RESUMO
Influenza virus epidemics potentially cause pneumonia, which is responsible for much of the mortality due to the excessive immune responses. The role of costimulatory OX40-OX40 ligand (OX40L) interactions has been explored in the non-infectious pathology of influenza pneumonia. Here, we describe a critical contribution of OX40L to infectious pathology, with OX40L deficiency, but not OX40 deficiency, resulting in decreased susceptibility to influenza viral infection. Upon infection, bronchiolar progenitors increase in number for repairing the influenza-damaged epithelia. The OX40L expression is induced on the progenitors for the antiviral immunity during the infectious process. However, these defense-like host responses lead to more extensive infection owing to the induced OX40L with α-2,6 sialic acid modification, which augments the interaction with the viral hemagglutinin. In fact, the specific antibody against the sialylated site of OX40L exhibited therapeutic potency in mitigating the OX40L-mediated susceptibility to influenza. Our data illustrate that the influenza-induced expression of OX40L on bronchiolar progenitors has pathogenic value to develop a novel therapeutic approach against influenza.
Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Ligante OX40/metabolismo , Infecções por Orthomyxoviridae/patologia , Pneumonia Viral/patologia , Células-Tronco/metabolismo , Ligação Viral , Animais , Suscetibilidade a Doenças , Camundongos Endogâmicos C57BL , Ligante OX40/química , Infecções por Orthomyxoviridae/virologia , Pneumonia Viral/virologia , Processamento de Proteína Pós-Traducional , Ácidos Siálicos/análise , Células-Tronco/virologiaRESUMO
KEY MESSAGE: The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements. Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.
Assuntos
Vacinas contra Cólera/genética , Oryza/genética , Plantas Geneticamente Modificadas/genética , Tecnologia Farmacêutica/métodos , Administração Oral , Animais , Western Blotting , Cólera/imunologia , Cólera/microbiologia , Cólera/prevenção & controle , Toxina da Cólera/toxicidade , Vacinas contra Cólera/administração & dosagem , Vacinas contra Cólera/imunologia , Análise Custo-Benefício , Diarreia/induzido quimicamente , Diarreia/imunologia , Diarreia/prevenção & controle , Embalagem de Medicamentos , Estabilidade de Medicamentos , Humanos , Imunização/métodos , Camundongos , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Pós , Reprodutibilidade dos Testes , Tecnologia Farmacêutica/economia , Vibrio cholerae/imunologiaRESUMO
A lipopolysaccharide from Pantoea agglomerans (LPSpa) has been applied to various fields for human use as a Toll-like receptor 4 ligand and its safety has been confirmed. Here, we showed for the first time the application of LPSpa as an effective mucosal adjuvant for activating vaccine-induced antigen specific immune responses. Mice sublingually immunized with influenza vaccine (HA split vaccine) with LPSpa induced both HA-specific IgG (systemic) and IgA (mucosal) antibody responses, which led to a significant increase in survival rate against lethal influenza virus challenge compared with subcutaneous vaccination. After sublingual administration of ovalbumin with LPSpa, ovalbumin-specific mucosal IgA responses were induced at both mucosal surfaces close to the immunized site and at remote mucosal surfaces. Sublingual administration of LPSpa evoked local antigen-uptake by dendritic cells in cervical lymph nodes. LPSpa induced cytokine production and the maturation and proliferation of innate immune cells via Toll-like receptor 4 in dendritic cells. Collectively, these results suggest that LPSpa can be used as an effective mucosal adjuvant to stimulate and activate local innate immune cells to improve and enhance mucosal vaccine potency against various pathogens.
Assuntos
Adjuvantes Imunológicos/farmacologia , Imunidade Humoral/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Lipopolissacarídeos/farmacologia , Pantoea/imunologia , Receptor 4 Toll-Like/fisiologia , Adjuvantes Imunológicos/administração & dosagem , Administração Sublingual , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Imunidade Humoral/imunologia , Imunidade nas Mucosas/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
GM1 gangliosidosis is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations in the ß-galactosidase (GLB1) gene. In feline GM1 gangliosidosis, a pathogenic mutation (c.1448G>C) in the feline GLB1 gene was identified in Siamese cats in the United States and Japan and in Korat cats in Western countries. The present study found the homozygous c.1448G>C mutation in 2 apparent littermate native kittens in Bangladesh that were exhibiting neurological signs. This is the first identification of GM1 gangliosidosis in native domestic cats in Southeast Asia. This pathogenic mutation seems to have been present in the domestic cat population in the Siamese region and may have been transferred to pure breeds such as Siamese and Korat cats originating in this region.
