RESUMEN
This work is aimed at investigating the effect of melittin on identified key genes in bladder cancer (BC) and further providing a theoretical basis for BC treatment. GSE35014 downloaded from the Gene Expression Omnibus (GEO) database was used to screen differentially expressed genes (DEGs) in BC cells and control. Results showed that a total of 389 upregulated and 169 downregulated genes were identified. Subsequently, GO analysis, KEGG pathway enrichment analysis, and PPI network analysis were employed to disclose the crucial genes and signaling pathways involved in BC. Fifteen module-related DEGs and their associated signaling pathways were obtained according to the PPI network and modular analyses. Based on the analysis of articles retrieved in the PubMed database, we found that melittin could induce apoptosis and constrain the progression of tumor cells as a result of regulating critical cancer-related signaling pathways, such as PI3K-Akt and TNF signaling pathways. Furthermore, PI3K-Akt and TNF signaling pathways were also found to be associated with module-related DEGs according to biological analyses. At last, qRT-PCR analysis demonstrated that melittin could constrain the expression of module-related DEGs (LPAR1, COL5A1, COL6A2, CXCL1, CXCL2, and CXCL3) associated with PI3K-Akt and TNF signaling pathways in BC cells. Functional assays revealed that melittin could constrain the proliferative and migrated abilities of BC cells. Conjointly, these findings provide a theoretical basis for these six genes as drug-sensitive markers of melittin in BC treatment.
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Medicina Tradicional China , Meliteno/uso terapéutico , Neoplasias de la Vejiga Urinaria/genética , Venenos de Abeja/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Bases de Datos de Ácidos Nucleicos , Conjuntos de Datos como Asunto , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/genética , Humanos , Meliteno/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Mapas de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Factores de Necrosis Tumoral/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
BACKGROUND/AIMS: Breast cancer is a common cause of cancer mortality throughout the world. The cross-talk between cancer cells and interstitial cells exerts significant effects on neoplasia and tumor development and is modulated in part by chemokines. CXC is one of four chemokine families involved in mediating survival, angiogenesis, and immunosensitization by chemoattracting leukocytes, and it incentivizes tumor cell growth, invasion and metastasis in the tumor microenvironment. However, the differential expression profiles and prognostic values of these chemokines remains to be elucidated. METHODS: In this study, we compared transcriptional CXC chemokines and survival data of patients with breast carcinoma (BC) using the ONCOMINE dataset, Kaplan-Meier Plotter, TCGA and cBioPortal. RESULTS: We discovered increased mRNA levels for CXCL8/10/11/16/17, whereas mRNA expression of CXCL1/2/3/4/5/6/7/12/14 was lower in BC patients compared to non-tumor tissues. Kaplan-Meier plots revealed that high mRNA levels of CXCL1/2/3/4/5/6/7/12/14 correlate with relapse-free survival (RFS) in all types of BC patients. Conversely, high CXCL8/10/11 predicted worse RFS in BC patients. Significantly, high transcription levels of CXCL9/12/13/14 conferred an overall survival (OS) advantage in BC patients, while high levels of CXCL8 demonstrated shorter OS in all BC sufferers. CONCLUSIONS: Integrative bioinformatics analysis suggests that CXCL8/12/14 are potential suitable targets for precision therapy in BC patients compared to other CXC chemokines.
