RESUMEN
Pain consists of sensory-discriminative and emotional-affective components. The anterior cingulate cortex (ACC) is a critical brain area in mediating the affective pain. However, the molecular mechanisms involved remain largely unknown. Our recent study indicated that C-X-C motif chemokine 13 (CXCL13) and its sole receptor CXCR5 are involved in sensory sensitization in the spinal cord after spinal nerve ligation (SNL). Whether CXCL13/CXCR5 signaling in the ACC contributes to the pathogenesis of pain-related aversion remains unknown. Here, we showed that SNL increased the CXCL13 level and CXCR5 expression in the ACC after SNL. Knockdown of CXCR5 by microinjection of Cxcr5 shRNA into the ACC did not affect SNL-induced mechanical allodynia but effectively alleviated neuropathic pain-related place avoidance behavior. Furthermore, electrophysiological recording from layer II-III neurons in the ACC showed that SNL increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), decreased the EPSC paired-pulse ratio, and increased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor/N-methyl-D-aspartate receptor ratio, indicating enhanced glutamatergic synaptic transmission. Finally, superfusion of CXCL13 onto ACC slices increased the frequency and amplitude of spontaneous EPSCs. Pre-injection of Cxcr5 shRNA into the ACC reduced the increase in glutamatergic synaptic transmission induced by SNL. Collectively, these results suggest that CXCL13/CXCR5 signaling in the ACC is involved in neuropathic pain-related aversion via synaptic potentiation.
Asunto(s)
Quimiocina CXCL13/metabolismo , Condicionamiento Psicológico/fisiología , Giro del Cíngulo/metabolismo , Giro del Cíngulo/fisiología , Neuralgia/metabolismo , Neuralgia/fisiopatología , Receptores CXCR5/metabolismo , Animales , Potenciales Postsinápticos Excitadores , Glutamatos/metabolismo , Hiperalgesia/metabolismo , Ligadura , Masculino , Ratones , Ratones Endogámicos ICR , Neuralgia/genética , Receptores CXCR5/antagonistas & inhibidores , Transducción de Señal , Médula Espinal , Raíces Nerviosas Espinales , Nervios Espinales/cirugía , Transmisión Sináptica/fisiologíaRESUMEN
Increasing evidence shows miR-155 plays an important role in regulating inflammatory processes in systemic lupus erythematosus (SLE), especially in lupus nephritis (LN). Because the chemokine CXCL13 is implicated in the pathogenesis of LN, here we examined whether miR-155 can modulate the activity of CXCL13 or its receptor CXCR5. We determined the expression of CXCL13 in normal and MRL/lpr mice and found elevated levels of CXCL13 in the kidneys of MRL/lpr mice compared with normal kidneys. Besides, CXCL13 expression was mainly detected in the glomerulus, specifically to mesangial areas. We then transfected a miR-155 mimic in human renal mesangial cells (HRMCs) to overexpress miR-155 and detected decreased protein levels of CXCR5 by western blot analysis. Transfection of the miR-155 mimic into CXCL13-treated HRMCs resulted in a significantly reduced proliferation rate of HRMCs as measured by the cell-counting assay and flow cytometry. Moreover, increased intracellular miR-155 also led to decreased phosphorylation of ERK and TGF-ß1 production. Together, these results revealed that miR-155 may play a role in the pathogenesis of LN.
Asunto(s)
Lupus Eritematoso Sistémico/metabolismo , Nefritis Lúpica/metabolismo , Células Mesangiales/citología , MicroARNs/farmacología , Factor de Crecimiento Transformador beta1/biosíntesis , Animales , Proliferación Celular , Células Cultivadas , Quimiocina CXCL13/análisis , Quimiocina CXCL13/farmacología , Humanos , Riñón/química , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Mesangiales/efectos de los fármacos , Ratones , Ratones Endogámicos MRL lpr , Fosforilación , Receptores CXCR5/antagonistas & inhibidores , Receptores CXCR5/metabolismoRESUMEN
Chemokine receptors undergo internalization and desensitization in response to ligand activation. Internalized receptors are either preferentially directed towards recycling pathways (e.g. CCR5) or sorted for proteasomal degradation (e.g. CXCR4). Here we describe a method for the analysis of receptor internalization and recycling based on specific Bir A-mediated biotinylation of an acceptor peptide coupled to the receptor, which allows a more detailed analysis of receptor trafficking compared to classical antibody-based detection methods. Studies on constitutive internalization of the chemokine receptors CXCR4 (12.1% ± 0.99% receptor internalization/h) and CCR5 (13.7% ± 0.68%/h) reveals modulation of these processes by inverse (TAK779; 10.9% ± 0.95%/h) or partial agonists (Met-CCL5; 15.6% ± 0.5%/h). These results suggest an actively driven internalization process. We also demonstrate the advantages of specific biotinylation compared to classical antibody detection during agonist-induced receptor internalization, which may be used for immunofluorescence analysis as well. Site-specific biotinylation may be applicable to studies on trafficking of transmembrane proteins, in general.
