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1.
PLoS One ; 15(4): e0232042, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324792

RESUMO

Forcipomyia taiwana is a tiny hematophagous midge that attacks en masse. It is responsible for the most prevalent biting insect allergy in Taiwan. For t 2 is its major allergen. The intense itchy reactions can prevent allergic individuals from performing their regular daily outdoor activities. This study aimed to investigate whether the For t 2 DNA vaccine was effective in treating mice with established biting midge allergy. Mice were sensitized with recombinant For t 2 proteins or whole midge extracts. Two to four consecutive shots of various concentrations of For t 2 DNA vaccine, with or without CpG adjuvants, were then administered to midge-sensitized mice. Mice that received two shots of 50-100 µg For t 2 DNA vaccine showed a significant reduction in allergen-induced bouts of scratching, For t 2-specific IgE, specific IgG1/IgG2a ratio in sera, skin eosinophil infiltration, and IL-31 production, as well as IL-4 and IL-13 production by splenocytes. Two doses of For t 2 DNA vaccine one week apart was sufficient to treat mice with established biting midge allergy. The treatment resulted in clinical, immunological, and histopathological improvements. We recommend that this low-cost, convenient treatment strategy be developed for use in humans who are allergic to biting midges.


Assuntos
Ceratopogonidae/imunologia , Hipersensibilidade/tratamento farmacológico , Mordeduras e Picadas de Insetos/tratamento farmacológico , Proteínas de Insetos/genética , Prurido/tratamento farmacológico , Vacinas de DNA/administração & dosagem , Animais , Modelos Animais de Doenças , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Hipersensibilidade/etiologia , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Mordeduras e Picadas de Insetos/imunologia , Proteínas de Insetos/imunologia , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Prurido/imunologia , Taiwan , Vacinas de DNA/imunologia
2.
Gene ; 742: 144583, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32184167

RESUMO

BACKGROUND: Studies showed that increased let-7b-5p microRNA during repeated electroacupuncture (EA) treatment was associated the formation of EA tolerance, which manifested as gradually decreased nociceptive threshold. Proenkephalin (PENK) is the precursor of enkephalin which is a pivot neuropeptide responsible for the decreased nociceptive threshold in EA. The aim of this study was to evaluate the relationship between let-7b-5p and PENK in EA tolerance. METHODS: The target gene of let-7b-5p microRNA was determined through the dual-luciferase reporter assay in cortical neurons. Seventy-two Sprague Dawley rats received a combination of EA and intracerebroventricular injection of microRNA (let-7b-5p agomir, antagomir or their controls). The nociceptive thresholds were assessed with radiant heat tail-flick method. PENK and let-7b-5p were measured with Western Blot and qPCR, respectively, after administration of let-7b-5p agomir, antagomir, and their controls at day 1, 4 and 7. RESULTS: Let-7b-5p targeted the 3' untranslated region of Penk1. The nociceptive thresholds in Let-7b-5p agomir + EA group were decreased (p < 0.05) compared with those in Let-7b-5p antagomir + EA group at day 1 to 7. Compared with Let-7b-5p agomir + EA group, the expression level of PENK in Let-7b-5p antagomir + EA group was increased at days 1, 4, and 7 (p < 0.05) CONCLUSION: Let-7b-5p may be a new potential target for decreasing the EA tolerance effect and facilitating the application of EA in treating chronic nociception of patients.


Assuntos
Eletroacupuntura , Encefalinas/genética , MicroRNAs/metabolismo , Dor Nociceptiva/terapia , Precursores de Proteínas/genética , Animais , Antagomirs/administração & dosagem , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Humanos , Injeções Intraventriculares , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Nociceptividade/efeitos dos fármacos , Dor Nociceptiva/diagnóstico , Dor Nociceptiva/genética , Dor Nociceptiva/imunologia , Limiar da Dor/efeitos dos fármacos , Ratos
3.
Artigo em Inglês | MEDLINE | ID: mdl-31747539