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Quimiocinas CXC/metabolismo , Biología Computacional/métodos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Quimiocinas CXC/antagonistas & inhibidores , Quimiocinas CXC/genética , Bases de Datos Factuales , Supervivencia sin Enfermedad , Femenino , Redes Reguladoras de Genes , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Estimación de Kaplan-Meier , Pronóstico , ARN Mensajero/metabolismo , Microambiente TumoralRESUMEN
AIMS: CXC ligand 16 (CXCL16) may be involved in inflammation and lipid metabolism, and we hypothesized a role for this chemokine in coronary artery disease (CAD). METHODS AND RESULTS: We performed clinical studies in CAD patients as well as experimental studies in cells with relevance to atherogenesis [i.e. endothelial cells, vascular smooth muscle cells (SMC), and peripheral blood mononuclear cells (PBMC)]. We also examined the ability of HMG-CoA reductase inhibitors (statins) to modulate CXCL16 levels both in vivo and in vitro. Our main findings were: (i) patients with stable (n = 40) and unstable (n = 40) angina had elevated plasma levels of CXCL16 compared with controls (n = 20); (ii) low-dose simvastatin (20 mg qd, n = 15) and high-dose atorvastatin (80 mg qd, n = 9) down-regulated plasma levels of CXCL16 during 6 months of therapy; (iii) in vitro, atorvastatin significantly decreased the interleukin (IL)-1beta-mediated release of CXCL16 from PBMC and endothelial cells; (iv) attenuating effect of atorvastatin on the IL-1beta-mediated release of CXCL16 in PBMC seems to involve post-transcriptional modulation as well as down-regulation of CXCL16 release through inhibition of the protease a disintegrin and metalloproteinase 10 (ADAM10); (v) soluble CXCL16 increased the release of IL-8, monocyte chemoattractant peptide 1, and matrix metalloproteinases in vascular SMC and increased the release of IL-8 and monocyte chemoattractant peptide 1 in PBMC, with particularly enhancing effects in cells from CAD patients. CONCLUSION: Our findings suggest that soluble CXCL16 could be linked to atherogenesis not only as a marker of inflammation, but also as a potential inflammatory mediator.
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Quimiocinas CXC/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Receptores Depuradores/metabolismo , Anciano , Atorvastatina , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL16 , Quimiocinas CXC/genética , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Enfermedad de la Arteria Coronaria/patología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Ácidos Heptanoicos/farmacología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Interleucina-8/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Pirroles/farmacología , Receptores Depuradores/genética , Simvastatina/farmacologíaRESUMEN
DNA microarray technology was used to identify new potential drug targets for myasthenia gravis (MG), to delineate genes involved in the pathogenesis of the disease and to possibly target their protein products for immunotherapy. In this study we compared the gene expression in lymph node cells (LNC) and muscles of rats with experimental autoimmune MG (EAMG) to those of control, healthy rats. Of the genes that were found to be deregulated in EAMG, we chose to elaborate on two gene systems: (a) The chemokine IFN-gamma-inducible protein 10 (IP-10, CXCL10), and its receptor (CXCR3) and (b) phosphodiesterases.
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Evaluación Preclínica de Medicamentos , Miastenia Gravis/tratamiento farmacológico , Miastenia Gravis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Corticoesteroides/uso terapéutico , Animales , Quimiocina CXCL10 , Quimiocinas CXC/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Miastenia Gravis/metabolismo , Miastenia Gravis/patología , Inhibidores de Fosfodiesterasa/uso terapéutico , Receptores CXCR3 , Receptores de Quimiocina/genéticaRESUMEN
PURPOSE: The aims of this study were to examine the in vivo effects of berberine, an alkaloid isolated from some medicinal herbs, on monocyte chemotactic protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) expression in rat lipopolysaccharide (LPS)-induced uveitis. METHODS: LPS was injected intraperitoneally. Berberine was orally administered. MCP-1 mRNA and CINC-1 mRNA were measured by semiquantitative reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. MCP-1 and CINC-1 protein concentration in the aqueous humor were measured by enzyme-linked immunosorbent assay. Histopathologic study was performed in the anterior ocular segments. RESULTS: Berberine dose-dependently inhibited LPS-induced MCP-1 mRNA and CINC-1 mRNA expression of the iris-ciliary body. The alkaloid inhibited chemokines, protein and cell levels in the aqueous humor in rats stimulated with LPS. On histopathologic study, the inflammatory cell infiltration was diminished by the berberine treatment. CONCLUSIONS: These findings indicate that berberine dose-dependently inhibited the expression of MCP-1 and CINC-1 induced by LPS and diminished the anterior uveitis.
