RESUMEN
Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.
Asunto(s)
Ceguera/genética , Glicoproteínas de Membrana/deficiencia , Lipofuscinosis Ceroideas Neuronales/genética , Epitelio Pigmentado de la Retina/patología , Animales , Atrofia/genética , Atrofia/patología , Autofagia , Ceguera/patología , Línea Celular , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Técnicas de Silenciamiento del Gen , Glucógeno/metabolismo , Humanos , Lisosomas/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Microscopía Electrónica , Chaperonas Moleculares/genética , Mutación , Lipofuscinosis Ceroideas Neuronales/complicaciones , Lipofuscinosis Ceroideas Neuronales/patología , ARN Interferente Pequeño/metabolismo , Epitelio Pigmentado de la Retina/ultraestructuraRESUMEN
In a consanguineous Pakistani family with two affected individuals, a homozygous variant Gly399Val in the eighth transmembrane domain of the taurine transporter SLC6A6 was identified resulting in a hypomorph transporting capacity of ~15% compared with normal. Three-dimensional modeling of this variant has indicated that it likely causes displacement of the Tyr138 (TM3) side chain, important for transport of taurine. The affected individuals presented with rapidly progressive childhood retinal degeneration, cardiomyopathy and almost undetectable plasma taurine levels. Oral taurine supplementation of 100 mg/kg/day resulted in maintenance of normal blood taurine levels. Following approval by the ethics committee, a long-term supplementation treatment was introduced. Remarkably, after 24-months, the cardiomyopathy was corrected in both affected siblings, and in the 6-years-old, the retinal degeneration was arrested, and the vision was clinically improved. Similar therapeutic approaches could be employed in Mendelian phenotypes caused by the dysfunction of the hundreds of other molecular transporters.
Asunto(s)
Cardiomiopatías/tratamiento farmacológico , Glicoproteínas de Membrana/deficiencia , Proteínas de Transporte de Membrana/deficiencia , Degeneración Retiniana/tratamiento farmacológico , Taurina/uso terapéutico , Adolescente , Transporte Biológico , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Niño , Femenino , Humanos , Masculino , Linaje , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patologíaRESUMEN
Mutations in NCSTN gene (encoding for nicastrin protein) are associated with hidradenitis suppurativa (HS), a chronic inflammatory disease involving hair follicles. HS is clinically handled with drugs but the most severe cases are treated with surgery. Photobiomodulation (PBM) therapy, already used in the treatment of skin diseases such as acne, herpes virus lesions, ultraviolet damage, vitiligo, hypertrophic scar, keloid, burn, psoriasis and diabetic chronic wounds, could be beneficial as an adjuvant supportive treatment to promote and foster the healing process after skin excision in HS. The effects of PBM therapy in promoting the wound closure are evaluated in a HaCaT cells NCSTN-/-, assessing cell metabolism, migration rate, proliferation and cell cycle progression. In our experimental model, PBM exerts a potent action on metabolism of mutated keratinocytes, incrementing adenosine triphosphate (ATP) production at 2 hours, while after 24 hours an increase of metabolism with a decrement of intracellular ATP levels were recorded. Moreover, PBM speeds up the wound closure, inducing cells' migration without affecting their proliferation.Based on our findings, we suggest the use of PBM in HS patients, who undergo major surgery with large skin excision.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Técnicas de Inactivación de Genes , Terapia por Luz de Baja Intensidad , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Cicatrización de Heridas/efectos de la radiación , Ciclo Celular/efectos de la radiación , Línea Celular , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Humanos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiaciónRESUMEN
UNLABELLED: In this study, we show that the HIV-1 Tat protein interacts with rapid kinetics to engage the Toll-like receptor 4 (TLR4) pathway, leading to the production of proinflammatory and anti-inflammatory cytokines. The pretreatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of the TLR4 pathway, leading to the production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Therefore, this study analyzed whether the HIV-1 Tat protein is able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient in the MyD88, TIRAP/MAL, or TRIF adaptor, biochemical analysis, and the use of specific small interfering RNAs (siRNAs), we demonstrated (i) that Tat was able to activate both the MyD88 and TRIF pathways, (ii) the capacity of