein-10 (CXCL10) from normal human astrocytes stimulated by interleukin 1ß (IL-1ß). ß-FNA decreased CXCL10 by an unknown action that did not involve the mu opioid receptor (MOR). As IL-1ß acts through its receptor to activate NF-κB/MAPK signaling pathways which leads to CXCL10 expression and release, key steps in the IL-1ß signaling pathways were examined following ß-FNA treatment. IL-1ß-induced activation of p38 mitogen-activated protein kinases (p38 MAPK) was inhibited by ß-FNA as shown by decreased p38 MAPK phosphorylation in treated cells. ß-FNA also decreased the levels of activated subunits of NF-κB (p50 and p65) in treated astrocytes. The impact of ß-FNA was also observed in proteins that act to negatively regulate NF-κB signaling. IL-1ß upregulated the expression of A20, a ubiquitin (Ub)-editing enzyme that dampens NF-κB signaling by altering ubiquination patterns on IL-1 receptor second messengers, and the increase in A20 was significantly inhibited by ß-FNA treatment. Inhibition of the Ub-activating enzyme E1 by the inhibitor PYR41 also decreased CXCL10 release, like ß-FNA, and concurrent treatment with both PYR41 and ß-FNA inhibited CXCL10 more than did either agent alone. In mice, lipopolysaccharide-induced CXCL10 expression in the brain was inhibited by treatment with ß-FNA. These findings suggest that ß-FNA exerts an anti-inflammatory action in vitro and in vivo that is MOR-independent and possibly due to the alkylating ability of ß-FNA.
Asunto(s)
Astrocitos/efectos de los fármacos , Quimiocina CXCL10/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , FN-kappa B/metabolismo , Naltrexona/análogos & derivados , Antagonistas de Narcóticos/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Astrocitos/citología , Astrocitos/metabolismo , Conducta Animal/efectos de los fármacos , Encéfalo/citología , Quimiocina CXCL10/genética , Humanos , Interleucina-1beta/farmacología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Naltrexona/farmacologíaRESUMEN
Development of tolerance to endotoxin prevents sustained hyper inflammation during systemic infections. Here we report for the first time that chronic morphine treatment tempers endotoxin tolerance resulting in persistent inflammation, septicemia and septic shock. Morphine was found to down-regulate endotoxin/LPS induced miR-146a and 155 in macrophages. However, only miR-146a over expression, but not miR-155 abrogates morphine mediated hyper-inflammation. Conversely, antagonizing miR-146a (but not miR-155) heightened the severity of morphine-mediated hyper-inflammation. These results suggest that miR-146a acts as a molecular switch controlling hyper-inflammation in clinical and/or recreational use of morphine.
Asunto(s)
Endotoxinas/toxicidad , MicroARNs/genética , Morfina/efectos adversos , Sepsis/fisiopatología , Animales , Línea Celular , Lipopolisacáridos/toxicidad , Ratones , Receptor Toll-Like 4/metabolismoRESUMEN
Neuroinflammation is an integral component of neurodegenerative disorders, CNS infection and trauma. Astroglial chemokines, such as CXCL10, are instrumental in neuroinflammatory signaling as well as neurotoxicity. We have utilized proinflammatory-induced CXCL10 expression in normal human astrocytes (NHA) as a model in which to assess the anti-inflammatory actions of the selective, mu-opioid receptor (MOR) antagonist, ß-funaltrexamine (ß-FNA). Interferon (IFN)γ+HIV-1 Tat-induced CXCL10 expression (secreted protein and mRNA) was inhibited by co-treatment with ß-FNA. Neither the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2 (CTAP) nor the nonselective opioid receptor antagonist, naltrexone inhibited IFNγ+HIV-1 Tat-induced CXCL10 expression. Furthermore, co-treatment with excess CTAP or naltrexone did not prevent ß-FNA mediated inhibition of IFNγ+HIV-1 Tat-induced CXCL10 expression. Additionally, we utilized an inhibitor of NF-κB activation (SN50) to demonstrate that IFNγ+HIV-1 Tat-induced CXCL10 expression is NF-κB-dependent in NHA. Subsequent experiments revealed that ß-FNA did not significantly affect NF-κB activation. Interestingly, we discovered that ß-FNA inhibited p38 activation as indicated by decreased expression of phospho-p38. Together, these findings suggest that the inhibitory actions of ß-FNA are MOR-independent and mediated, in part, via a transcriptional mechanism. These findings add to our understanding of the mechanism by which chemokine expression is inhibited by ß-FNA. In conjunction with future investigations, these novel findings are expected to provide insights into the development of safe and effective treatments for neuroinflammation.