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Berberina/uso terapéutico , Quimiocina CCL2/genética , Quimiocinas CXC/genética , Expresión Génica/efectos de los fármacos , ARN Mensajero/metabolismo , Uveítis Anterior/tratamiento farmacológico , Animales , Quimiocina CXCL1 , Cuerpo Ciliar/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Iris/metabolismo , Lipopolisacáridos/toxicidad , Masculino , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Uveítis Anterior/inducido químicamente , Uveítis Anterior/metabolismoRESUMEN
Epithelial neutrophil-activating peptide-78 (ENA-78), a member of the CXC chemokine subfamily, is induced by inflammatory cytokines in human colonic enterocyte cell lines and increased in the colon of patients with inflammatory bowel disease (IBD). Lipopolysaccharide-induced CXC-chemokine (LIX) was recently identified as the murine homolog of ENA-78. Here we show that, similar to ENA-78, inflammatory cytokine stimulation of a murine colonic epithelial cell line, MODE-K, results in increased LIX expression. Consistent with the expression pattern of ENA-78 in IBD, LIX expression is significantly increased in mice with colitis induced by the ingestion of dextran sodium sulfate (DSS). Treating mice with antisense oligonucleotides to LIX via rectal enema delivery before DSS treatment results in colonic enterocyte uptake and a significant reduction in neutrophil infiltration and severity of colitis. These findings indicate that LIX plays an integral role in the pathogenesis of DSS-induced colitis. Similarly, enterocyte-derived CXC chemokines may play a key role in regulating neutrophil recruitment and intestinal injury in IBD. The intracolonic administration of ENA-78 antisense oligonucleotides may be effective in treating distal ulcerative colitis in humans.
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Quimiocinas CXC/antagonistas & inhibidores , Colitis/tratamiento farmacológico , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Animales , Línea Celular , Quimiocina CXCL5 , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Colitis/inducido químicamente , Colitis/patología , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Enterocitos/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: We previously showed that local use of periodate oxidized ATP (oATP, a selective inhibitor of P2X7 receptors for ATP) in rat paw treated with Freund's adjuvant induced a significant reduction of hyperalgesia Herein we investigate the role of oATP, in the rat paws inflamed by carrageenan, which mimics acute inflammation in humans. RESULTS: Local, oral or intravenous administration of a single dose of oATP significantly reduced thermal hyperalgesia in hind paws of rats for 24 hours, and such effect was greater than that induced by diclofenac or indomethacin. Following oATP treatment, the expression of the pro-inflammatory chemokines interferon-gamma-inducible protein-10 (IP-10), mon ocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) within the inflamed tissues markedly decreased on vessels and infiltrated cells. In parallel, the immunohistochemical findings showed an impairment, with respect to the untreated rats, in P2X7 expression, mainly on nerves and vessels close to the site of inflammation. Finally, oATP treatment significantly reduced the presence of infiltrating inflammatory macrophages in the paw tissue. CONCLUSION: Taken together these results clearly show that oATP reduces carrageenan-induced inflammation in rats.
