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1.
Chin Med Sci J ; 34(3): 199-204, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31601303

RESUMO

Objective Psoriasis is an immune-mediated inflammatory disease. Despite advances in the study of its pathogenesis, the exact development mechanism of psoriasis remains to be fully elucidated. Hyperproliferative epidermis plays a crucial role in psoriasis. This study aimed to investigate the effects of interleukin-36ß (IL-36ß) on keratinocyte dysfunction in vitro. Methods Human keratinocyte cell lines, HaCaT cells, were treated with 0 (control), 50 or 100 ng/ml IL-36ß respectively for 24 h. Cell viability was determined with a cell counting kit-8 assay. Flow cytometry was used to assess the effects of IL-36ß on apoptosis and cell cycle distribution. Expressions of the differentiation markers, such as keratin 10 and involucrin, were evaluated by quantitative real-time polymerase chain reaction (RT-qPCR). Expressions of the inflammatory cytokines, IL-1ß and IL-6 were tested by ELISA. Results CCK8 assay showed the survival rate had no significant difference between the control and treated group (P > 0.05). Flow cytometry analysis showed cell cycle arrest at S phase in the IL-36ß-treated groups compared with the control group (P < 0.05). RT-qPCR verified the decreased mRNA expressions of keratin 10 and involucrin in the IL-36ß-treated groups compared with the negative control (P < 0.01). ELISA showed 100 ng/ml IL-36ß enhanced levels of IL-1ß and IL-6 in culture supernatants of HaCaT cells compared with the negative control (P < 0.05). Conclusion Taken together, these findings suggest that IL-36ß could induce cell cycle arrest at S phase, inhibit keratin 10 and involucrin expressions and promote inflammatory activity in HaCaT cell lines.


Assuntos
Apoptose/imunologia , Diferenciação Celular/imunologia , Interleucina-1/imunologia , Queratinócitos/imunologia , Pontos de Checagem da Fase S do Ciclo Celular/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Queratina-10/imunologia , Queratinócitos/patologia , Precursores de Proteínas/imunologia
2.
PLoS One ; 14(9): e0221550, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31504041

RESUMO

HIV envelope protein (Env) is the sole target of broadly neutralizing antibodies (BNAbs) that are capable of neutralizing diverse strains of HIV. While BNAbs develop spontaneously in a subset of HIV-infected patients, efforts to design an envelope protein-based immunogen to elicit broadly neutralizing antibody responses have so far been unsuccessful. It is hypothesized that a primary barrier to eliciting BNAbs is the fact that HIV envelope proteins bind poorly to the germline-encoded unmutated common ancestor (UCA) precursors to BNAbs. To identify variant forms of Env with increased affinities for the UCA forms of BNAbs 4E10 and 10E8, which target the Membrane Proximal External Region (MPER) of Env, libraries of randomly mutated Env variants were expressed in a yeast surface display system and screened using fluorescence activated cell sorting for cells displaying variants with enhanced abilities to bind the UCA antibodies. Based on analyses of individual clones obtained from the screen and on next-generation sequencing of sorted libraries, distinct but partially overlapping sets of amino acid substitutions conferring enhanced UCA antibody binding were identified. These were particularly enriched in substitutions of arginine for highly conserved tryptophan residues. The UCA-binding variants also generally exhibited enhanced binding to the mature forms of anti-MPER antibodies. Mapping of the identified substitutions into available structures of Env suggest that they may act by destabilizing both the initial pre-fusion conformation and the six-helix bundle involved in fusion of the viral and cell membranes, as well as providing new or expanded epitopes with increased accessibility for the UCA antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos , Proteína gp41 do Envelope de HIV/imunologia , Mutação , Precursores de Proteínas/imunologia , Anticorpos Antivirais/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estabilidade Proteica
3.
Nat Commun ; 9(1): 5238, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30531969

RESUMO

The skin immune system must discriminate between innocuous antigens and pathogens. Antigen applied topically using a Viaskin® patch elicits immune tolerance that can suppress colitis and food allergy. Here we show how topical antigen is acquired and presented by dendritic cells in the skin. Topical antigen is acquired by Langerhans cells (LC) and CD11b+ cDC2s but not cDC1s, and both  LCs and CD11b+ cDC2s reaching the lymph node can prime T cells and expand LAP+ Tregs. However, LCs are neither required nor sufficient for T cell priming, and have no role in tolerance induction. Conversely, IRF-4-dependent cDC2s are required for T cell priming. Acquisition of antigen in the dermis, delivery to the draining lymph node, and generation of tolerance are all absent in hairless mice. These results indicate an important function for hair follicle niche and CD11b+ cDC2s in antigen acquisition, and in generation of primary immune tolerance to topical antigens.