RESUMO

Rheumatoid arthritis (RA) has a negative impact on muscle mass, and reduces patient's mobility and autonomy. Furthermore, RA is associated with metabolic comorbidities, notably in lipid homeostasis by unknown mechanisms. To understand the links between the loss in muscle mass and the metabolic abnormalities, arthritis was induced in male Sprague Dawley rats (n = 11) using the collagen-induced arthritis model. Rats immunized with bovine type II collagen were compared to a control group of animals (n = 11) injected with acetic acid and complete Freund's adjuvant. The clinical severity of the ensuing arthritis was evaluated weekly by a semi-quantitative score. Skeletal muscles from the hind limb were used for the histological analysis and exploration of mitochondrial activity, lipid accumulation, metabolism and regenerative capacities. A significant atrophy in tibialis anterior muscle fibers was observed in the arthritic rats despite a non-significant decrease in the weight of the muscles. Despite moderate inflammation, accumulation of triglycerides (P < 0.05), reduced mitochondrial DNA copy number (P < 0.05) and non-significant dysfunction in mitochondrial cytochrome c oxidase activity were found in the gastrocnemius muscle. Concomitantly, our results suggested an activation of the muscle specific E3 ubiquitin ligases MuRF-1 and MAFbx. Finally, the adipose tissue from the arthritic rats exhibited decreased PPARγ mRNA suggesting reduced adipogenic capacities. In conclusion, the reduced adipose tissue adipogenic capacity and skeletal muscle mitochondrial capacity are probably involved in the activation of protein catabolism, inhibition of myogenesis, accumulation of lipids and fiber atrophy in the skeletal muscle during RA.


Assuntos
Artrite Experimental/complicações , Artrite Reumatoide/complicações , Mitocôndrias/patologia , Atrofia Muscular/metabolismo , Triglicerídeos/metabolismo , Tecido Adiposo/metabolismo , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Colágeno Tipo II/administração & dosagem , Colágeno Tipo II/imunologia , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Humanos , Masculino , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , PPAR gama/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
4.
J Pharm Biomed Anal ; 177: 112856, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31521020

RESUMO

Daphne genkwa Sieb. et Zucc., as a traditional oriental herb, has been widely distributed and employed in China. The major bioactive components in D. genkwa are flavonoid compounds, which showed pharmacological activities such as anti-inflammatory, analgesic, anti-tumor and immunomodulatory activities. In this study, we analyzed total flavonoids in D. genkwa and their metabolites in normal and adjuvant arthritis (AA) rat plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). A total of 4 metabolites in plasma, 9 metabolites in urine and 15 metabolites in feces were characterized respectively by LC-Q-TOF-MS technology in normal rat. And 9 of the metabolites were observed in the AA rat urine, while there was no prototype drug or its metabolites detected in plasma and fecal samples. The metabolic pathway mainly involves hydroxylation, methylation, glucuronide, sulfate conjugation, oxidation and reduction, during the phase I and phase II biotransformation pathway. All the information gained here will be greatly helpful in elucidating the potential biological and pharmacological mechanism of flavonoid in D. genkwa, thus providing new ideas for drug development.


Assuntos
Artrite Experimental/tratamento farmacológico , Daphne/química , Medicamentos de Ervas Chinesas/farmacocinética , Flavonoides/farmacocinética , Administração Oral , Animais , Artrite Experimental/sangue , Artrite Experimental/imunologia , Artrite Experimental/urina , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Fezes/química , Flavonoides/administração & dosagem , Flavonoides/química , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Glucuronosiltransferase/metabolismo , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Distribuição Tecidual
5.
J Immunol Methods ; 474: 112670, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31525365

RESUMO

Moraea pallida Bak. (yellow tulp) poisoning is the most important plant cardiac glycoside toxicosis in South Africa. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. The aim was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine, proscillaridin and bufalin, were successfully conjugated to hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH). There was a low immune response following vaccination of adult male New Zealand White rabbits with epoxyscillirosidine-OVA (n = 3) and OVA (n = 3) using Freund's adjuvant in Trial (T) 1. The immune response improved significantly in T2 following doubling of the dose to 0.8 mg/rabbit and changing the adjuvant to Montanide. In T3, the rabbits (n = 15), allocated into 5 equal groups, vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA and BSA respectively, using Montanide adjuvant, developed antibodies against the administered immunogens, with epoxyscillirosidine-KLH inducing the highest immune response. Proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine in an enzyme linked immunosorbent assay. The conjugation methodology will be adjusted in the future to target optimal conjugation efficiency. Additional vaccination will be conducted in search of neutralizing antibodies against the yellow tulp toxin. The cross-reactivity of proscillaridin and bufalin antibodies with epoxyscillirosidine could be studied in future to explore the potential to prevent yellow tulp poisoning.