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Adenosina Trifosfato/análogos & derivados , Analgésicos no Narcóticos/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Quimiocinas/biosíntesis , Hiperalgesia/tratamiento farmacológico , Adenosina Trifosfato/administración & dosificación , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/fisiología , Adenosina Trifosfato/uso terapéutico , Administración Cutánea , Administración Oral , Analgésicos no Narcóticos/administración & dosificación , Analgésicos no Narcóticos/farmacología , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Carragenina/toxicidad , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Quimiocina CXCL10 , Quimiocinas/genética , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Diclofenaco/administración & dosificación , Diclofenaco/uso terapéutico , Modelos Animales de Enfermedad , Miembro Posterior , Calor , Hiperalgesia/inducido químicamente , Hiperalgesia/etiología , Indometacina/administración & dosificación , Indometacina/uso terapéutico , Inyecciones Intravenosas , Interleucina-8/biosíntesis , Interleucina-8/genética , Macrófagos/efectos de los fármacos , Masculino , Antagonistas del Receptor Purinérgico P2 , Ratas , Ratas Wistar , Receptores Purinérgicos P2/biosíntesis , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Método Simple CiegoRESUMEN
BACKGROUND: Most array analyses of ulcerative colitis have focused on identifying susceptibility genes for ulcerative colitis. AIM: To clarify the changes in gene expression during inflammation in ulcerative colitis colon mucosa using cDNA macroarray. METHODS: From 23 ulcerative colitis patients, 16 each of inflamed and non-inflamed specimens (total 32 samples for individual analysis) were obtained by colonoscopic biopsy. Eighteen of the 32 samples, used for pairwise analysis, consisted of nine sample pairs, each pair being from the same patient. We examined expression profiles of approximately 1300 genes with cDNA macroarray. Comparisons were made using two kinds of statistics, t-test and significance analysis of microarray in both analyses. The reproducibility of significant genes from the macroarray analysis was confirmed by real-time ploymerase chain reaction. RESULTS: We detected five upregulated genes, categorized into proinflammatory genes (MRP14, GRO gamma and SAA1) and anti-inflammatory genes (TIMP1 and Elafin) in inflamed mucosa, and one upregulated gene (L-FABP) in non-inflamed mucosa. CONCLUSIONS: As the cDNA macroarray analysis in this study exactly reflects the total profile of gene expression in the clinical setting of ulcerative colitis, the genes identified will be directly applicable to diagnostics or as novel therapeutic targets in active ulcerative colitis.
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Colitis Ulcerosa/genética , ADN Complementario/análisis , Genes/genética , Acrosoma , Adulto , Antígenos/genética , Calgranulina B/genética , Proteínas Portadoras/genética , Quimiocina CXCL1 , Quimiocinas CXC/genética , ADN Complementario/genética , Proteínas de Unión a Ácidos Grasos , Humanos , Inflamación/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Isoantígenos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas/genética , ARN/análisis , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas de Plasma Seminal , Inhibidor Tisular de Metaloproteinasa-1/genética , Regulación hacia Arriba/genéticaRESUMEN
We investigated the expression and functions of chemokine receptors in neural progenitor cells isolated from embryonic and adult mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated mRNA expression for most known chemokine receptors in neural progenitor cells grown as neurospheres from embryonic (E17) and adult (4-week-old) mice. The expression of CXCR4 receptors was demonstrated further in E17 neurospheres using immunohistochemistry, in situ hybridization, Northern blot analysis and fura-2-based Ca(2+) imaging. Most neurospheres grown from E17 mice responded to stromal cell-derived factor-1 (SDF-1/CXCL12) in Ca(2+) imaging studies. In addition, immunohistochemical studies demonstrated that these neurospheres consisted of dividing cells that uniformly colocalized nestin and CXCR4 receptors. Differentiation of E17 neurospheres yielded astrocytes and neurons exhibiting several different phenotypes, including expression of calbindin, calretinin, gamma-aminobutyric acid (GABA), and glutamate, and many also coexpressed CXCR4 receptors. In addition, neurospheres grown from the subventricular zone (SVZ) of 4-week-old mice exhibited large increases in Ca(2+) in response to CXCL12 and several other chemokines. In comparison, neurospheres prepared from olfactory bulb of adult mice exhibited only small Ca(2+) responses to CXCL12, whereas neurospheres prepared from hippocampus were insensitive to CXCL12, although they did respond to other chemokines. Investigations designed to investigate whether CXCL12 can act as a chemoattractant demonstrated that cells dissociated from E17 or adult SVZ neurospheres migrated toward an CXCL12 gradient and this was blocked by the CXCR4 antagonist AMD3100. These results illustrate widespread chemokine sensitivity of embryonic and adult neural progenitor cells and support the view that chemokines may be of general importance in control of progenitor cell migration in embryonic and adult brain.
