RESUMEN
Prematurity and bronchopulmonary dysplasia (BPD) increase the risk of asthma later in life. Supplemental oxygen therapy is a risk factor for chronic respiratory symptoms in infants with BPD. Hyperoxia induces cell injury and release of damage-associated molecular patterns (DAMPs). Cytoskeletal filamentous actin (F-actin) is a DAMP which binds Clec9a, a C-type lectin selectively expressed on CD103+ dendritic cells (DCs). Co-stimulation of Clec9a and TLR3 induces maximal proinflammatory responses. We have shown that neonatal hyperoxia (a model of BPD) increases lung IL-12+Clec9a+CD103+ DCs, pro-inflammatory responses and airway hyperreactivity following rhinovirus (RV) infection. CD103+ DCs and Clec9a are required for these responses. Hyperoxia increases F-actin levels in bronchoalveolar lavage fluid (BALF). We hypothesized that the F-actin severing protein gelsolin attenuates neonatal hyperoxia-induced Clec9a+CD103+ DC-dependent pro-inflammatory responses to RV and preserves alveolarization. We exposed neonatal mice to hyperoxia and treated them with gelsolin intranasally. Subsequently we inoculated the mice with RV intranasally. Alternatively, we inoculated normoxic neonatal mice with BALF from hyperoxia-exposed mice (hyperoxic BALF), RV and gelsolin. We analyzed lung gene expression two days after RV infection. For in vitro studies, lung CD11c+ cells were isolated from C57BL/6J or Clec9agfp-/- mice and incubated with hyperoxic BALF and RV. Cells were analyzed by flow cytometry. In neonatal mice, gelsolin blocked hyperoxia-induced Il12p40, TNF-α and IFN-γ mRNA and protein expression in response to RV infection. Similar effects were observed when gelsolin was co-administered with hyperoxic BALF and RV. Gelsolin decreased F-actin levels in hyperoxic BALF in vitro and inhibited hyperoxia-induced D103lo DC expansion and inflammation in vivo. Gelsolin also attenuated hyperoxia-induced hypoalveolarization. Further, incubation of lung CD11c+ cells from WT and Clec9agfp-/- mice with hyperoxic BALF and RV, showed Clec9a is required for maximal hyperoxic BALF and RV induced IL-12 expression in CD103+ DCs. Finally, in tracheal aspirates from mechanically ventilated human preterm infants the F-actin to gelsolin ratio positively correlates with FiO2, and gelsolin levels decrease during the first two weeks of mechanical ventilation. Collectively, our findings demonstrate a promising role for gelsolin, administered by inhalation into the airway to treat RV-induced exacerbations of BPD and prevent chronic lung disease.
Asunto(s)
Displasia Broncopulmonar/tratamiento farmacológico , Gelsolina/administración & dosificación , Hiperoxia/fisiopatología , Lectinas Tipo C/metabolismo , Infecciones por Picornaviridae/tratamiento farmacológico , Receptores Inmunológicos/metabolismo , Administración por Inhalación , Animales , Animales Recién Nacidos/metabolismo , Antígenos CD/metabolismo , Displasia Broncopulmonar/virología , Femenino , Humanos , Recién Nacido , Cadenas alfa de Integrinas/metabolismo , Interleucina-12/metabolismo , Lectinas Tipo C/genética , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Terapia por Inhalación de Oxígeno/efectos adversos , Infecciones por Picornaviridae/virología , Receptores Inmunológicos/genética , Pruebas de Función Respiratoria , Rhinovirus/aislamiento & purificaciónRESUMEN
Therapy to enhance host immune defenses may improve outcomes in serious infections, especially for antibiotic-resistant pathogens. Recombinant human plasma gelsolin (rhu-pGSN), a normally circulating protein, has beneficial effects in diverse preclinical models of inflammation and injury. We evaluated delayed therapy (24-48 hours after challenge) with rhu-pGSN in a mouse model of pneumococcal pneumonia. rhu-pGSN without antibiotics increased survival and reduced morbidity and weight loss after infection with either penicillin-susceptible or penicillin-resistant pneumococci (serotypes 3 and 14, respectively). rhu-pGSN improves outcomes in a highly lethal pneumococcal pneumonia model when given after a clinically relevant delay, even in the setting of antimicrobial resistance.