Assuntos
Substituição de Aminoácidos , Doenças do Gato/enzimologia , Gangliosidose GM1/veterinária , Regulação Enzimológica da Expressão Gênica/fisiologia , beta-Galactosidase/metabolismo , Animais , Bangladesh/epidemiologia , Doenças do Gato/genética , Doenças do Gato/metabolismo , Gatos , Gangliosidose GM1/genética , Predisposição Genética para Doença , Mutação , beta-Galactosidase/genéticaRESUMO
Here we examined whether polymeric IgA (pIgA) and monomeric IgA (mIgA) antibodies differ in their ability to neutralize drift viruses within the same subtype. We used an IgA monoclonal antibody (mAb; H1-21) against influenza virus strain A/Hiroshima/52/2005 (A/Hiroshima; H3N2). The mAb was obtained after immunizing mice mucosally with a split-virion (SV) vaccine. The mAb contained both mIgA and pIgA forms. It reacted with the homologous virus and cross-reacted with drift viruses A/New York/55/2004 (H3N2) and A/Wyoming/3/2003 (H3N2) in hemagglutinin-inhibition (HI) and neutralizing Ab assays. The mAb also cross-reacted with A/Panama/2007/99 (H3N2) in an ELISA. We separated the mAb into pIgA and mIgA fractions by gel filtration, and then tested them for neutralizing Ab activity. The neutralizing activity for the A/Hiroshima/52/2005, A/New York/55/2004, and A/Wyoming/3/2003 viruses was lower for the mIgA than the pIgA fraction. However, the neutralizing efficiency for drift variants relative to that for the homotype did not differ between pIgA and mIgA, and pIgA only neutralized variants that could also be neutralized by mIgA. These results suggest that the polymerization of IgA enhances its antiviral immune responses, but does not increase the number of influenza virus strains neutralized by the IgA.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Imunoglobulina A/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Reações Cruzadas/imunologia , Feminino , Vacinas contra Influenza , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Toxina Pertussis/imunologia , Análise de Sequência de RNARESUMO
GM1 gangliosidosis is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations of the ß-galactosidase (GLB1) gene. In feline GM1 gangliosidosis, a pathogenic mutation (c.1448G>C) of the feline GLB1 gene was identified in Siamese and Korat cats previously diagnosed with the disease in the USA and Italy, respectively. The present study demonstrated the same mutation in a Siamese cat that had been diagnosed with GM1 gangliosidosis in Japan in the 1960s. The mutation was confirmed using DNA extracted from stored paraffin-embedded brain tissue by a direct sequencing method and a polymerase chain reaction-restriction fragment length polymorphism assay. This pathogenic mutation seems to have been distributed around the world.