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Neuronas/metabolismo , Receptores de Quimiocina/biosíntesis , Células Madre/metabolismo , Animales , Northern Blotting , Calcio/metabolismo , División Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas/fisiología , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Quimiotaxis/fisiología , Cartilla de ADN , ADN Complementario/biosíntesis , Embrión de Mamíferos/citología , Inmunohistoquímica , Hibridación in Situ , Ratones , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: Stromal-derived factor-1 (SDF-1/CXCL12) is chemotactic for lympho/hematopoietic stem cells. We have previously shown that increasing peripheral blood (PB) levels of SDF-1 with adenovectors expressing human SDF-1 complementary DNA (ad-SDF-1) leads to hematopoietic stem cell mobilization as well as migration of megakaryocytes and thrombocytosis in mice. Herein, we studied the in vivo effects of ad-SDF-1 and of an analogue peptide of SDF-1 (CTCE-0214) on human hematopoiesis in a xenotransplant model. MATERIALS AND METHODS: Sublethally irradiated (300 cGY) NOD/SCID mice transplanted with human cord blood mononuclear cells (CB MNC) were injected with ad-SDF-1 (10(9) plaque forming units, i.v., x 1) or CTCE-0214 (10 mg/kg/dose, i.v. q 24 hours x 7). Effects on megakaryocytopoiesis (CD41+ cells and platelets) as well as stem cell mobilization were monitored. RESULTS: CB MNC in NOD/SCID mice are able to differentiate into CD41+ cells and platelets, peaking at week 9 at a mean of 3.7 x 10(3)/microL. i.v. injection of ad-SDF-1 increased human CD41+ cells by day 4 in PB and was followed by an increase in human platelet production by day 5, with return to baseline by day 30. Human colony-forming cells (CFC) were mobilized from bone marrow to spleen (by day 6-13) and to PB (by day 13). Human CD34+ and CD33+ cells were mobilized by this treatment as well. A novel SDF-1 peptide agonist (CTCE-0214) also mobilized human CFC and enhanced human thrombopoiesis. CONCLUSION: SDF-1 and its analogue may be of clinical value in stimulating platelet recovery after chemo/radiation treatment as well as in stem cell mobilization.
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Plaquetas/citología , Quimiocinas CXC/farmacología , Hematopoyesis/efectos de los fármacos , Movilización de Célula Madre Hematopoyética/métodos , Megacariocitos/efectos de los fármacos , Adenoviridae/genética , Animales , Diferenciación Celular/efectos de los fármacos , Quimiocina CXCL12 , Quimiocinas CXC/agonistas , Quimiocinas CXC/sangre , Quimiocinas CXC/genética , Evaluación Preclínica de Medicamentos , Sangre Fetal/citología , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Quimera por Radiación , Proteínas Recombinantes de Fusión/fisiología , Trombopoyetina/genética , Trombopoyetina/farmacología , Transducción Genética , Trasplante HeterólogoRESUMEN
Recent in vitro studies suggest that the alpha chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor CXCR-4 may contribute to neuronal apoptosis in HIV infection of the brain. The cellular and regional expression of this chemokine and its relationship to the AIDS dementia complex (ADC), however, have remained undetermined. Using immunohistochemistry and semiquantitative RT-PCR, we examined the expression of SDF-1alpha in the frontal cortex (FC), the adjacent deep white matter (DWM). and the basal ganglia (BG) of 17 patients with ADC and 5 normal controls, and the FC and temporal cortex of 6 patients with Alzheimer disease (AD). Additionally, SDF-1alpha expression was studied in 3 different neuronal cultures: differentiated SK-N-MC cells, primary human fetal neuronal, and mouse hippocampal cultures. SDF-1alpha staining was predominantly localized to astrocytes in all 3 groups in the gray matter of the FC and the BG, often in the vicinity of cortical and basal ganglia neurons, but was generally absent in the DWM. Further, the number of positive neurons was significantly greater in the BG of AIDS subjects with advanced brain disease compared to subjects with lesser disease (p = 0.029). All cultures showed prominent SDF-1alpha staining of neurons within the cytoplasm and in neurites, whereas preferential expression in GABA-ergic neurons was found in hippocampal cultures. This is the first study to show that SDF-1alpha is constitutively expressed in astrocytes of the deep and cortical gray matter as well as in neurons of the human brain. Its increased expression in basal ganglia neurons of patients with advanced HIV CNS disease suggests it may also contribute to pathogenesis.