Tat to induce TIRAP/MAL degradation, (iii) the crucial role of the MyD88 pathway in the production of Tat-induced TNF-α and IL-10, (iv) a reduction but not abrogation of IL-10 and TNF-α by Tat-stimulated macrophages from mice deficient in TIRAP/MAL, and (v) the crucial role of the TRIF pathway in Tat-induced IL-10 production. Further, we showed that downstream of the MyD88 and TRIF pathways, the Tat protein activated the protein kinase C (PKC) ßII isoform, the mitogen-activated protein (MAP) kinases p38 and extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB in a TLR4-dependent manner. Collectively, our data show that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10. IMPORTANCE: In this study, we demonstrate that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC-ßII, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Thus, it may be interesting to target Tat as a pathogenic factor early after HIV-1 infection. This could be achieved either by vaccination approaches including Tat as an immunogen in potential candidate vaccines or by developing molecules capable of neutralizing the effect of the Tat protein.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , VIH-1/metabolismo , Interleucina-10/biosíntesis , Monocitos/virología , Factor 88 de Diferenciación Mieloide/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Animales , VIH-1/inmunología , Humanos , Interleucina-10/metabolismo , Cinética , Sistema de Señalización de MAP Quinasas/fisiología , Glicoproteínas de Membrana/deficiencia , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/inmunología , Factor 88 de Diferenciación Mieloide/deficiencia , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño , Receptores de Interleucina-1/deficiencia , Transducción de Señal , Receptor Toll-Like 4/metabolismoRESUMEN
Developing new adjuvants and vaccination strategies is of paramount importance to successfully fight against many life-threatening infectious diseases and cancer. Very few adjuvants are currently authorized for human use, and these mainly stimulate a humoral response. However, specific Abs are not sufficient to confer protection against persisting infections or cancer. Therefore, development of adjuvants and immunomodulators able to enhance cell-mediated immune responses represents a major medical need. We recently showed that papaya mosaic virus nanoparticles (PapMV), self-assembled from the coat protein of a plant virus and a noncoding ssRNA molecule, are highly immunogenic in mice. PapMV can be used either as a vaccine delivery platform, through fusion of various epitopes to the coat protein or as adjuvant to enhance humoral immune responses against coadministered Ags or vaccines. However, the mechanisms that confer these immunomodulatory properties to PapMV and its ability to enhance T cell vaccines remain unknown. Using immunization studies in mice, we demonstrate in this paper that PapMV represents a novel TLR7 agonist with strong immunostimulatory properties. More importantly, pretreatment with PapMV significantly improves effector and memory CD8(+) T cell responses generated through dendritic cell vaccination increasing protection against a Listeria monocytogenes challenge.
Asunto(s)
Adyuvantes Inmunológicos , Linfocitos T CD8-positivos/inmunología , Listeria monocytogenes/inmunología , Listeriosis/prevención & control , Glicoproteínas de Membrana/agonistas , Subgrupos de Linfocitos T/inmunología , Receptor Toll-Like 7/agonistas , Tymovirus/inmunología , Vacunación , Inmunidad Adaptativa , Animales , Células Dendríticas/inmunología , Evaluación Preclínica de Medicamentos , Femenino , Inmunoglobulina G/biosíntesis , Memoria Inmunológica , Interferón Tipo I/inmunología , Listeriosis/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/inmunología , Nanopartículas , Ovalbúmina/inmunología , ARN Viral/inmunología , Receptor de Interferón alfa y beta/deficiencia , Receptor Toll-Like 7/deficiencia , Receptor Toll-Like 7/inmunología , Tymovirus/genéticaRESUMEN
During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1-dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPS-mediated Ca2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1-/- mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3-dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release-activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Glicoproteínas de Membrana/antagonistas & inhibidores , NADPH Oxidasas/antagonistas & inhibidores , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Anilidas/farmacología , Animales , Canales de Calcio , Señalización del Calcio , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Técnicas de Silenciamiento del Gen , Receptores de Inositol 1,4,5-Trifosfato/deficiencia , Receptores de Inositol 1,4,5-Trifosfato/genética , Lipopolisacáridos/toxicidad , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Factores de Transcripción NFATC/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sepsis/complicaciones , Transducción de Señal , Molécula de Interacción Estromal 1 , Tiadiazoles/farmacologíaRESUMEN