Assuntos
Doenças do Gato/genética , Gangliosidose GM1/veterinária , beta-Galactosidase/genética , Animais , Gatos , Análise Mutacional de DNA/veterinária , Frequência do Gene , MutaçãoRESUMO
The antigenicity of seasonal human influenza virus changes continuously; thus, a cross-protective influenza vaccine design needs to be established. Intranasal immunization with an influenza split-virion (SV) vaccine and a mucosal adjuvant induces cross-protection; however, no mucosal adjuvant has been assessed clinically. Formalin-inactivated intact human and avian viruses alone (without adjuvant) induce cross-protection against the highly pathogenic H5N1 avian influenza virus. However, it is unknown whether seasonal human influenza formalin-inactivated whole-virion (WV) vaccine alone induces cross-protection against strains within a subtype or in a different subtype of human influenza virus. Furthermore, there are few reports comparing the cross-protective efficacy of the WV vaccine and SV vaccine-mucosal adjuvant mixtures. Here, we found that the intranasal human influenza WV vaccine alone induced both the innate immune response and acquired immune response, resulting in cross-protection against drift variants within a subtype of human influenza virus. The cross-protective efficacy conferred by the WV vaccine in intranasally immunized mice was almost the same as that conferred by a mixture of SV vaccine and adjuvants. The level of cross-protective efficacy was correlated with the cross-reactive neutralizing antibody titer in the nasal wash and bronchoalveolar fluids. However, neither the SV vaccine with adjuvant nor the WV vaccine induced cross-reactive virus-specific cytotoxic T-lymphocyte activity. These results suggest that the intranasal human WV vaccine injection alone is effective against variants within a virus subtype, mainly through a humoral immune response, and that the cross-protection elicited by the WV vaccine and the SV vaccine plus mucosal adjuvants is similar.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Proteção Cruzada , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Administração Intranasal , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
The levels and properties of neutralizing antibodies in nasal wash and serum collected from five healthy adults were examined after intranasal administration of an A/Uruguay/716/2007 (H3N2) split vaccine (45 µg hemagglutinin (HA) per dose; five doses, with an interval of 3 weeks between each dose). Prior to the assays, nasal wash samples were concentrated so that the total amount of antibodies was equivalent to about 1/10 of that found in the natural nasal mucus. Vaccination induced virus-specific neutralizing antibody responses, which increased with the number of vaccine doses given. Neutralizing antibodies were produced more efficiently in the nasal passages than in the serum: A ≥4-fold increase in nasal neutralization titres was observed after the second vaccination in four out of five subjects, whereas a rise in serum neutralization titres was observed only after the fifth vaccination. Nasal and serum neutralizing antibodies were mainly found in the polymeric IgA and monomeric IgG fractions, respectively, after gel filtration. Taken together, these results suggest that intranasal administration of an inactivated split vaccine induces high levels of nasal neutralizing antibodies (primarily polymeric IgA) and low levels of serum neutralizing antibodies (primarily monomeric IgG).
Assuntos
Anticorpos Neutralizantes/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Administração Intranasal , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Influenza Humana/imunologia , Masculino , Pessoa de Meia-Idade , Líquido da Lavagem Nasal/imunologia , Vacinação/métodos , Adulto JovemRESUMO
Effective pneumococcal vaccines are required for preventing secondary bacterial pneumonia, a life-threatening condition, during epidemics of influenza. We examined whether nasal administration of a low dose of pneumococcal surface protein A (PspA) plus polyinosinic-polycytidylic acid (poly(I:C)) could protect against a fatal secondary pneumococcal pneumonia after influenza A virus infection in mice. PspA-specific IgG but not IgA level was higher in the airways and blood of mice nasally administered a low dose of PspA plus poly(I:C) than in mice nasally administered PspA alone or poly(I:C) alone. Binding of PspA-specific IgG increased C3 deposition on the bacterial surface. The survival rate during secondary infection was higher in mice immunized with PspA plus poly(I:C) than in mice immunized with poly(I:C) alone. The significant reduction in bacterial density in the lung and blood was associated with increased survival of immunized mice with secondary pneumonia. Passive transfer of sera from mice immunized with PspA plus poly(I:C) increased the survival of mice infected with secondary pneumonia. Our data suggest that an intranasal PspA vaccine has promising protective effects against secondary pneumonia after influenza and that PspA-specific IgG plays a critical role in this protection.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Influenza Humana/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Pneumonia Pneumocócica/prevenção & controle , Poli I-C/administração & dosagem , Prevenção Secundária/métodos , Adjuvantes Imunológicos/uso terapêutico , Administração Intranasal , Animais , Humanos , Imunização Secundária/métodos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/complicações , Influenza Humana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/etiologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Pneumonia Pneumocócica/etiologia , Pneumonia Pneumocócica/imunologia , Poli I-C/imunologia , Poli I-C/uso terapêuticoRESUMO
The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K(+) current (I(A) ) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4-AP attenuated or blocked the CO(2) -induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch-clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT-PCR of mRNA and the expression of voltage-gated K(+) (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the I(A) by approximately 27% and the additional application of 4-AP (1 mM) further inhibited I(A) by 48%. The application of 0.1 µM α-dendrotoxin (α-DTX), a slow inactivating transient K(+) current (I(D) ) blocker, inhibited the baseline I(A) by approximately 27%, and the additional application of 1 mM AZ further decreased the I(A) by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage-gated K(+) channel proteins were present, and most (80-90%) of the four Kv channels immunoreactive neurons showed the co-expression of carbonic anhydrase-II (CA-II) immunoreactivity. These results indicate that the application of AZ causes the reduction in I(A) via the inhibition of four voltage-gated K(+) channel (Kv) proteins without affecting I(D).