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Complejo SIDA Demencia/metabolismo , Astrocitos/metabolismo , Quimiocinas CXC/metabolismo , Neuronas/metabolismo , Complejo SIDA Demencia/genética , Complejo SIDA Demencia/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Astrocitos/patología , Astrocitos/virología , Ganglios Basales/metabolismo , Ganglios Basales/patología , Ganglios Basales/virología , Northern Blotting/métodos , Southern Blotting/métodos , Recuento de Células/métodos , Células Cultivadas/virología , Quimiocina CXCL12 , Quimiocinas CXC/genética , Feto , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Lóbulo Frontal/virología , Regulación de la Expresión Génica , Productos del Gen tat/metabolismo , VIH/patogenicidad , Proteína gp120 de Envoltorio del VIH/metabolismo , Compuestos Heterocíclicos con 1 Anillo/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/virología , Humanos , Inmunohistoquímica/métodos , Técnicas In Vitro , Ratones , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuronas/patología , Neuronas/virología , ARN Mensajero/biosíntesis , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Altered gene expression in endothelial cells interacting with sickle red blood cells (RBC) and other blood components, in particular the pro-inflammatory cytokines, is an essential step in the pathogenesis of vasoocclusive crises in sickle cell disease (SCD). Using cDNA arrays, we monitored gene expression profiles of human lung microvascular endothelial cells (HMVEC-L) after stimuli that are likely involved in the pathogenesis of vasoocclusion. We detected increased expression of multiple genes in HMVEC-L after their exposure to pro-inflammatory cytokines TNFalpha and IL-1beta and to RBC with increased external exposure of phosphatidylserine and RBC from a patient with SC hemoglobin. While some of these genes have previously been implicated in vascular damage, many represent new targets for investigation of the pathogenesis of vasoocclusion in SCD.
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Endotelio Vascular/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Interleucina-1/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL5/genética , Quimiocina CXCL6 , Quimiocinas CXC/genética , Técnicas de Cocultivo , ADN Complementario/análisis , ADN Complementario/genética , Selectina E/genética , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Eritrocitos/citología , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Hemoglobina Falciforme/farmacología , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interleucina-6/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fosfatidilserinas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
BACKGROUND: Chemokines act as chemoattractants and activators of specific leukocytes at the site of inflammation. In this study, we investigated serial expression of chemokines and chemokine receptors in the eye with experimental autoimmune uveoretinitis (EAU) using RNAse protection assay, and confirmed their expression by immunohistochemical staining. METHODS: B10.A mice were immunized with 50 micro g of interphotoreceptor retinoid binding protein (IRBP) emulsified in complete Freund's adjuvant in order to induce EAU. The eyes were enucleated 0, 7, 14 and 21 days after IRBP immunization to analyze mRNA expression of chemokines and chemokine receptors in the posterior segment. In addition, expression of IP-10 and CXCR3 was analyzed by immunohistochemistry. RESULTS: The gene expression of RANTES, IP-10, and MCP-1 was upregulated on day 14 after immunization (early stage of EAU). The expression of chemokine receptors (CCR2 and CCR5) associated with Th1-type T cells correlated with their appropriate ligands. Furthermore, immunohistochemical study showed that IP-10 and CXCR3, the receptor for IP-10, were strongly expressed in the posterior segment of the eyes from mice with EAU. CONCLUSION: These results suggest that RANTES, IP-10 and MCP-1 may contribute to the recruitment of Th1-type T cells into the eye during the development of EAU in mice.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Quimiocinas/genética , Proteínas del Ojo , ARN Mensajero/metabolismo , Receptores de Quimiocina/genética , Retinitis/metabolismo , Uveítis Posterior/metabolismo , Animales , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/patología , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiocina CXCL10 , Quimiocinas/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Técnicas para Inmunoenzimas , Ratones , Receptores de Quimiocina/metabolismo , Retina/metabolismo , Retinitis/inducido químicamente , Retinitis/patología , Proteínas de Unión al Retinol , Organismos Libres de Patógenos Específicos , Regulación hacia Arriba , Uveítis Posterior/inducido químicamente , Uveítis Posterior/patologíaRESUMEN
