RESUMEN
AMG X, a human neutralizing monoclonal antibody (mAb) against a soluble human protein, caused thrombocytopenia, platelet activation, reduced mean arterial pressure, and transient loss of consciousness in cynomolgus monkeys after first intravenous administration. In vitro, AMG X induced activation in platelets from macaque species but not from humans or baboons. Other similar mAbs against the same pharmacological target failed to induce these in vivo and in vitro effects. In addition, the target protein was known to not be expressed on platelets, suggesting that platelet activation occurred through an off-target mechanism. AMG X bound directly to cynomolgus platelets and required both the Fab and Fc portion of the mAb for platelet activation. Binding to platelets was inhibited by preincubation of AMG X with its pharmacological target or with anti-human Fc antibodies or by preincubation of platelets with AMG X F(ab')(2) or human immunoglobulin (IVIG). AMG X F(ab')(2) did not activate platelets. Thus, platelet activation required both recognition/binding of a platelet ligand with the Fab domain and interaction of platelet Fc receptors (i.e., FcγRIIa) with the Fc domain. These findings reflect the complexity of the mechanism of action of mAbs and the increasing awareness of potential for unintended effects in preclinical species.
Asunto(s)
Anticuerpos Monoclonales/toxicidad , Plaquetas/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Administración Intravenosa , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacocinética , Plaquetas/metabolismo , Humanos , Hipotensión/sangre , Hipotensión/inducido químicamente , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Macaca fascicularis , Masculino , Papio , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Serotonina/metabolismo , Síncope/sangre , Síncope/inducido químicamente , Trombocitopenia/sangre , Trombocitopenia/inducido químicamente , Tromboxano B2/metabolismoRESUMEN
BACKGROUND: Autoimmune pulmonary alveolar proteinosis (aPAP) is caused by granulocyte/macrophage-colony stimulating factor (GM-CSF) autoantibodies in the lung. Previously, we reported that GM-CSF inhalation therapy improved alveolar-arterial oxygen difference and serum biomarkers of disease severity in these patients. It is plausible that inhaled GM-CSF improves the dysfunction of alveolar macrophages and promotes the clearance of the surfactant. However, effect of the therapy on components in bronchoalveolar lavage fluid (BALF) remains unclear. OBJECTIVES: To figure out changes in surfactant clearance during GM-CSF inhalation therapy. METHODS: We performed retrospective analyses of BALF obtained under a standardized protocol from the same bronchus in each of 19 aPAP patients before and after GM-CSF inhalation therapy (ISRCTN18931678, JMA-IIA00013; total dose 10.5-21 mg, duration 12-24 weeks). For evaluation, the participants were divided into two groups, high responders with improvement in alveolar-arterial oxygen difference ≥13 mmHg (n = 10) and low responders with that < 13 mmHg (n = 9). RESULTS: Counts of both total cells and alveolar macrophages in BALF did not increase during the therapy. However, total protein and surfactant protein-A (SP-A) were significantly decreased in high responders, but not in low responders, suggesting that clearance of surfactant materials is correlated with the efficacy of the therapy. Among 94 biomarkers screened in bronchoalveolar lavage fluid, we found that the concentration of interleukin-17 and cancer antigen-125 were significantly increased after GM-CSF inhalation treatment. CONCLUSIONS: GM-CSF inhalation decreased the concentration of total protein and SP-A in BALF, and increase interleukin-17 and cancer antigen-125 in improved lung of autoimmune pulmonary alveolar proteinosis.
Asunto(s)
Líquido del Lavado Bronquioalveolar , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Pulmón/metabolismo , Proteinosis Alveolar Pulmonar/metabolismo , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/metabolismo , Terapia Respiratoria , Administración por Inhalación , Autoanticuerpos/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Medicina Basada en la Evidencia , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Inmunohistoquímica , Interleucina-17/inmunología , Pulmón/inmunología , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Proteinosis Alveolar Pulmonar/tratamiento farmacológico , Proteinosis Alveolar Pulmonar/inmunología , Proteinosis Alveolar Pulmonar/patología , Proteína A Asociada a Surfactante Pulmonar/inmunología , Surfactantes Pulmonares/inmunología , Terapia Respiratoria/métodos , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Interleukin-1 (IL-1) has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified an isoform of the IL-1 receptor (IL-1R) accessory protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1R but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild-type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild-type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest that it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival.
Asunto(s)
Empalme Alternativo , Sistema Nervioso Central/inmunología , Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Interleucina-1/metabolismo , Neuronas/inmunología , Secuencia de Aminoácidos , Animales , Astrocitos/inmunología , Secuencia de Bases , Encéfalo/citología , Encéfalo/inmunología , Línea Celular Tumoral , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Inflamación/inmunología , Proteína Accesoria del Receptor de Interleucina-1/química , Proteína Accesoria del Receptor de Interleucina-1/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal/inmunologíaRESUMEN
IP-10 secretion is induced by pro-inflammatory cytokines and mediates the migration of CXCR3+ cells. Its elevation in clinical samples has been associated with multiple inflammatory diseases and its antagonism has been reported to be effective in several animal models of inflammatory disease. We generated a mouse anti-mouse IP-10 monoclonal antibody (mAb; Clone 20A9) that specifically bound murine IP-10 with high affinity and inhibited in vitro IP-10 induced BaF3/mCXCR3 cell migration with an IC(50) of approximately 4 nM. The 20A9 mAb was completely absorbed in vivo and had dose proportional pharmacokinetic exposure with a serum half life of 2.4-6 days. The 20A9 mAb inhibited IP-10 mediated T-cell recruitment to the airways, indicating that it is effective in vivo. However, administration of the 20A9 mAb had no significant effect on disease in mouse models of delayed type hypersensitivity, collagen induced arthritis, cardiac allograft transplantation tolerance, EAE or CD4+ CD45RBHi T-cell transfer-induced IBD. These data suggest that the 20A9 mAb can antagonize IP-10 mediated chemotaxis in vitro and in vivo and that this is insufficient to cause a therapeutic benefit in multiple mouse models of inflammatory disease.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL10/antagonistas & inhibidores , Quimiocina CXCL10/inmunología , Animales , Anticuerpos Monoclonales/farmacocinética , Artritis Experimental/patología , Artritis Experimental/terapia , Líquido del Lavado Bronquioalveolar/citología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Movimiento Celular/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/terapia , Femenino , Rechazo de Injerto/prevención & control , Trasplante de Corazón/inmunología , Inflamación/patología , Inflamación/terapia , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/terapia , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ratones Endogámicos , Ratones SCID , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/efectos de los fármacos , Resultado del TratamientoRESUMEN
Thymocytes interact with various subpopulations of thymic epithelial cells (TECs) at different stages of their development. To identify new molecules specifically expressed in TECs and/or thymic nurse cells (TNCs), we used representational difference analysis. We identified a LIM protein located on mouse chromosome 17 (m17TLP) and belonging to the family of the LIM-only proteins (LIMo). We found a new splice variant in addition to the two described A and B isoforms. The three alternative species of m17TLP are found strictly in the thymic stroma. This protein is expressed on a subpopulation of TECs and TNCs. Strikingly, we found that the human ortholog of m17TLP, located on chromosome 6 (h6LIMo), is expressed in most tissues, but not in skeletal muscle. We have identified four human splice variants of h6LIMo which differ in their carboxy-terminal regions. The sequence comprising the genomic structure suggests that CRP2 is the closest known relative of m17TLP. Although the human and mouse nucleotide sequences are 88-97% homologous, this homology is reduced to 47% in the promoter regions, which strongly suggests that their differential expression is related to their promoter regulatory activity.