Assuntos
Acetazolamida/farmacologia , Potenciais de Ação/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Gânglio Nodoso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Animais , Inibidores da Anidrase Carbônica/farmacologia , Células Cultivadas , Interações Medicamentosas , Masculino , Neurônios/classificação , Gânglio Nodoso/citologia , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Subunidades Proteicas , Ratos , Ratos WistarRESUMO
A variety of different vaccine types are available for H1N1 influenza A virus infections; however, their immunological mechanisms of action remain unclear. Here, we show that plasmacytoid dendritic cells (pDCs) and type I interferon (IFN)-mediated signaling delineate the immunogenicity of live attenuated virus, inactivated whole-virus (WV), and split-virus vaccines. Although Toll-like receptor 7 acted as the adjuvant receptor for the immunogenicity of both live virus and WV vaccines, the requirement for type I IFN production by pDCs for the immunogenicity of the vaccines was restricted to WV. A split vaccine commonly used in humans failed to immunize naïve mice, but a pDC-activating adjuvant could restore immunogenicity. In blood from human adults, however, split vaccine alone could recall memory T cell responses, underscoring the importance of this adjuvant pathway for primary, but not secondary, vaccination.
Assuntos
Células Dendríticas/imunologia , Vacinas contra Influenza/imunologia , Adulto , Animais , Feminino , Citometria de Fluxo , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/classificação , Masculino , Camundongos , Camundongos Mutantes , Pessoa de Meia-Idade , Receptor 7 Toll-Like/imunologia , Vacinas Atenuadas/imunologiaRESUMO
Adding poly(gamma-glutamic acid) nano-particles (gamma-PGA-NPs), a safe, natural material, to subcutaneous immunization with influenza virus hemagglutinin (HA) vaccine increases the protective immune responses against influenza virus in mice. Here, we examined whether intranasal administration of the HA vaccine with gamma-PGA-NPs would induce protection from influenza virus challenge in mice. Intranasal immunization with the mixture of gamma-PGA-NPs and HA vaccine from an influenza virus strain A/PR/8/34 (H1N1) or A/New Caledonia/20/99 (H1N1) enhanced protection of mice from A/PR/8/34 infection. Intranasal immunization with A/New Caledonia/20/99 HA vaccine and gamma-PGA-NPs induced cell-mediated immune responses and neutralizing antibody production for both A/New Caledonia/20/99 and A/PR/8/34. These data suggest that gamma-PGA-NPs may have potential for clinical applications as a mucosal adjuvant.
