RESUMEN
Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner. CNTO2424 down-regulates poly(I:C)-induced production of IL-6, IL-8, MCP-1, RANTES, and IP-10 in human lung epithelial cells. In addition, mAb CNTO2424 was able to interfere with the known TLR3-dependent signaling pathways, namely NF-kappaB, IRF-3/ISRE, and p38 MAPK. The generation of this neutralizing anti-TLR3 mAb provides a unique tool to better understand TLR3 signaling and potential cross-talk between TLR3 and other molecules.
Asunto(s)
Anticuerpos Monoclonales , Receptor Toll-Like 3/antagonistas & inhibidores , Receptor Toll-Like 3/inmunología , Animales , Anticuerpos Bloqueadores/metabolismo , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Línea Celular , Línea Celular Transformada , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Proyectos Piloto , Receptor Toll-Like 3/metabolismoRESUMEN
Jimpy (Plp(jp)) is an X-linked recessive mutation in mice that causes CNS dysmyelination and early death in affected males. It results from a point mutation in the acceptor splice site of myelin proteolipid protein (Plp) exon 5, producing transcripts that are missing exon 5, with a concomitant shift in the downstream reading frame. Expression of the mutant PLP product in Plp(jp) males leads to hypomyelination and oligodendrocyte death. Expression of our Plp-lacZ fusion gene, PLP(+)Z, in transgenic mice is an excellent readout for endogenous Plp transcriptional activity. The current studies assess expression of the PLP(+)Z transgene in the Plp(jp) background. These studies demonstrate that expression of the transgene is decreased in both the central and peripheral nervous systems of affected Plp(jp) males. Thus, expression of mutated PLP protein downregulates Plp gene activity both in oligodendrocytes, which eventually die, and in Schwann cells, which are apparently unaffected in Plp(jp) mice.
Asunto(s)
Sistema Nervioso Central/metabolismo , Proteína Proteolipídica de la Mielina/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Sistema Nervioso Periférico/metabolismo , Animales , Western Blotting , Sistema Nervioso Central/crecimiento & desarrollo , Regulación hacia Abajo , Femenino , Regulación del Desarrollo de la Expresión Génica , Operón Lac/genética , Masculino , Ratones , Ratones Jimpy , Ratones Transgénicos , Proteína Proteolipídica de la Mielina/genética , Proteínas del Tejido Nervioso/genética , Oligodendroglía/metabolismo , Células de Schwann/metabolismo , Transgenes/genética , beta-Galactosidasa/biosíntesisRESUMEN
The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the drug in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Herein we describe a novel immunization method that utilizes type 1 interferons (IFNs) as immunomodulators coupled with an agonistic anti-CD40 mAb as a B cell proliferative agent to drive immune responses. This novel protocol allows for rapid and robust mAb reagent generation without the use of conventional protein-denaturing adjuvants. The use of IFNs allowed for the generation of comparable and in some cases, increased numbers of anti-variable region mAbs in a dramatically shorter timeframe. This IFN based, immunostimulatory approach utilizing a non-denaturing adjuvant, likely presents conformational epitopes and may optimize the humoral response for the rapid generation of anti-variable region reagents to therapeutic mAb candidates.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Anticuerpos Monoclonales/biosíntesis , Interferones/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos BALB C , Sensibilidad y Especificidad , Bazo/inmunologíaRESUMEN
We describe the genetic and neurological features of toppler, a spontaneous autosomal mutation that appeared in a colony of FVB/N mice and that manifests as severe ataxia appearing at around 12 days of age, worsening with age. The lifespan of affected mice is 8-12 months, with occasional mice living longer. Both homozygous males and females are fertile, and females are able to nurture litters. Histological examination of brain revealed no striking abnormalities other than the loss of cerebellar Purkinje cells. The toppler mutation was mapped to mouse chromosome 8, and to assess whether it was novel or a recurrence of a previously described chromosome 8 mouse mutant, toppler mice were crossed with the nervous and tottering mouse mutants. These studies demonstrate that toppler is a unique mouse mutation. Purkinje cell abnormalities in toppler mice were obvious around postnatal day (P) 14, i.e., toppler Purkinje cells already exhibited abnormal morphology. Staining for calbindin, a calcium binding protein enriched in Purkinje cells, showed altered dendritic morphology. Between P14 and P30, dramatic Purkinje cell loss occurred, although there were differences in the degree of Purkinje cell loss in each lobule. At P30, the surviving Purkinje cells expressed zebrin II. From P30 through 6 months, many of the remaining Purkinje cells gradually degenerated. Purkinje cell loss was analyzed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL), and Purkinje cells were TUNEL-positive most abundantly at P21. In addition, Bergmann glia were TUNEL positive at P21, and they expressed activated caspase-3 at earlier time points. Interestingly, despite the apparent death of some Bergmann glia, there was up-regulation of glial fibrillary acidic protein, expressed in astrocytes as well as Bergmann glia. Given the changes in both Purkinje cells and glia in toppler cerebellum, this may be a very useful model in which to investigate the developmental interaction of Purkinje cells and Bergmann glia.
Asunto(s)
Ataxia/genética , Ataxia/patología , Cerebelo/patología , Ratones Mutantes Neurológicos , Células de Purkinje/patología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Ataxia/metabolismo , Ataxia/fisiopatología , Muerte Celular , Sistema Nervioso Central/patología , Cerebelo/fisiopatología , Mapeo Cromosómico , Cruzamientos Genéticos , Femenino , Fructosa-Bifosfato Aldolasa/metabolismo , Expresión Génica , Inmunohistoquímica , Masculino , Ratones , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/patología , Fenotipo , Células de Purkinje/metabolismo , Retina/patología , Degeneraciones Espinocerebelosas/genéticaRESUMEN
The factors that regulate oligodendrogenesis have been studied extensively in optic nerve, where oligodendrocyte production and myelination quickly follow colonization of the nerve by progenitor cells. In contrast, oligodendrocyte production in the cerebral cortex begins approximately 1 week after progenitor cell colonization and continues for 3-4 weeks. This and other observations raise the possibility that oligodendrogenesis is regulated by different mechanisms in white and gray matter. The present study examined oligodendrocyte production in the developing cerebral cortex of jimpy (jp) and jimpy(msd) (msd) mice, which exhibit hypomyelination and oligodendrocyte death due to mutations in and toxic accumulations of proteolipid protein, the major structural protein of CNS myelin. Proliferation of oligodendrocyte progenitors and production of myelinating oligodendrocytes was reduced in jp cerebral cortex when compared to wild-type (wt) and msd mice. The incidence of oligodendrocyte cell death was similar in jp and msd cortex, but total dying oligodendrocytes were greater in msd. We confirm previous reports of increased oligodendrocyte production in white matter of both jp and msd mice. The jp mutation, therefore, reduces oligodendrocyte production in cerebral cortex but not in white matter. These data provide additional evidence that oligodendrogenesis is differentially regulated in white matter and gray matter and implicate PLP/DM20 as a modulator of these differences.