Emerging evidence indicates that NADPH oxidase (NOX) and its reactive oxygen species (ROS) products modulate a variety of cellular events, including proliferation, differentiation, and apoptosis. In this study, we investigated the functions of NOX2 and ROS in immune modulation using NOX2 knockout (KO) mice. Interestingly, NOX2 KO mice spontaneously developed arthritis with onset at 6-7 wk of age and high incidence (60%) at 15-18 wk of age. Arthritis severity in NOX2 KO mice was proportionally increased with age and higher in females than in males. Bone destruction was confirmed by microcomputed tomography scanning and histological analyses of joints. Inflammatory factors, including TNF-α, IL-1ß, and RANKL, and serum level of anti-type II collagen IgG were significantly increased in NOX2 KO mice. In addition, NOX2 deficiency perturbed the immune system upon aging. NOX2 KO mice demonstrated preferred development of CD11b+Gr-1+ myeloid cells with profound production of proinflammatory cytokines and augmented expression of IL-17 through the activation of STAT3 and RORγt in vivo. NOX2 deficiency increased differentiation of effector Th cells in vitro and decreased CD25+FoxP3+ Treg cells both in vitro and in vivo. Furthermore, adoptive transfer of NOX2-deficient CD4(+) T cells into RAG KO mice increased arthritic inflammation compared with WT cells. These results demonstrated that NOX2 deficiency affected the development of CD11b+ myeloid cells and Th17/Treg cells, and thus promoted inflammatory cytokine production and inflammatory arthritis development, strongly supporting a crucial role for ROS generation in the modulation of Th17/Treg cell development and its related inflammatory immune response upon aging.
Asunto(s)
Envejecimiento/inmunología , Artritis/inmunología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Glicoproteínas de Membrana/inmunología , NADPH Oxidasas/inmunología , Traslado Adoptivo , Animales , Artritis/genética , Artritis/metabolismo , Densidad Ósea , Huesos/anomalías , Huesos/metabolismo , Antígeno CD11b/inmunología , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Células Cultivadas , Femenino , Citometría de Flujo , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-17/inmunología , Interleucina-17/metabolismo , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Microtomografía por Rayos XRESUMEN
BACKGROUND: In addition to its well-known expression in dorsal telencephalic progenitor cells, where it regulates cell proliferation and identity, the transcription factor Pax6 is expressed in some ventral telencephalic cells, including many postmitotic neurons. Its functions in these cells are unknown. RESULTS: We generated a new floxed allele of Pax6 and tested the consequences of a highly specific ventral telencephalic depletion of Pax6. We used the Six3A1A2-Cre allele that drives production of Cre recombinase in a specific region of Pax6-expression close to the internal capsule, through which thalamic axons navigate to cerebral cortex. Depletion in this region caused many thalamic axons to take aberrant routes, either failing to turn normally into ventral telencephalon to form the internal capsule or exiting the developing internal capsule ventrally. We tested whether these defects might have resulted from abnormalities of two structural features proposed to guide thalamic axons into and through the developing internal capsule. First, we looked for the early pioneer axons that project from the region of the future internal capsule to the thalamus and are thought to guide thalamocortical axons to the internal capsule: we found that they are present in conditional mutants. Second, we examined the development of the corridor of Islet1-expressing cells that guides thalamic axons through ventral telencephalon and found that it was broader and less dense than normal in conditional mutants. We also examined corticofugal axons that are thought to interact with ascending thalamocortical axons, resulting in each set providing guidance to the other, and found that some are misrouted to lateral telencephalon. CONCLUSION: These findings indicate that ventral telencephalic Pax6 is important for formation of the Islet1-expressing corridor and the thalamic and cortical axons that grow through it. We suggest that Pax6 might affect thalamic axonal growth indirectly via its effect on the corridor.