Assuntos
Anticorpos Neutralizantes/sangue , Colenos/imunologia , Iridaceae/imunologia , Extratos Vegetais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Especificidade de Anticorpos , Colenos/administração & dosagem , Colenos/envenenamento , Reações Cruzadas , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Hemocianinas/administração & dosagem , Hemocianinas/imunologia , Iridaceae/envenenamento , Masculino , Manitol/administração & dosagem , Manitol/análogos & derivados , Manitol/imunologia , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/imunologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/envenenamento , Envenenamento/imunologia , Envenenamento/prevenção & controle , Coelhos , Vacinação
6.
J Ethnopharmacol ; 236: 183-195, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-30849505

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Paullinia pinnata L. (Sapindaceae) is an African woody vine, traditionally used for the treatment of itch and pain-related conditions such as rheumatoid arthritis. AIM: This work evaluates, in vitro and in vivo, the anti-inflammatory and analgesic effects of aqueous (AEPP) and methanol (MEPP) extracts from Paullinia pinnata leaves. METHODS: AEPP and MEPP (100, 200 and 300 mg/kg/day) were administered orally in monoarthritic rats induced by a unilateral injection of 50 µl of Complete Freund's Adjuvant (CFA) in the ankle joint. During the 14 days of treatment, pain and inflammation were evaluated alternatively in both ankle and paw of the CFA-injected leg. Malondialdehyde (MDA) and glutathione (GSH) levels were assessed in serum and spinal cord. Histology of soft tissue of the ankle was also analyzed. For in vitro studies, AEPP and MEPP (10, 30 and 100 µg/ml) were evaluated against nitric oxide (NO) production by macrophages that were either non-stimulated or stimulated with LPS, 8-Br-AMPc and the mixture of both substances after 8 h exposure. These extracts were also evaluated on TNF-α and IL-1ß production in cells stimulated with LPS for 8 h. Finally, the ability of the extracts to bind to neuroactive receptors was evaluated in vitro using competitive binding assays with >45 molecular targets. RESULTS: AEPP and MEPP significantly reduced by 20-98% (p < 0.001) the inflammation and pain sensation in both the ankle and paw. AEPP significantly increased glutathione levels (p < 0.05) in serum. Both extracts reduced MDA production in serum and spinal cord (p < 0.001), and significantly improved tissue reorganization in treated arthritic rats. P. pinnata extracts did not affect NO production in non-stimulated macrophages but significantly reduced it by 47-88% in stimulated macrophages. AEPP and MEPP also significantly inhibited TNF-α (35-68%) and IL-1ß (31-36%) production in LPS stimulated macrophages. No cytotoxic effect of plant extracts was observed. MEPP showed concentration-dependent affinity for Sigma 2 receptors with an IC50 of 50 µg/ml. CONCLUSION: These results demonstrate the analgesic and anti-inflammatory effects of P. pinnata extracts on monoarthritis and further support its traditional use for pain and inflammation. These activities are at least partly due to the ability of these extracts to inhibit the production of NO, TNF-α, IL-1ß and to likely modulate Sigma 2 receptors.


Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Artralgia/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Medicina Tradicional Africana/métodos , Paullinia/química , Extratos Vegetais/farmacologia , Analgésicos/isolamento & purificação , Analgésicos/uso terapêutico , Animais , Articulação do Tornozelo/efeitos dos fármacos , Articulação do Tornozelo/imunologia , Articulação do Tornozelo/patologia , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/uso terapêutico , Artralgia/etiologia , Artralgia/patologia , Artrite Experimental/complicações , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/complicações , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Humanos , Masculino , Metanol/química , Óxido Nítrico/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Ratos , Ratos Wistar , Resultado do Tratamento , Água/química
7.
Int Immunopharmacol ; 70: 225-234, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30851702

RESUMO

Thymulin is a peptide hormone which is mainly produced by thymic epithelial cells and it has immune-modulatory and anti-inflammatory effects. In this study, we investigated the effects of different doses and various timings of thymulin intraperitoneal administration on spinal microglial activity and intracellular pathways in an inflammatory rat model of Complete Freund's adjuvant (CFA). Thymulin treatment was implemented following CFA-induced inflammation for 21 days. After conducting behavioral tests (edema and hyperalgesia), the cellular and molecular aspects were examined to detect the thymulin effect on inflammatory factors and microglial activity. We demonstrated that thymulin treatment notably reduced thermal hyperalgesia and paw edema induced by CFA. Furthermore, molecular investigations showed that thymulin reduced CFA-induced activation of microglia cells, phosphorylation of p38 MAPK and the production of spinal pro-inflammatory cytokines (TNF-α, IL-6) during the study. Our results suggest that thymulin treatment attenuates CFA-induced inflammation. This effect may be mediated by inhibition of spinal microglia and production of central inflammatory mediators which seems to be associated with the ability of thymulin to reduce p38 MAPK phosphorylation. These data provide evidence of the anti-hyperalgesic effect of thymulin on inflammatory pain and characterize some of the underlying spinal mechanisms.


Assuntos
Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Microglia/fisiologia , Dor/tratamento farmacológico , Medula Espinal/patologia , Fator Tímico Circulante/uso terapêutico , Animais , Modelos Animais de Doenças , Adjuvante de Freund/imunologia , Humanos , Injeções Intraperitoneais , Interleucina-6/metabolismo , Masculino , Microglia/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Nat Immunol ; 20(2): 195-205, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30643267

RESUMO

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.