Assuntos
Adjuvantes Imunológicos/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Nanopartículas , Infecções por Orthomyxoviridae/prevenção & controle , Ácido Poliglutâmico/análogos & derivados , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Proliferação de Células , Citocinas/imunologia , Feminino , Imunidade Celular , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Ácido Poliglutâmico/imunologia , Baço/citologia , Baço/imunologiaRESUMO
Cell-mediated immunity (CMI) is critical for the prevention and control of varicella-zoster virus (VZV)-related disease. To assess CMI to VZV, a varicella skin test and interferon-gamma enzyme-linked immunospot (ELISPOT) assay were both performed in healthy volunteers, and the results were compared. A total of 151 subjects were examined: 16 aged 20-29 years, 26 aged 30-39 years, 18 aged 40-49 years, 73 aged 50-59 years, and 18 aged 60-69 years. All were seropositive by a glycoprotein antigen-based enzyme-linked immunosorbent assay (gpELISA). Skin test reactivity was significantly correlated with the ELISPOT count, and both decreased with increasing age, indicating an age-dependent decline in CMI to VZV. In contrast, the antibody titer obtained by the gpELISA did not correlate with skin test reactivity. The results suggest that the skin test and ELISPOT assay are both reliable for assessing CMI to VZV and can easily be applied to screen individuals susceptible to the development of herpes zoster.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Herpesvirus Humano 3/imunologia , Imunidade Celular/fisiologia , Interferon gama/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes CutâneosRESUMO
Voltage-gated K+ (Kv) channels are one of the important physiological regulators of the membrane potentials in excitable cells, including sensory ganglion neurons. The aim of the present study was to investigate whether temporomandibular joint (TMJ) inflammation alters expression of Kv channel subtype 1.4 (Kv1.4) of trigeminal ganglion (TRG) neurons innervating TMJ relating allodynia (pain caused by normally innoxious stimulation), by using both behavioral and immunohistochemical techniques. TMJ inflammation was induced by injection of Complete Freund's Adjuvant (CFA) into the rat TMJ. The threshold for escape from mechanical stimulation applied to the orofacial area in TMJ inflamed rats was significantly lower than that in naïve rats. TMJ afferents were identified by fluorogold (FG) labeling. The mean numbers of Kv1.4-/neurofilament (NF) 200(myelinated fiber marker) positive- and negative-immunoreactivities FG-labeled small-/medium-diameter TRG neurons in inflamed rats were significantly decreased when compared with those in the naïve rats. These findings suggest that TMJ inflammation reduces the expression of Kv1.4 subunits in the small-/medium sized (Adelta-/C-) TRG neurons and this may contribute to trigeminal inflammatory allodynia in TMJ disorder. These results lead us to suggest that Kv channel openers may be a potential therapeutic agents for prevention of mechanical allodynia.
Assuntos
Artrite/metabolismo , Canal de Potássio Kv1.4/metabolismo , Neurônios/metabolismo , Articulação Temporomandibular , Gânglio Trigeminal/metabolismo , Animais , Artrite/induzido quimicamente , Artrite/patologia , Tamanho Celular , Adjuvante de Freund , Hiperalgesia/induzido quimicamente , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar , Articulação Temporomandibular/inervação , Gânglio Trigeminal/patologiaRESUMO
The innate immune system recognizes influenza A virus via TLR 7 or retinoic acid-inducible gene I in a cell-type specific manner in vitro, however, physiological function(s) of the MyD88- or interferon-beta promoter stimulator 1 (IPS-1)-dependent signaling pathways in antiviral responses in vivo remain unclear. In this study, we show that although either MyD88- or IPS-1-signaling pathway was sufficient to control initial antiviral responses to intranasal influenza A virus infection, mice lacking both pathways failed to show antiviral responses, resulting in increased viral load in the lung. By contrast, induction of B cells or CD4 T cells specific to the dominant hemagglutinin or nuclear protein Ags respectively, was strictly dependent on MyD88 signaling, but not IPS-1 signaling, whereas induction of nuclear protein Ag-specific CD8 T cells was not impaired in the absence of either MyD88 or IPS-1. Moreover, vaccination of TLR7- and MyD88-deficient mice with inactivated virus failed to confer protection against a lethal live virus challenge. These results strongly suggest that either the MyD88 or IPS-1 signaling pathway is sufficient for initial antiviral responses, whereas the protective adaptive immune responses to influenza A virus are governed by the TLR7-MyD88 pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Vacinação , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos/imunologia , Células Cultivadas , Imunidade Inata/imunologia , Interferon Tipo I/biossíntese , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Receptores de Superfície Celular , Receptores Toll-Like/deficiência , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Peripheral nerve injury activates satellite cells to produce interleukin 1beta (IL-1beta) which mediates inflammation and hyperalgesia. This study investigated the hypothesis that activation of satellite glial cells modulates the excitability of trigeminal ganglion (TRG) neurons via IL-1beta following inflammation. Inflammation was induced by injection of complete Freund's adjuvant (CFA) into the whisker pad area. The threshold for escape from mechanical stimulation applied to the whisker pad in inflamed rats was significantly lower than that in control. Two days post-CFA injection, the mean percentage of TRG neurons encircled by glial fibrillary acidic protein (GFAP)-/IL-1beta-immunoreactive cells was significantly increased compared to controls. GFAP and IL-1beta immunoreactivities were coexpressed in the same cells. Fluorogold (FG) labeling identified the site of inflammation. The number of FG-labeled IL-receptor type I (IL-1RI) TRG neurons in inflamed rats was significantly greater than in controls. In FG-labeled small TRG neurons, the size of IL-1beta (1 nM) induced-depolarization in inflamed rats was larger than in controls. IL-1beta application significantly increased firing rates evoked by depolarizing pulses in the neurons of inflamed rats, compared to controls. The response to IL-1beta was abolished by treatment with the IL-1RI antagonist. These results suggest that activation of satellite glial cells modulates the excitability of small-diameter TRG neurons via IL-1beta following inflammation, and that the upregulation of IL-1RI in the soma may contribute to the mechanism underlying inflammatory hyperalgesia. Therefore IL-1beta blockers are potential therapeutic agents for prevention of trigeminal hyperalgesia.