Asunto(s)
Axones/fisiología , Corteza Cerebral/embriología , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Neuronas/citología , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Telencéfalo , Tálamo/embriología , Factores de Edad , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Corteza Cerebral/citología , Proteínas del Huevo/genética , Embrión de Mamíferos , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Humanos , Intrones/genética , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Vías Nerviosas/embriología , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/deficiencia , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Telencéfalo/citología , Telencéfalo/embriología , Telencéfalo/metabolismo , Tálamo/citología , Glicoproteínas de la Zona Pelúcida , Proteína Homeobox SIX3RESUMEN
Although corneal transplantation is one of the most common tissue transplantations and is known to have a high graft acceptance rate, occasional corneal graft rejection remains a cause of blindness. OX40, a member of the TNF receptor superfamily, is expressed on activated T cells, and transmits a costimulatory signal by binding to OX40 ligand (OX40L) expressed on several cells with antigen-presenting functions. Using a blocking monoclonal antibody (mAb) against murine OX40L, we investigated the role of OX40 in a murine model of corneal transplantation. C3H/He mouse corneas were transplanted to BALB/c mice orthotopically. Administration of anti-OX40L mAb significantly reduced allograft rejection, and increased graft survival rate to 40% at 8 weeks after transplantation, while all corneas were rejected within 5 weeks in control IgG-treated mice. Similar reduced rejection was observed when wild-type donor corneas were transplanted to OX40L-deficient recipients. In vitro study revealed that the anti-OX40L mAb treatment reduced proliferative response and IFN-gamma production of draining lymph node cells in response to stimulation with donor alloantigen. These results demonstrate that OX40L blockade is effective for prolongation of corneal allograft survival by inhibiting recipient T cell activation.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Trasplante de Córnea , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/efectos de los fármacos , Glicoproteínas de Membrana/antagonistas & inhibidores , Inhibidores del Factor de Necrosis Tumoral , Animales , Anticuerpos Monoclonales/inmunología , Evaluación Preclínica de Medicamentos , Proteínas del Ojo/inmunología , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Isoantígenos/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Noqueados , Ligando OX40 , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Donantes de Tejidos , Trasplante Homólogo , Factores de Necrosis Tumoral/deficiencia , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/fisiologíaRESUMEN
Translocon-associated protein complex (TRAP) is thought to be required for efficient protein-specific translocation across the endoplasmic reticulum membrane. We created a mutation in the Trapalpha gene that leads to the synthesis of a truncated TRAPalpha protein fused to ShBle-beta-galactosidase. Analysis of Trapalpha cDNAs reveals that among three different messenger RNAs expressed in the mouse, one of them encodes a slightly larger protein that differs in its C-terminal end. This mRNA, specific for skeletal muscle and heart, is only expressed after birth. Homozygous Trapalpha mutant pups die at birth, likely as a result of severe cardiac defects. Indeed, the septation of the proximal part of the outflow tract is absent, resulting in a double-outlet right ventricle. Studies of protein secretion in transfected embryonic fibroblasts reveal that the TRAP complex does not function properly in homozygous mutant cells and confirm, in vivo, the involvement of TRAP in substrate-specific translocation. Our results provide the first in vivo demonstration that a member of the TRAP complex plays a crucial role in mammalian heart development and suggest that TRAPalpha could be involved in translocation of factors necessary for maturation of endocardial cushions.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Envejecimiento/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Movimiento Celular , Secuencia Conservada , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Corazón/embriología , Homocigoto , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación/genética , Miocardio/metabolismo , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/genética , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Péptidos/química , Receptores de Péptidos/deficiencia , Receptores de Péptidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de SecuenciaRESUMEN
Actin-bundling proteins organize actin filaments into densely packed bundles. In Dictyostelium discoideum two abundant proteins display calcium-regulated bundling activity, fimbrin and the 34-kDa protein (ABP34). Using a GFP fusion we observed transient localization of fimbrin at the phagocytic cup and macropinosomes. The distribution of truncated constructs encompassing the EF hands and the first actin-binding domain (EA1) or both actin-binding domains devoid of EF hands (A1A2) was indistinguishable from that of the full length protein. The role of fimbrin and a possible functional overlap with ABP34 was investigated in fim- and double 34-/fim- mutants. Except for a moderate cell size defect, fim- mutants did not show defects in growth, endocytosis, exocytosis, and chemotaxis. Double mutants were characterized by a small cell size and a defect in morphogenesis resulting in small fruiting bodies and a low spore yield. The cell size defect could not be overcome by expression of fimbrin fragments EA1 or A1A2, suggesting that both bundling activity and regulation by calcium are important. Induction of filopod formation in 34-/fim- cells was not impaired, indicating that both proteins are dispensable for this process. We searched in the Dictyostelium genome database for fimbrin-like proteins that could compensate for the fimbrin defect and identified three unconventional fimbrins and two more proteins with actin-binding domains of the type present in fimbrins.