Assuntos
Diferenciação Celular/imunologia , Encefalomielite Autoimune Experimental/imunologia , Células Progenitoras Linfoides/fisiologia , Linfócitos T Reguladores/fisiologia , Timo/crescimento & desenvolvimento , Animais , Autoantígenos/imunologia , Colite/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Humanos , Tolerância Imunológica/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células Progenitoras Linfoides/transplante , Camundongos , Camundongos Transgênicos , Mycobacterium tuberculosis/imunologia , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Transdução de Sinais , Organismos Livres de Patógenos Específicos , Timo/citologia , Timo/imunologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-30457056

RESUMO

BACKGROUND: Garcinia indica also known as kokum is used in traditional system of medicine for relieving inflammation and rheumatic pain. Garcinol, a benzophenone obtained from its fruit rind is reported to have anti-inflammatory effect via modulating arachidonic acid metabolism, suppressing iNOS expression, NF-κB activation and COX-2 expression. It has also been studied for antioxidant and anticancer activity. Apart from these, few patents claim that garcinol also has anti-obesity and hepatoprotective effect and has a potential to be used for the treatment of renal disorders, endometriosis and cardiac dysfunction. OBJECTIVE: Garcinol Enriched Fraction (GEF) from the fruit rind of Garcinia indica should be effective in the treatment of arthritis, one of the chronic inflammatory disorder owing to its anti-inflammatory property as indicated by earlier experiments. METHODS: GEF was prepared from the fruit rind of Garcinia indica and quantified using LC-MS/MS. It was found to contain 89.4% w/w of garcinol. GEF was evaluated at the dose of 10mg/kg for its efficacy against Complete Freund's Adjuvant (CFA) induced arthritis in Wistar albino rats. Paw volumes of both sides were measured by Plethysmometer and body weight was recorded on 0, 1, 5, 12 and 21st day. The hyperalgesic response was also measured by motility test and stair climbing test. RESULTS: GEF showed a significant reduction in paw swelling (p < 0.0001) and arthritis index (p < 0.0001) exhibiting anti-inflammatory potential. It also improves the motility and stair climbing ability of experimental animals (p < 0.05), thus reducing hyperalgesia. CONCLUSION: Garcinol enriched fraction shows anti-arthritic activity in experimental animals.


Assuntos
Anti-Inflamatórios/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Experimental/terapia , Artrite Reumatoide/terapia , Hiperalgesia/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Terpenos/uso terapêutico , Animais , Modelos Animais de Doenças , Adjuvante de Freund/imunologia , Garcinia/imunologia , Humanos , Masculino , Ratos , Ratos Wistar
10.
Monoclon Antib Immunodiagn Immunother ; 37(5): 195-199, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30281392

RESUMO

Adjuvant can play an important role in vaccine formulation by aiding in the development of a robust immune response. In our hybridoma development work, we typically use both Freund's and AddaVax™ adjuvant regimens for mouse immunizations. While we have repeatedly shown success with our protocols, we continually seek to improve upon the titer and affinity of the serum antibody response. To that end, we evaluated the use of CpG oligodeoxynucleotides (CpG-DNA), a B cell stimulant, in our adjuvant regimens. Mice were immunized using our standard Freund's protocol (Adjulite Complete Freund's Adjuvant for the primary immunization followed by Adjulite Incomplete Freund's Adjuvant (AIFA) for all additional immunizations) or a test protocol using AIFA supplemented with CpG-DNA for all immunizations. A second group of mice were immunized with antigen emulsified in AddaVax adjuvant alone or AddaVax supplemented with CpG-DNA. Our results show a trend toward a higher titer response when CpG-DNA was used with either adjuvant. In addition, AIFA+CpG-DNA mice trended toward a higher relative affinity versus mice immunized using our standard Freund's methodology. Additional antigens will need to be studied to determine whether these observations are limited to the proteins (antigens) studied or whether this is a generalized response to any immunogen.


Assuntos
Hibridomas/imunologia , Oligodesoxirribonucleotídeos/imunologia , Esqualeno/imunologia , Animais , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Lipídeos/administração & dosagem , Lipídeos/imunologia , Camundongos , Oligodesoxirribonucleotídeos/administração & dosagem , Polissorbatos/administração & dosagem , Esqualeno/administração & dosagem , Vacinação
11.
Nat Commun ; 9(1): 3151, 2018 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-30089795