Assuntos
Citocinas/farmacologia , Neuroglia/química , Neurônios/fisiologia , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Gânglio Trigeminal/patologia , Animais , Reação de Fuga/efeitos dos fármacos , Reação de Fuga/fisiologia , Adjuvante de Freund , Lateralidade Funcional , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/induzido quimicamente , Inflamação/complicações , Masculino , Potenciais da Membrana/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Doenças do Sistema Nervoso Periférico/etiologia , Ratos , Ratos Wistar , Limiar Sensorial/efeitos dos fármacos , Limiar Sensorial/fisiologia , Pele/inervação , Estilbamidinas/metabolismoRESUMO
We examined whether 8-bromo-cAMP (8-Br-cAMP)-induced modification of tetrodotoxin-resistant (TTX-R) sodium current in neonatal rat nodose ganglion neurons is mediated by the activation of protein kinase A (PKA) and/or protein kinase C (PKC). In 8-Br-cAMP applications ranging from 0.001 to 1.0mM, 8-Br-cAMP at 0.1mM showed a maximal increase in the peak TTX-R Na(+) (Nav1.8) current and produced a hyperpolarizing shift in the conductance-voltage (G-V) curve. The PKC inhibitor bisindolylmaleimide Ro-31-8425 (Ro-31-8425, 0.5microM) decreased the peak Nav 1.8 current. The Ro-31-8425-induced modulation of the G(V)(1/2) baseline (a percent change in G at baseline V1/2) was not affected by additional 8-Br-cAMP application (0.1mM). The maximal increase in Nav 1.8 currents was seen at 0.1microM after the application of a PKC activator, phorbol 12-myristate 13-acetate (PMA) and forskolin. The PMA-induced increase in Nav 1.8 currents was not significantly affected by additional 0.1mM 8-Br-cAMP application. Intracellular application of a PKA inhibitor, protein kinase inhibitor (PKI, 0.01mM), inhibited the baseline Nav 1.8 current, significantly attenuated the 8-Br-cAMP-and PMA-induced increase in the peak Nav 1.8 current, and caused a significant increase in the slope factor of the inactivation curve. The PKI application at a higher concentration (0.5mM) greatly inhibited the PMA (0.1microM)-induced increase in the peak Nav 1.8 current amplitude and further enhanced the Ro-31-8425-induced decrease in the current. These results suggest that the 8-Br-cAMP-induced increase in Nav 1.8 currents may be mediated by activation of both PKA and PKC.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/efeitos dos fármacos , Gânglio Nodoso/citologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/fisiologia , Tetrodotoxina/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Expressão Gênica/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.8 , Inibição Neural/efeitos dos fármacos , Inibição Neural/efeitos da radiação , Neurônios/citologia , Técnicas de Patch-Clamp , RNA Mensageiro/biossíntese , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The aim of the present study was to determine whether there is a convergence of inputs from tooth pulp (TP) and the superior sagittal sinus (SSS) on rat C1 spinal neurons, and to examine the effects of iontophoretically applied N-methyl-D: -aspartate (NMDA) and non-NMDA receptor antagonists on the SSS-evoked activity of C1 neurons. Extracellular single unit-recordings were made from 20 C1 units responding to TP electrical stimulation with a constant temporal relationship to a digastric electromyogram signal, using a multibarrel electrode in pentobarbital-anesthetized rats. Ninety percent of C1 neurons (18/20) responding to TP stimulation also responded to the SSS stimulation. These neurons were considered to be SSS-afferent inputs from Adelta-fibers (5.8 +/- 0.6 m/s; n = 18), based on the calculation of nerve conduction velocity. After the iontophoretic application (30, 50, and 70 nA) of an NMDA receptor blocker (5R-10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cycloheptene-5,10-imine hydrogen maleate (MK801) or a non-NMDA receptor blocker (6-cyano-7-nitroquinoxaline-2,3-dione) (CNQX), the mean number of spikes responding to the SSS stimulation significantly decreased (30, 50, and 70 nA; P < 0.05). These results suggest that there is a convergence of inputs from SSS and TP afferents on C1 neurons; it is possible that both NMDA and non-NMDA receptors located on C1 neurons may be targets for the treatment of the trigeminal referred pain associated with migraine.