Asunto(s)
Actinas/genética , Proteínas de Unión al Calcio/genética , Dictyostelium/genética , Glicoproteínas de Membrana/genética , Proteínas de Microfilamentos/genética , Mutación , Actinas/química , Actinas/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Tamaño de la Célula , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Motivos EF Hand/genética , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/metabolismo , Indoles , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Peso Molecular , Fagosomas/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Seudópodos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.
Asunto(s)
Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Animales , Membrana Basal/metabolismo , Membrana Basal/patología , Proteínas de Unión al Calcio , Moléculas de Adhesión Celular , Diferenciación Celular/genética , Corazón/embriología , Riñón/citología , Riñón/embriología , Pulmón/embriología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , Ratones , Mutación , Isoformas de Proteínas/fisiologíaRESUMEN
Borna disease virus (BDV) can persistently infect the central nervous system and induce CD8+ T-cell-mediated neurological disease in MRL mice. To determine whether specific immune priming would prevent disease, a prime-boost immunization protocol was established in which intramuscular injection of a recombinant parapoxvirus expressing BDV nucleoprotein (BDV-N) was followed by intraperitoneal infection with vaccinia virus expressing BDV-N. Immunized wild-type and perforin-deficient mice remained healthy after intracerebral infection with BDV and contained almost no virus in the brain at 5 weeks post-challenge. Immunization failed to induce resistance against BDV in mice lacking mature CD8+ T cells. Immunization of perforin-deficient mice with a poxvirus vector expressing mutant BDV-N lacking the known CD8+ T-cell epitope did not efficiently block multiplication of BDV in the brain and did not prevent neurological disease, indicating that vaccine-induced immunity to BDV in wild-type and perforin-deficient mice resulted from the action of CD8+ T cells.
Asunto(s)
Enfermedad de Borna/prevención & control , Virus de la Enfermedad de Borna , Vacunación , Vacunas Virales/administración & dosificación , Animales , Enfermedad de Borna/inmunología , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/inmunología , Encéfalo/virología , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Vectores Genéticos , Inyecciones Intramusculares , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Mutación , Nucleoproteínas/biosíntesis , Nucleoproteínas/genética , Parapoxvirus/genética , Perforina , Proteínas Citotóxicas Formadoras de Poros , Vacunas Sintéticas/administración & dosificación , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
Ginseng radix, the dried root of Panax ginseng C. A. Meyer, has been shown to enhance the ability to resist microbial infections. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and interferon-gamma (IFN-gamma) are produced by macrophages treated with ginseng radix extract (GRE) in vitro as well as in vivo. However, the molecular mechanisms of the production are still not clear. In the present study, we demonstrated that production of TNF-alpha and IFN-gamma was induced by GRE in spleen cells and peritoneal macrophages from C3H/HeN mice but was impaired in C3H/HeJ mice carrying a defective toll-like receptor-4 (TLR-4) gene. In addition to these cytokines, the expression of IFN-beta and inducible nitric oxide synthase (iNOS) mRNAs was also increased in GRE-treated C3H/HeN spleen cells. We investigated the possibility that GRE contains a lipopolysaccharide (LPS)-like component. However, GRE induced production of TNF-alpha and IFN-gamma in the presence of polymyxin B, an LPS inhibitor. Furthermore, a Limulus amebocyte lysate assay showed that the endotoxin content of GRE was below the threshold level of 1 ng/ml LPS. These results suggest that GRE contains a non-LPS agent that enhances innate immunity through production of proinflammatory cytokines via TLR-4.