RESUMO

γδ T cells have many known functions, including the regulation of antibody responses. However, how γδ T cells control humoral immunity remains elusive. Here we show that complete Freund's adjuvant (CFA), but not alum, immunization induces a subpopulation of CXCR5-expressing γδ T cells in the draining lymph nodes. TCRγδ+CXCR5+ cells present antigens to, and induce CXCR5 on, CD4 T cells by releasing Wnt ligands to initiate the T follicular helper (Tfh) cell program. Accordingly, TCRδ-/- mice have impaired germinal center formation, inefficient Tfh cell differentiation, and reduced serum levels of chicken ovalbumin (OVA)-specific antibodies after CFA/OVA immunization. In a mouse model of lupus, TCRδ-/- mice develop milder glomerulonephritis, consistent with decreased serum levels of lupus-related autoantibodies, when compared with wild type mice. Thus, modulation of the γδ T cell-dependent humoral immune response may provide a novel therapy approach for the treatment of antibody-mediated autoimmunity.


Assuntos
Diferenciação Celular , Imunidade Humoral/imunologia , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/fisiologia , Compostos de Alúmen , Animais , Formação de Anticorpos , Autoanticorpos/sangue , Galinhas , Feminino , Adjuvante de Freund/imunologia , Glomerulonefrite , Imunização , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Linfonodos/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes/imunologia , Modelos Animais , Modelos Imunológicos , Ovalbumina/sangue , Ovalbumina/imunologia , Receptores CXCR5/metabolismo
12.
Infect Immun ; 86(10)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30104212

RESUMO

Novel adjuvants are in demand for improving the efficacy of human vaccines. The immunomodulatory properties of Mycobacterium tuberculosis cell wall components have been highlighted in the formulation of complete Freund's adjuvant (CFA). We have explored the adjuvant potential of poly-α-l-glutamine (PLG), a lesser-known constituent of the pathogenic mycobacterial cell wall. Immune parameters indicated that the adjuvant potency of PLG was statistically comparable to that of CFA and better than that of alum in the context of H1 antigen (Ag85B and ESAT-6 fusion). At 1 mg/dose, PLG augmented the immune response of Ag85B, BP26, and protective antigen (PA) by increasing serum antibodies and cytokines in the culture supernatant of antigen-stimulated splenocytes. PLG modulated the humoral response of vaccine candidate ESAT-6, eliciting significantly higher levels of total IgG and isotypes (IgG1, IgG2a, and IgG2b). Additionally, the splenocytes from PLG-adjuvanted mice displayed a robust increase in the Th1-specific gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), Th2-specific IL-6 and IL-10, and Th17-specific IL-17A cytokines upon antigenic stimulation. PLG improved the protective efficacy of ESAT-6 by reducing bacillary load in the lung and spleen as well as granuloma formation, and it helped in maintaining vital health parameters of mice challenged with M. tuberculosis The median survival time of PLG-adjuvanted mice was 205 days, compared to 146 days for dimethyl-dioctadecyl ammonium bromide-monophosphoryl lipid A (DDA-MPL)-vaccinated groups and 224 days for Mycobacterium bovis BCG-vaccinated groups. PLG enhanced the efficiency of the ESAT-6 vaccine to the level of BCG and better than that of DDA-MPL (P < 0.05), with no ill effect in C57BL/6J mice. Our results propose that PLG is a promising adjuvant candidate for advanced experimentation.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Parede Celular/imunologia , Mycobacterium tuberculosis/imunologia , Peptídeos/imunologia , Tuberculose/microbiologia , Aciltransferases/administração & dosagem , Aciltransferases/genética , Aciltransferases/imunologia , Animais , Anticorpos Antibacterianos , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Parede Celular/genética , Feminino , Adjuvante de Freund/imunologia , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Células Th1/imunologia , Tuberculose/genética , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia
13.
AAPS PharmSciTech ; 19(7): 3187-3198, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30143947

RESUMO

Celecoxib (CXB), a COX-2 inhibitor, is primarily indicated for long-term treatment of rheumatoid arthritis (RA). The effective therapeutic efficacy of CXB on RA via oral administration shows adverse systemic complications, and therefore, local application of CXB has been recommended. The aim of the present study was to develop and characterize solid lipid nanoparticles (SLNs) with enhanced skin permeation potential of CXB. The particle size, polydispersity index (PDI), and percentage drug entrapment (PDE) of the developed SLNs (CXB-SLNs) were found to be 240 nm, < 0.3, and ~ 86% respectively. The developed SLNs exhibited sustained release up to 70% at the end of 48 h. Drug permeation was found to be 45% for SLN gel and 31% for conventional gel. The dermatokinetic studies also confirmed enhanced permeation of CXB in the epidermis and dermis and revealed superiority of the developed SLN gel vis-à-vis the conventional gel. Further, in the CFA-induced arthritis rat model, % arthritis index (AI) of the CXB-SLN gel formulation was found to be very less (18.54%) as compared to untreated (187.34%) and conventional gel-treated (91.61%) animals. In conclusion, the current study can provide a suitable alternative for the development of an effective topical formulation of CXB in lipid nanocarriers.


Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Celecoxib/administração & dosagem , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Nanopartículas/administração & dosagem , Animais , Celecoxib/química , Celecoxib/farmacocinética , Portadores de Fármacos , Adjuvante de Freund/imunologia , Lipídeos/química , Masculino , Ratos , Ratos Wistar , Pele/metabolismo
14.
J Pharm Biomed Anal ; 158: 361-369, 2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-29935325

RESUMO

Prostate-Specific Antigen (PSA) is a crucial biomarker for screening prostate cancer, but a sensitive and selective immunosensor for rapid quantification of serum PSA remains to be developed. In this study, a sandwich pair of nanobodies (Nbs) (i.e., Nb2 and Nb40) against PSA surface antigen was obtained from an alpaca-derived immune phage display library. A sandwich-type immunosensor for the sensitive and selective detection of PSA in serum samples was ingeniously designed based on the pair of Nbs. The small size of Nb40 allowed high capture densities on the surface of reduced graphene oxide (rGO) nanocomposed with massive Au nanoparticles (rGO@AuNPs), which significantly improved the conductivity and provided a large area to anchor many primary antibodies. The secondary antibody Nb2 fused with streptavidin -binding peptide (SBP) cooperated with Nb40 for PSA sandwiching. Accompanying introduction of horseradish peroxidase-streptavidin (HRP-SA) coupled with Nb2-SBP, the faradaic current was linearly correlated with the logarithm of PSA concentration in a range of 0.1-100 ng mL-1. More importantly, this immunosensor exhibited excellent selectivity, stability, and reproducibility due to the sandwich pair Nbs. The proposed immunosensor was successfully applied in determining PSA in serum samples and could be used for the sensitive and specific detection of PSA.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Calicreínas/sangue , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Animais , Camelídeos Americanos , Adjuvante de Freund/imunologia , Ouro/química , Grafite , Humanos , Calicreínas/imunologia , Limite de Detecção , Masculino , Nanopartículas Metálicas/química , Biblioteca de Peptídeos , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Anticorpos de Domínio Único/imunologia
15.
Eur J Immunol ; 48(9): 1580-1587, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29932463

RESUMO

Vaccination with MHC-II-restricted peptides from Apolipoprotein B (ApoB) with complete and incomplete Freund's adjuvant (CFA/IFA) is known to protect mice from atherosclerosis. This vaccination induces antigen-specific IgG1 and IgG2c antibody responses and a robust CD4 T cell response in lymph nodes. However, CFA/IFA cannot be used in humans. To find a clinically applicable adjuvant, we tested the effect of vaccinating Apoe-deficient mice with ApoB peptide P6 (TGAYSNASSTESASY). In a broad screening experiment, Addavax, a squalene-based oil-in-water adjuvant similar to MF59, was the only adjuvant that showed similar efficacy as CFA/IFA. This was confirmed in a confirmation experiment for both the aortic arch and whole aorta analyzed by en face analysis after atherosclerotic lesion staining. Mechanistically, restimulated peritoneal cells from mice immunized with P6 in Addavax released significant amounts of IL-10. Unlike P6 in CFA/IFA, vaccination with P6 in Addavax did not induce any detectable IgG1 or IgG2c antibodies to P6. These data suggest that squalene-based adjuvants such as MF59 are good candidate adjuvants for developing a clinically effective atherosclerosis vaccine.


Assuntos
Adjuvantes Imunológicos/farmacologia , Apolipoproteínas B/imunologia , Aterosclerose/prevenção & controle , Polissorbatos/farmacologia , Esqualeno/farmacologia , Vacinas/imunologia , Animais , Apolipoproteínas B/administração & dosagem , Aterosclerose/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Imunoglobulina G/imunologia , Lipídeos/administração & dosagem , Lipídeos/imunologia , Camundongos , Camundongos Knockout , Vacinação
16.
Cell Physiol Biochem ; 47(2): 842-850, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29807368

RESUMO

BACKGROUND/AIMS: Paeoniflorin (PF) is known to have anti-inflammatory and paregoric effects, but the mechanism underlying its analgesic effect remains unclear. The aim of this study was to clarify the effect of PF on Freund's complete adjuvant (CFA)-induced inflammatory pain and explore the underlying molecular mechanism. METHODS: An inflammatory pain model was established by intraplantar injection of CFA in C57BL/6J mice. After intrathecal injection of PF daily for 8 consecutive days, thermal and mechanical withdrawal thresholds, the levels of inflammatory factors TNF-α, IL-1ß and IL-6, microglial activity, and the expression of Akt-NF-κB signaling pathway in the spinal cord tissue were detected by animal ethological test, cell culture, enzyme-linked immunosorbent assay, immunofluorescence histochemistry, and western blot. RESULTS: PF inhibited the spinal microglial activation in the CFA-induced pain model. The production of proinflammatory cytokines was decreased in the central nervous system after PF treatment both in vivo and in vitro. PF further displayed a remarkable effect on inhibiting the activation of Akt-NF-κB signaling pathway in vivo and in vitro. CONCLUSION: These results suggest that PF is a potential therapeutic agent for inflammatory pain and merits further investigation.