Asunto(s)
Basófilos/metabolismo , Biotina/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Escherichia coli/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR5/metabolismo , Proteínas Represoras/metabolismo , Amidas/farmacología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Basófilos/citología , Basófilos/efectos de los fármacos , Biotina/química , Biotinilación , Antagonistas de los Receptores CCR5/farmacología , Ligasas de Carbono-Nitrógeno/genética , Línea Celular Tumoral , Quimiocina CCL5/farmacología , Proteínas de Escherichia coli/genética , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Ratones , Transporte de Proteínas/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Ratas , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Receptores CXCR5/antagonistas & inhibidores , Receptores CXCR5/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , TransfecciónRESUMEN
A 256-compound library was evaluated in an anti-HIV screen to identify structural "mimics" of the fused tetracyclic indole compound 1 (IDC16) that conserve its anti-HIV activity without associated cytotoxicity. Four diheteroarylamide-type compounds, containing a common 5-nitroisobenzothiazole motif, were identified as active. In subsequent screens, the most potent compound 9 (1C8) was active against wild-type HIV-1IIIB (subtype B, X4-tropic) and HIV-1 97USSN54 (subtype A, R5-tropic) with EC50's of 0.6 and 0.9 µM, respectively. Compound 9 also inhibited HIV strains resistant to drugs targeting HIV reverse transcriptase, protease, integrase, and coreceptor CCR5 with EC50's ranging from 0.9 to 1.5 µM. The CC50 value obtained in a cytotoxicity assay for compound 9 was >100 µM, corresponding to a therapeutic index (CC50/EC50) of approximately 100. Further comparison studies revealed that, whereas the anti-HIV activity for compound 9 and the parent molecule 1 are similar, the cytotoxic effect for compound 9 was, as planned, markedly suppressed.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Benzotiazoles/farmacología , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Piridonas/farmacología , Precursores del ARN/metabolismo , ARN Viral/metabolismo , Replicación Viral/efectos de los fármacos , Empalme Alternativo/genética , Fármacos Anti-VIH/química , Benzotiazoles/síntesis química , Benzotiazoles/química , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , Humanos , Integrasas/metabolismo , Estructura Molecular , Péptido Hidrolasas/metabolismo , Piridonas/síntesis química , Piridonas/química , Precursores del ARN/genética , ARN Viral/genética , Receptores CXCR5/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
OBJECTIVE: To compare the efficacy, safety, and anti-inflammatory effects of cenicriviroc (CVC), an oral, once-daily C-C chemokine receptor types 5 and 2 antagonist, with those of efavirenz (EFV) in treatment-naive, HIV-1-infected adults. DESIGN: A 48-week, randomized, double-blind, double-dummy phase 2b trial at 43 institutions (USA and Puerto Rico). METHODS: Study participants (HIV-1 RNA ≥1000 copies/ml, CD4 cell count ≥200 cells/µl, C-C chemokine receptor type 5-tropic virus) were randomized 2â:â2â:â1 to CVC 100âmg (CVC100), CVC 200âmg (CVC200), or EFV 600âmg, each administered with emtricitabine/tenofovir disoproxil fumarate. Key end points were virologic success (HIV-1 RNA <50 copies/ml) at week 24 (primary) and week 48 (secondary), safety/tolerability at weeks 24 and 48. Study sites and patients remained blinded until week 48. RESULTS: A total of 143 patients were randomized (CVC100, nâ=â59; CVC200, nâ=â56; EFV, nâ=â28). Virologic success was obtained at week 24 in 76, 73, and 71% of study participants for CVC100, CVC200, and EFV, respectively (all Pâ>â0.05 versus EFV), and at week 48 in 68, 64, and 50%, respectively (all Pâ>â0.05 versus EFV). Resistance mutations emerged in five and zero CVC and EFV-treated study participants, respectively. Virologic nonresponse and nucleoside reverse transcriptase inhibitor resistance decreased when CVC minimum plasma concentration was at least 47.8âng/ml. Treatment-related adverse events of at least grade 2 and discontinuations because of adverse events were less frequent in CVC-treated study participants. Total and low-density lipoprotein cholesterol decreased with CVC, but increased with EFV. C-C chemokine ligand type 2 (CCL2) (aka monocyte chemotactic protein-1) increased in a dose-dependent manner, whereas soluble CD14 levels decreased with CVC. CONCLUSION: CVC showed efficacy and favorable safety in treatment-naive HIV-1-infected study participants, supporting selection of CVC200 for phase 3 studies. TRIAL REGISTRATION: NCT01338883.