Accumulation of neutrophils is a prominent feature of gouty arthritis in which CXC chemokines may play a role. Recently, we have shown that IL-8 (CXCL8) contributes to neutrophil influx in a rabbit model of gouty arthritis. Here, we demonstrate that growth-related oncogene-alpha (GROalpha) (CXCL1), a prototype of CXC chemokine, is also involved in this process. GROalpha level in the joints peaked at 2 hours after intra-articular injection of monosodium urate crystals, at a time before the neutrophil influx reached the maximal level (9 hours). Once decreased, the level increased and reached the second peak at 9 hours. The kinetics was comparable to that of IL-8. Administration of anti-GROalpha mAb attenuated the neutrophil influx at the same level as did the anti-IL-8 IgG, and combination of these antibodies enhanced the inhibition, resulting in a 33% reduction. Interaction of GROalpha with TNFalpha, IL-1beta, and IL-8 was next investigated by injecting antibodies or receptor antagonist with monosodium urate crystals. Administration of anti-TNFalpha mAb did not alter GROalpha level at 2 hours, but inhibited the levels 9 hours after the injection. Treatment with either IL-1 receptor antagonist or anti-IL-8 IgG resulted in decreased levels of GROalpha at 2 and 9 hours. Neutralization of GROalpha with anti-GROalpha mAb did not alter TNFalpha, IL-1beta, and IL-8 levels at their peak (2 hours), but decreased the second peak of IL-1beta (9 hours) and IL-8 (12 hours). These results provide evidence that GROalpha as well as IL-8 are involved ad eundem in the neutrophil infiltration in this model. IL-1 and IL-8, but not TNFalpha, are responsible in part for the initial phase of GROalpha, whereas these cytokines induce GROalpha in a late phase. GROalpha does not seem to initiate TNFalpha, IL-1beta, and IL-8 in an early phase, but induces IL-1beta and IL-8 in a late phase.
Asunto(s)
Artritis Experimental/inmunología , Quimiocinas CXC/fisiología , Oncogenes , Ácido Úrico/toxicidad , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/genética , División Celular , Quimiocinas CXC/análisis , Quimiocinas CXC/genética , Interleucina-8/fisiología , ConejosAsunto(s)
Quimiocinas CC/genética , Quimiocinas CXC/genética , Expresión Génica , Granuloma/metabolismo , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/fisiología , Quimiocinas CC/fisiología , Quimiocinas CXC/fisiología , Aceite de Crotón , Modelos Animales de Enfermedad , Femenino , Adyuvante de Freund , Granuloma/inducido químicamente , Ratones , Peroxidasa/metabolismo , ARN Mensajero/análisis , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/fisiologíaRESUMEN
STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an alpha (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4(+) T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19(+) B cells and CD14(+) monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.
Asunto(s)
Quimiocinas CC/química , Quimiocinas CX3C/química , Quimiocinas CXC/química , Quimiocinas CXC/genética , Clonación Molecular/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Receptores de Citocinas/metabolismo , Receptores Acoplados a Proteínas G , Receptores Inmunológicos , Receptores Virales , Secuencia de Aminoácidos , Southern Blotting , Línea Celular , Membrana Celular/genética , Membrana Celular/inmunología , Membrana Celular/metabolismo , Quimiocina CXCL16 , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/fisiología , ADN Complementario/aislamiento & purificación , Glicosilación , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Ligandos , Activación de Linfocitos , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , ARN/biosíntesis , Receptores CXCR6 , Receptores de Quimiocina , Receptores Depuradores , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , TransfecciónRESUMEN
Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.