Assuntos
Antígenos/imunologia , Células Dendríticas/imunologia , Folículo Piloso/imunologia , Linfócitos T Reguladores/imunologia , Animais , Apresentação do Antígeno/imunologia , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Células Dendríticas/metabolismo , Derme/citologia , Derme/imunologia , Derme/metabolismo , Folículo Piloso/metabolismo , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/imunologia , Peptídeos/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Pele/imunologia , Pele/metabolismo , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo
4.
Vaccine ; 36(52): 8008-8018, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30416020

RESUMO

We previously reported the development of an oral vaccine for diabetes based on live attenuated Salmonella-expressing preproinsulin (PPI) as the autoantigen. When combined with host cell-expressed TGFß, the vaccine prevented the onset of diabetes in non-obese diabetic (NOD) mice. Herein, we investigated factors that could affect vaccine efficacy including vaccination number, optimization of the autoantigen codon sequence, Salmonella SPI2-TTSS promoter/effector combinations, concurrent short-course low-dose anti-CD3. We also evaluated autoantigen GAD65 and cytokine IL10 treatment upon vaccine efficacy. T-cells we employed to elucidate the mechanism of the vaccine action. Our results showed that GAD65+TGFß or PPI+TGFß+IL10 prevented the onset of diabetes in the NOD mice and maintained glucose tolerance. However, increasing the number of vaccine doses, codon-optimization of the autoantigen(s) or use of other Salmonella promoter/effector combinations had no in vivo effect. Interestingly, two doses of vaccine (PPI+TGFß+IL10) combined with a sub-therapeutic dose of anti-CD3 prevented diabetes and decreased hyperglycemia in mice. The combined therapy also increased splenic Tregs and local Tregs in pancreatic lymph nodes (PLN) and increased regulatory (IL10 and IL2) but reduced inflammatory (IFNγ and TNFα) cytokines. Together, these results indicate that the combination of low vaccine dose number, less vaccine autoantigen expression and short-course low-dose anti-CD3 can increase regulatory mechanisms and suppress autoimmunity.


Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Imunoterapia/métodos , Insulina/imunologia , Precursores de Proteínas/imunologia , Animais , Autoantígenos/administração & dosagem , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Quimioterapia Combinada , Feminino , Insulina/genética , Interleucina-10/administração & dosagem , Interleucina-10/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Precursores de Proteínas/genética , Salmonella , Baço/imunologia , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/administração & dosagem , Fator de Crescimento Transformador beta/imunologia
5.
J Microbiol Biotechnol ; 28(8): 1376-1383, 2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-30301315

RESUMO

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity (KD) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Biblioteca de Peptídeos , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Bacteriófagos/genética , Genótipo , Células HEK293 , Células Hep G2 , Anticorpos Anti-Hepatite B/imunologia , Anticorpos Anti-Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Testes de Neutralização , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo
6.
Vaccine ; 36(38): 5789-5795, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30082163

RESUMO

Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S) envelope protein are currently used in universal vaccination programs and achieve protective immune response in more than 90% of recipients. However, new vaccination strategies are necessary for successful immunization of the remaining non- or low-responders. We have previously characterized a novel HBV chimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L) envelope protein (genotype D). The S/preS121-47 chimera produced in mammalian cells and Nicotiana benthamiana plants, induced a significantly stronger immune response in parenterally vaccinated mice than the S protein. Here we describe the transient expression of the S/preS121-47 antigen in an edible plant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral administration of adjuvant-free Lactuca sativa expressing the S/preS121-47 antigen, three times, at 1 µg/dose, was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies were able to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) more efficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results support the S/preS121-47 antigen as a promising candidate for future development as an edible HBV vaccine.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/prevenção & controle , Precursores de Proteínas/imunologia , Administração Oral , Animais , Linhagem Celular Tumoral , Feminino , Células Hep G2 , Vacinas contra Hepatite B/administração & dosagem , Humanos , Alface/genética , Alface/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Proteínas do Envelope Viral/imunologia
7.
PLoS One ; 13(6): e0199079, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29927948