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Benzoxazinas/administración & dosificación , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Imidazoles/administración & dosificación , Receptores CXCR5/antagonistas & inhibidores , Tropismo Viral , Adulto , Alquinos , Fármacos Anti-VIH/efectos adversos , Benzoxazinas/efectos adversos , Ciclopropanos , Método Doble Ciego , Femenino , Genotipo , VIH-1/clasificación , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Imidazoles/efectos adversos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/efectos adversos , Masculino , Persona de Mediana Edad , Puerto Rico , Receptores del VIH/antagonistas & inhibidores , Sulfóxidos , Resultado del Tratamiento , Estados Unidos , Carga Viral , Adulto JovenRESUMEN
Bispecific antibodies are promising agents for immunotherapy. Here, we describe a quadroma-based trifunctional bispecific antibody binding the chemokine receptor CXCR5 and the T-cell antigen CD3 that efficiently prevents tumor growth in a mouse B-cell lymphoma model. CXCR5 regulates the tissue homeostasis of mature B cells and is highly expressed on B-cell non-Hodgkin and lymphocyte-predominant Hodgkin lymphoma, as well as on a subset of CD4(+) T cells known as follicular T-helper cells. In vitro, the bispecific CXCR5::CD3 antibody efficiently recruited effector T cells to CXCR5 expressing B cells and induced a co-stimulation-independent activation of CD8(+) and CD4(+) T cells as demonstrated by the de novo expression of CD25 and CD69, and secretion of the cytokines IFN-γ, TNF-α, IL-6 and IL-10 by peripheral blood mononuclear cells. Notably, at low antibody concentrations, CXCR5::CD3 displayed a significantly higher cytotoxic activity against autologous B cells than its parental antibodies or rituximab. In vivo imaging revealed that CXCR5::CD3 and its parental CXCR5 antibody efficiently prevent tumor growth in a xenograft model of B-cell lymphoma in mice and prolong their survival. Taken together, our results identify CXCR5 as a promising target for antibody-based therapies in the treatment of B-cell malignancies.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3/química , Modelos Animales de Enfermedad , Inmunoterapia , Linfoma de Células B/terapia , Receptores CXCR5/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Complejo CD3/inmunología , Femenino , Humanos , Linfoma de Células B/inmunología , Linfoma de Células B/mortalidad , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores CXCR5/inmunología , Tasa de Supervivencia , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
We investigated the expression of -CXC chemokine ligand 13 (CXCL13) and its receptor -CXC chemokine receptor 5 (CXCR5) in 98 breast cancer (BC) patients with infiltrating duct carcinoma, out of which 56 were found lymph node metastasis (LNM) positive. Interestingly, co-expression of CXCL13 and CXCR5 showed a significant correlation with LNM. Since, epithelial to mesenchymal transition (EMT) is highly associated with metastasis we investigated EMT-inducing potential of CXCL13 in BC cell lines. In CXCL13-stimulated BC cells, expression of various mesenchymal markers (Vimentin, N-cadherin), EMT regulators (Snail, Slug), and matrix metalloproteinase-9 (MMP9) was increased, whereas the expression of epithelial marker E-cadherin was found to be decreased. In addition, expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which is known to regulate MMP9 expression via Src activation, was also significantly increased after CXCL13 stimulation. Using specific protein kinase inhibitors, we confirmed that CXCL13 stimulated EMT and MMP9 expression via RANKL-Src axis in BC cell lines. To further validate this observation, we examined gene expression patterns in primary breast tumors and detected significantly higher expression of various mesenchymal markers and regulators in CXCL13-CXCR5 co-expressing patients. Therefore, this study showed the EMT-inducing potential of CXCL13 as well as demonstrated the prognostic value of CXCL13-CXCR5 co-expression in primary BC. Moreover, CXCL13-CXCR5-RANKL-Src axis may present a therapeutic target in LNM positive BC patients.