Assuntos
Anti-Inflamatórios/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Glucosídeos/farmacologia , Monoterpenos/farmacologia , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Adjuvante de Freund/imunologia , Glucosídeos/uso terapêutico , Inflamação , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Monoterpenos/uso terapêutico , Dor/patologia , Dor/prevenção & controle , Fator de Necrose Tumoral alfa/metabolismo
17.
Microb Pathog ; 119: 12-18, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29626658

RESUMO

Klebsiella pneumoniae is a Gram-negative bacterium that is increasingly reported as a serious nosocomial and community-acquired pathogen. In the current study, two K. pneumoniae antigens, OmpK17 and OmpK36, as well as their fusion protein cognate F36/17 were investigated as potential vaccine candidates in a murine infection model. Three immunoadjuvants, namely the Gram-positive Enhancer Matrix (GEM) adjuvant, synthetic hemozoin (Hz) adjuvant and incomplete Freund's adjuvant (IFA) were evaluated. Genes of OmpK17 and OmpK36 antigens as well as their fusion protein were cloned in Escherichia coli for recombinant expression. Mice were immunized thrice with the individual recombinant purified antigens adjuvanted with one of the three adjuvants. Two weeks after the last booster, animals were challenged with a lethal dose of K. pneumoniae and immune protection parameters were assessed. Animals immunized with GEM- or Hz-adjuvanted K. pneumoniae antigens did not show significant protection upon bacterial challenge. Animals immunized with subcutaneous IFA-adjuvanted antigens showed the best results with survival percentages of 50, 60 and 50% for groups immunized with OmpK17, OmpK36 and F36/17, respectively. Serum IgG1, rather than IgG2a, antibodies were the most prevalent following vaccination indicating bias towards T helper type 2 (Th2) immune response. Opsonophagocytic assays demonstrated significant percentage killing in case of animals immunized with IFA-adjuvanted antigens. Overall, OmpK17 and OmpK36 are promising vaccine antigens which are worthy of further optimization of the immunization conditions, particularly the used immunoadjuvants, in order to achieve full protection against K. pneumoniae.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Imunização , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/prevenção & controle , Klebsiella pneumoniae/imunologia , Porinas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Vacinas Bacterianas/administração & dosagem , Clonagem Molecular , Modelos Animais de Doenças , Escherichia coli/genética , Feminino , Adjuvante de Freund/imunologia , Hemeproteínas/imunologia , Imunoglobulina G/sangue , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/patogenicidade , Lipídeos/imunologia , Camundongos , Porinas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Taxa de Sobrevida , Vacinação
18.
Mol Immunol ; 99: 9-18, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29649688

RESUMO

Development of a safe and efficacious vaccine for brucellosis is a long standing challenge for scientists. Recognizing potential antigens towards developing vaccine candidate is crucial. Omp25c, a porin protein of Brucella, is a paralog of two previously identified promising vaccine candidates namely, Omp25 and Omp31, with notable sequence identity. Also, Omp25c is conserved in all major Brucella species. This highlights the possibility of employing this protein in multivalent subunit vaccine based approach of Brucella management. In this study, we were interested in examining the immunogenicity and protective efficacy of Omp25c against Brucella infections. Recombinant unlipidated form of this antigen (rOmp25c) produced, upon intraperitoneal immunization in BALB/c mice along with Freund's adjuvant, was confirmed to be highly immunogenic; leading to high IgG antibody titers during the study duration. The IgG2a/IgG2b ratio of anti-rOmp25c antibodies revealed elicitation of Th2 based humoral immunity. Lymphocyte proliferation study divulged induction of specific memory response and secretion of both Th1-type (IFN-γ, GM-CSF and TNF-α) and Th2-type cytokine (IL-5) from restimulated splenocytes of rOmp25c immunized mice. CD4 T-cell subpopulation was comparatively increased than total B cell subpopulation in case of immunized mice, indicating the induction of strong cell-mediated (Th1 biased) immunity than humoral (Th2) immunity. The collective Th1 plus Th2 immune response specific to rOmp25c could be the reason for protection against Brucella challenge observed in mice groups that was comparable with S19 vaccine strain. Thus, the study encourages rOmp25c as a potent candidate vaccine against brucellosis.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Brucella abortus/imunologia , Brucelose/imunologia , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Vacina contra Brucelose/imunologia , Modelos Animais de Doenças , Feminino , Adjuvante de Freund/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunidade Humoral/imunologia , Imunização/métodos , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa/imunologia , Vacinação/métodos
19.
Vaccine ; 36(17): 2314-2320, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29567034