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Astrocitos/efectos de los fármacos , Quimiocina CXCL10/metabolismo , Naltrexona/análogos & derivados , Astrocitos/metabolismo , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Activación Enzimática , Productos del Gen tat/metabolismo , Humanos , Interferón gamma/metabolismo , FN-kappa B/metabolismo , Naltrexona/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Withdrawal stress is a common occurrence in opioid users, yet very few studies have examined the effects of morphine withdrawal (MW) on immune functioning or the role of glucocorticoids in MW-induced immunomodulation. This study investigated for the first time the role of glucocorticoids in MW modulation of LPS-induced IL-12p40, a key cytokine playing a pivotal role in immunoprotection. Using WT and µ-opioid receptor knock-out mice, we show that MW in vivo significantly attenuated LPS-induced IL-12p40 mRNA and protein expression. The role of glucocorticoids in MW modulation of IL-12p40 was investigated using a murine macrophage cell line, CRL2019, in an in vitro MW model. Interestingly, MW alone in the absence of glucocorticoids resulted in a significant reduction in IL-12p40 promoter activity and mRNA and protein expression. EMSA revealed a concurrent decrease in consensus binding to transcription factors NFκB, Activator Protein-1, and CCAAT/enhancer-binding protein and Western blot analysis demonstrated a significant activation of LPS-induced ERK1/2 phosphorylation. Interestingly, although glucocorticoid treatment alone also modulated these transcription factors and ERK1/2 activation, the addition of glucocorticoids to MW samples resulted in a greater than additive reduction in the transcription factors and significant hyperactivation of LPS-induced ERK1/2 phosphorylation. ERK inhibitors reversed MW and MW plus corticosterone inhibition of LPS-induced IL-12p40. The potentiating effects of glucocorticoids were non-genomic because nuclear translocation of glucocorticoid receptor was not significantly different between MW and corticosterone treatment. This study demonstrates for the first time that MW and glucocorticoids independently modulate IL-12p40 production through a mechanism involving ERK1/2 hyperactivation and that glucocorticoids can significantly augment MW-induced inhibition of IL-12p40.
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Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Subunidad p40 de la Interleucina-12/biosíntesis , Lipopolisacáridos/farmacología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Morfina , Estrés Fisiológico , Síndrome de Abstinencia a Sustancias/metabolismo , Animales , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Línea Celular , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Subunidad p40 de la Interleucina-12/genética , Masculino , Ratones , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Síndrome de Abstinencia a Sustancias/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Infection rate among intravenous drug users (IDU) is higher than the general public, and is the major cause of morbidity and hospitalization in the IDU population. Epidemiologic studies provide data on increased prevalence of opportunistic bacterial infections such as TB and pneumonia, and viral infections such as HIV-1 and hepatitis in the IDU population. An important component in the intravenous drug abuse population and in patients receiving medically indicated chronic opioid treatment is opioid withdrawal. Data on bacterial virulence in the context of opioid withdrawal suggest that mice undergoing withdrawal had shortened survival and increased bacterial load in response to Salmonella infection. As the body of evidence in support of opioid dependency and its immunosuppressive effects is growing, it is imperative to understand the mechanisms by which opioids exert these effects and identify the populations at risk that would benefit the most from the interventions to counteract opioid immunosuppressive effects. Thus, it is important to refine the existing animal model to closely match human conditions and to cross-validate these findings through carefully controlled human studies. Better understanding of the mechanisms will facilitate the search for new therapeutic modalities to counteract adverse effects including increased infection rates. This review will summarize the effects of morphine on innate and adaptive immunity, identify the role of the mu opioid receptor in these functions and the signal transduction activated in the process. The role of opioid withdrawal in immunosuppression and the clinical relevance of these findings will also be discussed.
Asunto(s)
Analgésicos Opioides/inmunología , Sistema Inmunológico/efectos de los fármacos , Infecciones Oportunistas/inducido químicamente , Infecciones Oportunistas/inmunología , Trastornos Relacionados con Sustancias/inmunología , Analgésicos Opioides/farmacología , Animales , Humanos , Morfina/farmacología , Dependencia de Morfina/inmunología , Dependencia de Morfina/microbiología , Infecciones Oportunistas/metabolismo , Receptores Opioides mu/metabolismo , Síndrome de Abstinencia a Sustancias/inmunología , Síndrome de Abstinencia a Sustancias/metabolismo , Trastornos Relacionados con Sustancias/metabolismoRESUMEN
A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34-41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%-16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life=7.3 h) than that from SBL11 (half-life=6.4 h at 50 degrees C), while both showed the same temperature optimum of 37 degrees C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.