Asunto(s)
Gelsolina/administración & dosificación , Factores Inmunológicos/administración & dosificación , Neumonía Neumocócica/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Neumonía Neumocócica/patología , Proteínas Recombinantes/administración & dosificación , Análisis de Supervivencia , Resultado del Tratamiento , Pérdida de PesoRESUMEN
Influenza viruses are one of the most important respiratory pathogens worldwide, causing both epidemic and pandemic infections. The aim of the study was to evaluate the effect of FPR2 antagonists PBP10 and BOC2 on influenza virus replication. We determined that these molecules exhibit antiviral effects against influenza A (H1N1, H3N2, H6N2) and B viruses. FPR2 antagonists used in combination with oseltamivir showed additive antiviral effects. Mechanistically, the antiviral effect of PBP10 and BOC2 is mediated through early inhibition of virus-induced ERK activation. Finally, our preclinical studies showed that FPR2 antagonists protected mice from lethal infections induced by influenza, both in a prophylactic and therapeutic manner. Thus, FPR2 antagonists might be explored for novel treatments against influenza.
Asunto(s)
Antivirales/farmacología , Gelsolina/antagonistas & inhibidores , Virus de la Influenza A/efectos de los fármacos , Oligopéptidos/antagonistas & inhibidores , Fragmentos de Péptidos/antagonistas & inhibidores , Receptores de Formil Péptido/antagonistas & inhibidores , Receptores de Lipoxina/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Células A549/efectos de los fármacos , Animales , Antivirales/administración & dosificación , Supervivencia Celular , Perros , Combinación de Medicamentos , Femenino , Gelsolina/administración & dosificación , Haplorrinos , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/administración & dosificación , Oseltamivir/farmacología , Fragmentos de Péptidos/administración & dosificación , Alineación de SecuenciaRESUMEN
This study was to explore the sequential signaling of disorganization of the actin cytoskeletal architecture by phloretin. RAW 264.7 macrophages were incubated with 1-20 µM phloretin for 5 days in the presence of RANKL. C57BL/6 mice were ovariectomized (OVX) and orally treated with 10 mg/kg phloretin once a day for 8 weeks. Phloretin allayed RANKL stimulated formation of actin podosomes with the concomitant retardation of the vinculin activation. Oral administration of phloretin suppressed the induction of femoral gelsolin and vinculin in OVX mice. The RANK-RANKL interaction resulted in the αvß3 integrin induction, which was demoted by phloretin. The RANKL induction of actin rings and vacuolar-type H(+)-ATPase entailed Pyk2 phosphorylation and c-Src and c-Cbl induction, all of which were blunted by phloretin. Similar inhibition was also observed in phloretin-exposed OVX mouse femoral bone tissues with decreased trabecular collagen formation. Phloretin suppressed the paxillin induction in RANKL-activated osteoclasts and in OVX epiphyseal bone tissues. Also, phloretin attenuated the Syk phosphorylation and phospholipase Cγ induction by RANKL in osteoclasts. These results suggest that phloretin was an inhibitor of actin podosomes and sealing zone, disrupting αvß3 integrin-c-Src-Pyk2/Syk signaling pathway for the regulation of actin cytoskeletal organization in osteoclasts.