Asunto(s)
Neoplasias de la Mama/patología , Quimiocina CXCL13/metabolismo , Transición Epitelial-Mesenquimal , Metástasis Linfática/patología , Receptores CXCR5/metabolismo , Adulto , Anciano , Antígenos CD/biosíntesis , Biomarcadores de Tumor/metabolismo , Cadherinas/biosíntesis , Línea Celular Tumoral , Movimiento Celular , Quimiocina CXCL13/antagonistas & inhibidores , Quimiocina CXCL13/biosíntesis , Femenino , Furanos/farmacología , Humanos , Indoles/farmacología , Metaloproteinasa 9 de la Matriz/biosíntesis , Persona de Mediana Edad , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacología , Pirimidinas/farmacología , Ligando RANK/biosíntesis , Ligando RANK/genética , ARN Mensajero/biosíntesis , Receptores CXCR5/antagonistas & inhibidores , Receptores CXCR5/biosíntesis , Transducción de Señal , Factores de Transcripción de la Familia Snail , Sulfonamidas/farmacología , Factores de Transcripción/biosíntesis , Vimentina/biosíntesis , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
BACKGROUND.: Recruitment of recipient mononuclear cells to the donor heart is a central event in the development of cardiac allograft vasculopathy (CAV). The role of individual chemokine receptors in this process is incompletely defined. TAK-779 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetra-hydro-2H-pyran-4-aminium chloride) is a synthetic nonpeptide antagonist of CCR5 and CXCR3. The purpose of this study is to determine if combined CCR5 and CXCR3 blockade by TAK-779 would attenuate CAV in an experimental model. METHODS.: We used a previously characterized murine model of CAV. Recipient mice were treated with TAK-779 or vehicle control. Donor hearts were harvested on day 24 posttransplantation and analyzed for intimal thickening and cellular infiltration. Recipient splenocytes were analyzed for proliferative response and interferon (IFN)-gamma production by enzyme-linked immunosorbent spot assay. The donor hearts were also examined for heme oxygenase-1 (HO-1) gene induction. RESULTS.: CCR5 and CXCR3 blockade by TAK-779 reduced the severity of intimal lesions (53+/-10% vs. 16+/-2%; P<0.05) and decreased the number of graft infiltrating CCR5 and CXCR3 CD4 and CD8 lymphocytes. Moreover, treatment with TAK-779 (a) decreased alloantigen-specific T-lymphocyte proliferation and number of IFN-gamma producing cells and (b) increased HO-1 gene transcript level in the allografts. CONCLUSIONS.: Antagonism of CCR5 and CXCR3 by TAK-779 is effective in controlling CAV. The beneficial effects of TAK-779 may be because of (a) reduction in CCR5 and CXCR3 T-lymphocyte subset infiltration into the graft, (b) attenuation of alloantigen-specific T-lymphocyte proliferative response and IFN-gamma production, and (c) induction of HO-1 gene. This compound may offer a novel approach in clinical management of CAV.
Asunto(s)
Amidas/uso terapéutico , Antagonistas de los Receptores CCR5 , Trasplante de Corazón/fisiología , Compuestos de Amonio Cuaternario/uso terapéutico , Receptores CXCR5/antagonistas & inhibidores , Amidas/administración & dosificación , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Trasplante de Corazón/patología , Hemo-Oxigenasa 1/genética , Inmunohistoquímica , Inyecciones Intraperitoneales , Complejo Mayor de Histocompatibilidad/genética , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Miocardio/citología , Compuestos de Amonio Cuaternario/administración & dosificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HomólogoRESUMEN
Lupus is an Ab-mediated autoimmune disease. One of the potential contributors to the development of systemic lupus erythematosus is a defect in naturally occurring CD4(+)CD25(+) regulatory T cells. Thus, the generation of inducible regulatory T cells that can control autoantibody responses is a potential avenue for the treatment of systemic lupus erythematosus. We have found that nasal administration of anti-CD3 mAb attenuated lupus development as well as arrested ongoing lupus in two strains of lupus-prone mice. Nasal anti-CD3 induced a CD4(+)CD25(-)latency-associated peptide (LAP)(+) regulatory T cell that secreted high levels of IL-10 and suppressed disease in vivo via IL-10- and TFG-beta-dependent mechanisms. Disease suppression also occurred following adoptive transfer of CD4(+)CD25(-)LAP(+) regulatory T cells from nasal anti-CD3-treated animals to lupus-prone mice. Animals treated with nasal anti-CD3 had less glomerulonephritis and diminished levels of autoantibodies as measured by both ELISA and autoantigen microarrays. Nasal anti-CD3 affected the function of CD4(+)ICOS(+)CXCR5(+) follicular helper T cells that are required for autoantibody production. CD4(+)ICOS(+)CXCR5(+) follicular helper T cells express high levels of IL-17 and IL-21 and these cytokines were down-regulated by nasal anti-CD3. Our results demonstrate that nasal anti-CD3 induces CD4(+)CD25(-)LAP(+) regulatory T cells that suppress lupus in mice and that it is associated with down-regulation of T cell help for autoantibody production.