Asunto(s)
Citocinas/genética , Glicoproteínas de Membrana/fisiología , Panax , Extractos Vegetales/farmacología , Receptores de Superficie Celular/fisiología , Células 3T3 , Animales , Carcinoma de Células Escamosas , Línea Celular Tumoral , Humanos , Inflamación/fisiopatología , Interferón gamma/genética , Interleucina-1/genética , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C3H , Raíces de Plantas , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Nitric oxide (NO) production is known to be impaired in heart failure. A new compound (NCX 899), a NO-releasing derivative of enalapril was characterized, and its actions were evaluated in Bio 14.6 cardiomyopathic (CM) hamsters with heart failure. The hamsters were randomized to oral treatment for 4 weeks with vehicle (n=11), NCX 899 (NCX, 25 mg/kg, n=10), or enalapril (25 mg/kg, n=10). In the vehicle group, fractional shortening by echocardiography decreased (-23.6+/-2.0%) and LV end-diastolic dimension) increased (+10.9+/-1.0%), whereas fractional shortening increased (+17.5+/-4.4%) in NCX and was unchanged in the enalapril group (both P<0.01 vs. vehicle). End-diastolic dimension decreased only in NCX. LV contractility (LVdP/dt max and Emax) was significantly greater in NCX than in enalapril or vehicle, while relaxation (Tau) was shortened in both NCX and enalapril vs. vehicle. ACE activity was inhibited equally by NCX and enalapril in the CM hamster, and plasma nitrate levels were increased only in NCX (P<0.05 vs. enalapril and vehicle). In aortic strips endothelium-independent relaxation occurred only with NCX. The superior effects of NO-releasing enalapril (NCX) vs. enalapril alone to enhance vascular effects, increase LV contractility and prevent unfavorable remodeling and are consistent with vascular delivery of exogenous NO. NCX 899 may hold promise for the future treatment of heart failure.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Cardiomiopatía Dilatada/complicaciones , Enalapril/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Donantes de Óxido Nítrico/uso terapéutico , Óxido Nítrico/metabolismo , Remodelación Ventricular/efectos de los fármacos , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Aorta , Cardiomiopatía Dilatada/diagnóstico por imagen , Cardiomiopatía Dilatada/genética , Cricetinae , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Enalapril/análogos & derivados , Enalapril/farmacología , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Insuficiencia Cardíaca/diagnóstico por imagen , Insuficiencia Cardíaca/etiología , Hemodinámica/efectos de los fármacos , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Mesocricetus , Contracción Miocárdica/efectos de los fármacos , Nitratos/sangre , Donantes de Óxido Nítrico/farmacología , Nitritos/sangre , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Conejos , Sarcoglicanos , Ultrasonografía , Vasoconstricción/efectos de los fármacosRESUMEN
Recent studies indicated that the nicotinamide dinucleotide phosphate oxidase (NADPH) oxidase-derived oxygen radicals plays a deleterious role in arthritis. To study this in more detail, gonarthritis was induced in NADPH oxidase-deficient mice. Mice received an intraarticular injection of either zymosan, to elicit an irritant-induced inflammation, or poly-L-lysine coupled lysozyme, to evoke an immune-complex mediated inflammation in passively immunized mice. In contrast to wild-type mice, arthritis elicited in both p47phox(-/-) and gp91(-/-) mice showed more severe joint inflammation, which developed into a granulomatous synovitis. Treatment with either Zileuton or cobra venom factor showed that the chemokines LTB4 and complement C3 were not the driving force behind the aggravated inflammation in these mice. Arthritic NADPH oxidase-deficient mice showed irreversible cartilage damage as judged by the enhanced aggrecan VDIPEN expression, and chondrocyte death. Furthermore, only in the absence of NADPH oxidase-derived oxygen radicals, the arthritic joints showed osteoclast-like cells, tartrate-resistant acid phosphatase (TRAP)-positive/multinucleated cells, extensive bone erosion, and osteolysis. The enhanced synovial gene expression of tumor necrosis factor-alpha, interleukin-1alpha, matrix metalloproteinase (MMP)-3, MMP-9 and receptor activator of NF-kappaB ligand (RANKL) might contribute to the aggravated arthritis in the NADPH oxidase-deficient mice. This showed that the involvement of NADPH oxidase in arthritis is probably far more complex and that oxygen radicals might also be important in controlling disease severity, and reducing joint inflammation and connective tissue damage.