Asunto(s)
Quinasa 2 de Adhesión Focal/metabolismo , Integrina alfaVbeta3/metabolismo , Osteoclastos/metabolismo , Floretina/administración & dosificación , Familia-src Quinasas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/genética , Animales , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Proteína Tirosina Quinasa CSK , Quinasa 2 de Adhesión Focal/genética , Gelsolina/administración & dosificación , Regulación del Desarrollo de la Expresión Génica , Integrina alfaVbeta3/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Ratones , Osteoclastos/efectos de los fármacos , Fosforilación , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Ligando RANK/metabolismo , Transducción de Señal/efectos de los fármacos , Quinasa Syk , Vinculina/administración & dosificaciónRESUMEN
BACKGROUND: Gelsolin is an actin-binding protein found in the cytoplasm and in extracellular fluids including blood plasma. Plasma gelsolin concentration decreases after a wide range of injuries. We hypothesized that the repletion of gelsolin would limit inflammation and tissue injury in a rat model of sepsis using cecal ligation and double puncture (2CLP). METHODS: Human plasma gelsolin (pGSN, 10mg in 1ml saline) was administered once immediately following surgery, and control 2CLP (2CLP Alb) and sham animals were injected with 1ml saline containing equimolar albumin. Treatments were administered intraperitoneally (IP), intravenously (IV), or subcutaneously (SC). RESULTS: Gelsolin levels in the 2CLP Alb group were lower than in sham animals. Administration of pGSN increased levels when administered IV and SC, but not IP. Morbidity scores were significantly less severe in the 2CLP pGSN group than in the 2CLP Alb group when pGSN was administered IV and SC, but not IP. Furthermore, enzymatic activity indicative of tissue damage (lactate dehydrogenase and alanine transaminase) was significantly lower in 2CLP pGSN group when treated SC compared to 2CLP Alb group. CONCLUSION: These data provide further evidence that exogenous gelsolin can reduce morbidity from sepsis.
Asunto(s)
Gelsolina/administración & dosificación , Gelsolina/sangre , Sepsis/tratamiento farmacológico , Alanina Transaminasa/metabolismo , Animales , Citocinas/biosíntesis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación , Infusiones Intravenosas , Infusiones Parenterales , L-Lactato Deshidrogenasa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Prostate cancer (PC) growth is dependent on the androgen-androgen receptor (AR) axis. Because current androgen ablation therapies of PC lead to resistance, novel approaches to block AR activity are urgently needed. METHODS: We inhibited AR function beyond the level of hormone binding by blockade of the coactivator groove in the ligand-binding domain (LBD) using a high-affinity gelsolin FxxFF peptide. Following peptide selection, the effect of the gelsolin FxxFF peptide on AR functions was determined in Hep3B cells that were transiently transfected with pM-peptide expression vectors or were incubated with synthetic gelsolin FxxFF peptide coupled to the TAT cell-penetrating peptide. Lentiviruses expressing the gelsolin FxxFF peptide were used to study endogenous AR target gene expression in LNCaP cells. RESULTS: pM-Gelsolin FxxFF efficiently interfered with AR N/C interaction and specifically inhibited AR-regulated reporter gene activity. The peptide did not inhibit progesterone receptor (PR) and glucocorticoid receptor (GR) activity, nor constitutively active gene promoters. The peptide also specifically blocked in vitro interactions of AR LBD with peptides. Like the gelsolin FxxFF peptide expressed by an expression vector, synthetic TAT-gelsolin FxxFF peptide efficiently blocked AR N/C interaction and inhibited full-length AR-regulated reporter gene activity. It hardly affected PR and GR activity, but the effect on constitutively active promoters was variable. Lentiviral gelsolin FxxFF peptide inhibited expression of KLK2 and NDRG1, but hardly affected PSA and TMPRSS2. CONCLUSIONS: Our results show that the AR coactivator groove may function as a target to overcome therapeutic failure that arises during current androgen ablation therapies.
Asunto(s)
Antagonistas de Receptores Androgénicos/administración & dosificación , Péptidos de Penetración Celular/administración & dosificación , Gelsolina/administración & dosificación , Lentivirus/genética , Receptores Androgénicos/fisiología , Transfección , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Gelsolina/genética , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores Androgénicos/químicaRESUMEN
Plaques containing beta-amyloid (Abeta) peptides are one of the pathological features of Alzheimer's disease, and the reduction of Abeta is considered a primary therapeutic target. Amyloid clearance by anti-Abeta antibodies has been reported after immunization, and recent data have shown that the antibodies may act as a peripheral sink for Abeta, thus altering the periphery/brain dynamics. Here we show that peripheral treatment with an agent that has high affinity for Abeta (gelsolin or GM1) but that is unrelated to an antibody or immune modulator reduced the level of Abeta in the brain, most likely because of a peripherally acting effect. We propose that in general, compounds that sequester plasma Abeta could reduce or prevent brain amyloidosis, which would enable the development of new therapeutic agents that are not limited by the need to penetrate the brain or evoke an immune response.