Assuntos
Polpa Dentária/inervação , Dura-Máter/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Dor Referida/fisiopatologia , Células do Corno Posterior/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Odontalgia/fisiopatologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Maleato de Dizocilpina/farmacologia , Estimulação Elétrica , Eletromiografia , Iontoforese , Masculino , Músculos do Pescoço/inervação , Nociceptores/efeitos dos fármacos , Nociceptores/fisiologia , Ratos , Ratos Wistar , Neuralgia do Trigêmeo/fisiopatologiaRESUMO
The effect of iontophoretic application of the 5-HT3 receptor agonist, phenylbiguanide (PBG), on the excitation of the trigeminal spinal nucleus oralis (TSNO) neurons to tooth-pulp (TP) stimulation was examined. The PBG application inhibited the TP-evoked TSNO neuronal excitation, and this inhibition was completely blocked by co-application of a GABAA receptor antagonist, bicuculline. The results suggest that the activation of 5-HT3 receptors elicits GABA release in the TSNO.
Assuntos
Polpa Dentária/inervação , Interneurônios/fisiologia , Receptores 5-HT3 de Serotonina/fisiologia , Núcleo Espinal do Trigêmeo/citologia , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bicuculina/farmacologia , Biguanidas/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Antagonistas GABAérgicos/farmacologia , Indóis/farmacologia , Interneurônios/efeitos dos fármacos , Iontoforese/métodos , Masculino , Ratos , Ratos Wistar , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , TropizetronaRESUMO
The combined effects of ouabain (Na(+)-K(+) ATPase inhibitor) and hyperinflation (inflation volume=three tidal volumes) on slowly adapting pulmonary stretch receptors (SARs) were studied before and after administration of nifedipine (an L-type Ca(2+) channel blocker) and KB-R7943 (a reverse-mode Na(+)-Ca(2+) exchanger blocker) in anesthetized, artificially ventilated rabbits after bilateral vagotomy. Before ouabain administration, hyperinflation stimulated SAR activity. After 20 min of ouabain administration (30 microg/kg) the SARs increased discharge rates in normal inflation. Under these conditions, hyperinflation initially stimulated SAR activity but subsequently inhibited the activity at peak inflation. Additional administration of 60 microg/kg ouabain (total dose=90 microg/kg) caused a further stimulation of SAR activity, but 20 min later both normal inflation and hyperinflation resulted in a greater inhibition of the receptor activity. The hyperinflation-induced SAR inhibition in the presence of ouabain (30 microg/kg) was not significantly altered by administration of either nifedipine (2 and 4 mg/kg) or KB-R7943 (1 and 3 mg/kg). In another series of experiments, we further examined the combined effects of ouabain and hyperinflation in veratridine (a Na(+) channel opener, 40 microg/kg)-treated animals. After recovery from the veratridine effect on SAR activity, which vigorously stimulated the receptor activity, ouabain treatment (30 microg/kg) that silenced the receptor activity at peak inflation greatly inhibited hyperinflation-induced SAR stimulation. These results suggest that hyperinflation-induced SAR inhibition in the presence of ouabain may be related to a Na(+) overload, but not to a Ca(2+) influx via activation of L-type Ca(2+) channels, in the SAR endings.