Asunto(s)
Artritis/metabolismo , Tejido Conectivo/patología , Granuloma/patología , Glicoproteínas de Membrana/deficiencia , Fosfoproteínas/deficiencia , Sinovitis/patología , Animales , Artritis/inducido químicamente , Artritis/diagnóstico por imagen , Artritis/inmunología , Artrografía , Cartílago Articular/patología , Combinación de Medicamentos , Granuloma/inducido químicamente , Granuloma/inmunología , Inmunización Pasiva , Inyecciones Intraarticulares , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/genética , Articulación de la Rodilla/diagnóstico por imagen , Articulación de la Rodilla/patología , Metaloproteinasas de la Matriz/genética , Ratones , Ratones Noqueados , Muramidasa/administración & dosificación , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , Polilisina/administración & dosificación , ARN Mensajero/metabolismo , Sialoglicoproteínas/genética , Membrana Sinovial/metabolismo , Sinovitis/inducido químicamente , Sinovitis/inmunología , Inhibidores Tisulares de Metaloproteinasas/genética , Zimosan/administración & dosificaciónRESUMEN
We investigated the mechanism of the immunomodulatory action of polysaccharide (ASP) isolated from a cell culture of Acanthopanax senticosus. ASP was found to directly increase the proliferation and differentiation of B cells, and the cytokine production of macrophage, but not the proliferation and cytokine production of T cells. Since ASP cannot penetrate the cell membrane due to its large molecular mass, such cellular activation may be caused by the surface binding of ASP to receptors expressed on B cells and macrophages. The possibility that TLRs, which are known to be involved in immune-related responses, may be the receptor(s) of ASP was investigated. The immunomodulating activities of ASP on the B cells and macrophages of C3H/HeJ mice, expressing a defective toll-like receptor (TLR)-4, were decreased versus the corresponding cells from C3H/HeN mice. In addition, the activities of ASP on B cells and macrophages were significantly reduced by treating the cells with antibodies to TLR4 and TLR2 prior to ASP, suggesting that both of them are the possible receptors of ASP. The ligation of TLRs induced by ASP was able to activate mitogen-activated protein kinases (MAPKs), such as Erk1/2, p38 and JNK, and the transcription factor NF-kappaB. Although ASP was shown to activate the TLR signaling cascades in the same manner as lipopolysaccharide (LPS), these two could be differentiated by the finding that polymyxin B (PMB), a specific inhibitor of LPS, did not significantly affect the activities of ASP on B cells and macrophages. Taken together, our results demonstrate that ASP, isolated from a cell culture of A. senticosus, activates B cells and macrophages by interacting with TLRs and leading to the subsequent activation of mitogen-activated protein kinases and NF-kappaB.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B/efectos de los fármacos , Eleutherococcus/química , Activación de Linfocitos/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Glicoproteínas de Membrana/efectos de los fármacos , Polisacáridos/farmacología , Receptores de Superficie Celular/efectos de los fármacos , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión , Células Cultivadas/química , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , FN-kappa B/metabolismo , Nitritos/metabolismo , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Polisacáridos/aislamiento & purificación , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Transcripción Genética/efectos de los fármacosRESUMEN
To investigate the role of the Toll-like receptor (TLR) family in host defense against Toxoplasma gondii, we infected TLR2-, TLR4- and MyD88-deficient mice with the avirulent cyst-forming Fukaya strain of T. gondii. All TLR2- and MyD88-deficient mice died within 8 days, whereas all TLR4-deficient and wild-type mice survived after i.p. infection with a high dose of T. gondii. Peritoneal macrophages from T. gondii-infected TLR2- and MyD88-deficient mice did not produce any detectable levels of NO. T. gondii loads in the brain tissues of TLR2- and MyD88-deficient mice were higher than in those of TLR4-deficient and wild-type mice. Furthermore, high levels of IFN-gamma and IL-12 were produced in peritoneal exudate cells (PEC) of TLR4-deficient and wild-type mice after infection, but low levels of cytokines were produced in PEC of TLR2- and MyD88-deficient mice. On the other hand, high levels of IL-4 and IL-10 were produced in PEC of TLR2- and MyD88-deficient mice after infection, but low levels of cytokines were produced in PEC of TLR4-deficient and wild-type mice. The most remarkable histological changes with infiltration of inflammatory cells were observed in lungs of TLR2-deficient mice infected with T. gondii, where severe interstitial pneumonia occurred and abundant T. gondii were found.