RESUMO

Potent adjuvant systems are required for subunit and single antigen based vaccines to provide sufficient immunogenicity. Furthermore, adjuvants can reduce the required number of immunisations and the antigen amount. Squalene nanoemulsions, like MF59® and AddaVax™, are potent, safe and well characterised adjuvant systems and approved for use in humans. Here, we developed squalene containing solid lipid nanoparticles, which can be sterilised by steam sterilisation and stored as freeze-dried power together with a yeast-based vaccine. Detailed size measurements using dynamic and static light scattering were applied, as the immune stimulating effect of squalene emulsions is mainly dependent on the particle size. The size range of AddaVax™ (120-170 nm) was favoured for the developed squalene containing solid lipid nanoparticles. Differential scanning calorimetry (DSC) and H NMR studies were performed to characterise the interactions of the incorporated liquid squalene with the solid hard fat matrix. A homogeneous distribution as liquid domains in the solid glyceride structure was suggested for the liquid squalene. The developed adjuvant was compared with Freund's adjuvant and a commercially available squalene nanoemulsion in a vaccine trial in the mouse model with a yeast-based vaccine directed against the infectious bursal disease virus. All squalene-based adjuvants showed excellent biocompatibility and provided immune stimulating properties comparable to Freund's adjuvant.


Assuntos
Adjuvantes Imunológicos/química , Adjuvante de Freund/química , Lipídeos/química , Nanopartículas/química , Esqualeno/química , Vacinas/química , Fermento Seco/química , Animais , Infecções por Birnaviridae/imunologia , Emulsões/química , Feminino , Adjuvante de Freund/imunologia , Imunização/métodos , Vírus da Doença Infecciosa da Bursa/imunologia , Lipídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Esqualeno/imunologia , Vacinas/imunologia , Fermento Seco/imunologia
20.
Eur J Pharmacol ; 826: 85-95, 2018 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-29501867

RESUMO

Levetiracetam (LEV), a novel anti-epileptic drug that has been demonstrated with an anti-inflammatory effect, but the exact mechanisms of its action remain to be fully defined. The present study aimed to evaluate the possible effects of LEV on lipopolysaccharides (LPS)-induced Janus kinase-signal transducers and activators of transcription (JAK/STAT) as well as toll-like receptor 4 (TLR4)/ mitogen activated protein kinase (MAPK) signaling pathways activation in adjuvant induced arthritis (AIA). Rats were allocated into normal control, three arthritic control groups: Complete Freund's Adjuvant (CFA) (0.4 ml/3days/12days), LPS (100 µg/kg/day), CFA+LPS, and three treated groups: CFA+LEV, LPS+LEV and CFA+LPS+LEV. LEV was administered in a dose 50 mg/kg/day for 15 day. After 28 days, tissue samples were collected for assessment of phosphorylated JAK2, STAT3, TLR4, MAPK and cathepsin K quantitative expression in synovium. Additionally, Serum samples were used for biochemical evaluation of interleukin-6 (IL-6), interleukin-1beta (IL-1B), LPS, anti-citrullinated protein antibody (ACPA) and 8-hydroxydeoxyguanosine (8-OHdG). Histopathological and macroscopical examinations of joints were also performed to support our study. Results indicated that LEV exerted its anti-inflammatory effect through inhibiting LPS-dependent phosphorylation of JAK2/STAT3 signaling. It significantly suppressed TLR4 and MAPK expressions, thereby decreasing release of inflammatory cytokines IL-1ß, IL-6.LEV exhibited a potent inhibitory effect on cathepsin K and 8-OHdG parallel to confirmatory histopathological and macroscopical findings. In conclusion, LEV has a powerful therapeutic effect on adjuvant induced arthritis in rats and its mechanisms are strongly related to inhibiting excessive activation of JAK2-STAT3 and TLR4 pathways. This may add a new approach for treatment of RA.


Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Lipopolissacarídeos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Piracetam/análogos & derivados , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Catepsina K/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Adjuvante de Freund/imunologia , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Janus Quinase 2/metabolismo , Levetiracetam , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Piracetam/farmacologia , Piracetam/uso terapêutico , Ratos , Fator de Transcrição STAT3/metabolismo , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Receptor 4 Toll-Like/metabolismo
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