Asunto(s)
Astrocitoma/inmunología , Astrocitoma/metabolismo , Receptores CXCR4/biosíntesis , Receptores CXCR4/fisiología , Adyuvantes Inmunológicos/farmacología , Butadienos/farmacología , Calcio/metabolismo , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Quimiocina CXCL10 , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inhibidores , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Quimiocinas CXC/fisiología , Activación Enzimática/inmunología , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Interleucina-1/farmacología , Interleucina-8/antagonistas & inhibidores , Interleucina-8/biosíntesis , Interleucina-8/genética , Líquido Intracelular/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Nitrilos/farmacología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Receptores CXCR4/genética , Células del Estroma/inmunología , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/inmunologíaRESUMEN
Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock. Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages. Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins. The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock. Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14. In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required. Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages correlated with a marked inhibition of NF-kappa B nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappa B nuclear translocation in response to LPS. These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage.
Asunto(s)
Antígenos CD11/fisiología , Antígenos CD18/fisiología , Proteínas de Drosophila , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/fisiología , Paclitaxel/farmacología , Receptores de Superficie Celular/fisiología , Transporte Activo de Núcleo Celular/inmunología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Animales , Antígenos CD11/biosíntesis , Antígenos CD11/genética , Antígenos CD18/biosíntesis , Antígenos CD18/genética , Células Cultivadas , Quimiocina CXCL10 , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Factores Reguladores del Interferón , Interferón gamma/metabolismo , Interferón gamma/fisiología , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Interleucina-12/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
STATEMENT OF FINDINGS: Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and beta-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).
Asunto(s)
Células Sanguíneas/citología , Mesodermo/citología , Células Madre/citología , Fosfatasa Alcalina/sangre , Antígenos CD34/análisis , Células de la Médula Ósea/citología , Recuento de Células , Separación Celular , Quimiocina CXCL12 , Quimiocinas CXC/genética , Humanos , Inmunohistoquímica , Microscopía por Video , Valores de ReferenciaRESUMEN
To investigate lymphocyte and monocyte recruitment-specific chemokine expression in synovial tissues from patients with rheumatoid arthritis (RA), psoriatic arthritis (PA) and osteoarthritis (OA), synovial membranes and cytocentrifuge preparations of 7 RA, 8 PA and 10 OA patients were examined by in situ hybridisation with antisense probes of Mig, GRO alpha and RANTES and by immunohistochemistry. Patients' local disease activity (swelling and tenderness) in order to was graded and histological evaluation was performed compare these data with their chemokine expression profiles. Mig and RANTES hybridisation signals were detected in the synovial lining layer and in cellular infiltrates, whereas GRO alpha expression was localised exclusively in the lining layer of PA and RA. Cytological analysis revealed Mig and GRO alpha mRNA mainly in monocytic cells expressing KIM6, while RANTES mRNA was demonstrated predominantly in lymphocytic cells expressing CD3. In OA synovial membranes, significantly fewer hybridisation signals were present than in RA and PA synovial membranes. Patients with PA and RA had mild to severe local disease activity, whereas OA patients showed only mild disease activity. Histologically, PA and RA inflammatory scores ranged from 1 to 5, while OA synovium was consistently graded 1. Therefore, we conclude that the differential expression of Mig, GRO alpha and RANTES in resident and in inflammatory cells has an important role in regulating leucocyte traffic in inflammatory arthropathies. The diverse leucocyte specificity of Mig, GRO alpha and RANTES may thus regulate the recruitment of different leucocyte populations, as detected in PA and RA. Therefore, the pattern of cellular infiltration in human synovitis and the corresponding clinical signs of inflammation basically reflect the localisation and expression intensity of chemokines, which may be an important target for future disease modulation.