Asunto(s)
Arginina/análogos & derivados , Citocinas/biosíntesis , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/inmunología , Arginina/farmacología , Pulmón/inmunología , Pulmón/parasitología , Pulmón/patología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide , Óxido Nítrico/biosíntesis , Receptores de Superficie Celular/deficiencia , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Th2/inmunología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/patologíaRESUMEN
Toll-like receptor 2 (TLR2) is a transmembrane signal transducer for tripalmitoyl-S-glyceryl-cysteine (Pam(3)Cys)-modified lipoproteins, including OspA from the Lyme disease spirochete Borrelia burgdorferi. The Pam(3)Cys modification provides adjuvant activity for inducing humoral responses, suggesting that TLR2 could function as the adjuvant receptor for the OspA vaccine. The importance of TLR2 in the humoral response to OspA was confirmed, because overall levels of immunoglobulin G (IgG) were reduced in TLR2-deficient mice, when compared with those in wild-type mice. However, the levels of production of IgG1 were similar in both mouse strains, and the levels of induction of protective immunity were comparable. Unlipidated OspA was not immunogenic in wild-type or TLR2-deficient mice, indicating the lipid modification was active in the absence of TLR2. These findings indicate that the Pam(3)Cys modification of bacterial lipoprotein has adjuvant properties independent of TLR2 signaling.
Asunto(s)
Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Cisteína/análogos & derivados , Cisteína/fisiología , Lipoproteínas , Enfermedad de Lyme/prevención & control , Glicoproteínas de Membrana/deficiencia , Receptores de Superficie Celular/deficiencia , Animales , Anticuerpos Antibacterianos/sangre , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C3H , Receptores de Superficie Celular/fisiología , Receptor Toll-Like 2 , Receptores Toll-Like , VacunaciónRESUMEN
It has been of much interest whether there is functional redundancy between the constitutively signaling pre-Talpha/TCRbeta (pre-TCR) and ligated TCRalphabeta complexes, which independently operate the two distinct checkpoints during thymocyte development, i.e., the pre-TCR involved in beta-selection at the CD4(-)CD8(-) double-negative stage and the TCRalphabeta being crucial for positive/negative selection at the CD4(+)CD8(+) double-positive stage. We found that the pre-TCR expressed on double-positive cells in TCRalpha-deficient (TCRalpha(-/-)) mice produced a small number of mature CD8(+) T cells. Surprisingly, when pre-Talpha was overexpressed, resulting in augmentation of pre-TCR expression, there was a striking increase of the CD8(+) T cells. In addition, even in the absence of up-regulation of pre-TCR expression, a similar increase of CD8(+) T cells was also observed in TCRalpha(-/-) mice overexpressing Egr-1, which lowers the threshold of signal strength required for positive selection. In sharp contrast, the CD8(+) T cells drastically decreased in the absence of pre-Talpha on a TCRalpha(-/-) background. Thus, the pre-TCR appears to functionally promote positive selection of CD8(+) T cells. The biased production of CD8(+) T cells via the pre-TCR might also support the potential involvement of signal strength in CD4/CD8 lineage commitment.