RESUMO

Many neurodegenerative retinal diseases are treated with monoclonal antibodies (mAb) delivered by invasive intravitreal injection (IVT). In Diabetic Retinopathy there is a scarcity of effective agents that can be delivered using non-invasive methods, and there are significant challenges in the validation of novel therapeutic targets. ProNGF represents a potential novel target, and IVT administration of a function-blocking anti-proNGF mAb is therapeutic in a mouse model of DR. We therefore compared invasive IVT to less invasive systemic intravenous (IV) and local subconjunctival (SCJ) administration, for therapy of Diabetic Retinopathy. The IV and SCJ routes are safe, afford sustained pharmacokinetics and tissue penetration of anti-proNGF mAb, and result in long-term therapeutic efficacy that blocks retinal inflammation, edema, and neuronal death. SCJ may be a more convenient and less-invasive approach for ophthalmic use and may enable reduced frequency of intervention for the treatment of retinal pathologies.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Retinopatia Diabética/tratamento farmacológico , Fator de Crescimento Neural/imunologia , Precursores de Proteínas/imunologia , Administração Intravenosa , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Modelos Animais de Doenças , Injeções Intravítreas , Camundongos , Tomografia de Coerência Óptica , Resultado do Tratamento
8.
Sci Rep ; 8(1): 8021, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29789580

RESUMO

CD4+latency-associated peptide (LAP)+ T cells are a newly discovered T cell subset with suppressive function on immune responses. In this study, we investigate the role of CD4+LAP+ T cells on mice corneal allograft survival by down-regulating their expression using anti-LAP mAb. We show that a blockage of LAP leads to a decrease in the percentage of T cells expressing CD4+Foxp3+, CD4+GARP+, CD4+LAP+ and CD4+IL-10+ in the lymph nodes and spleens of mice undergoing orthotopic penetrating transplantation of corneal allograft, without affecting corneal graft survival. In addition, higher percentages of CD4+IFN-γ+ and CD4+IL-17A+ T cells in the lymph nodes and spleens, as well as TNF, IFN-γ, IL-17A and IL-6 levels in the aqueous humor, significantly increase in mice with rejected corneal grafts. The expression of TGF-ß1 decreases in corneal grafts during corneal rejection period. It is therefore possible that anti-LAP mAb can down-regulate the regulatory T cell subsets with its immunosuppressive effects. The rejection of corneal grafts seems to mainly be associated with the up-regulation of Th1 and Th17 cell subsets in peripheral lymph nodes.


Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Transplante de Córnea/métodos , Peptídeos/imunologia , Peptídeos/metabolismo , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Córnea/efeitos dos fármacos , Córnea/imunologia , Transplante de Córnea/efeitos adversos , Regulação para Baixo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Imunologia de Transplantes/efeitos dos fármacos , Transplante Homólogo
9.
Turk J Haematol ; 35(2): 116-121, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29589834

RESUMO

OBJECTIVE: Plasma cell dyscrasias (PCDs) are disorders of plasma cells having in common the production of a monoclonal M-protein. They include a spectrum of conditions that may represent a natural progression of the same disease from monoclonal gammopathy of unknown significance to asymptomatic and symptomatic multiple myeloma, plasma cell leukemia, and Waldenström's macroglobulinemia. In PCDs, the immune system is actively suppressed through the secretion of suppressive factors and the recruitment of immune suppressive subpopulations. In this study, we examined the expression of two subpopulations of cells with immunosuppressive activity, monocytic myeloid-derived suppressor cells (MDSCs) and monocytes expressing latency-associated peptide (LAP), in patients with different PCDs and in healthy volunteers. MATERIALS AND METHODS: A total of 27 consecutive patients with PCDs were included in this study. Nineteen healthy volunteers served as controls. RESULTS: We observed a hierarchical correlation between disease activity and the presence of monocytes with immunosuppressive activity. CONCLUSION: These results suggest that MDSCs and monocytes expressing LAP have diverging roles in PCDs and may perhaps serve as biomarkers of tumor activity and bulk.


Assuntos
Monócitos/patologia , Células Supressoras Mieloides/patologia , Paraproteinemias/patologia , Peptídeos/análise , Precursores de Proteínas/análise , Fator de Crescimento Transformador beta/análise , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Tolerância Imunológica , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Células Supressoras Mieloides/imunologia , Paraproteinemias/imunologia , Peptídeos/imunologia , Precursores de Proteínas/imunologia , Fator de Crescimento Transformador beta/imunologia
10.
Sci Rep ; 8(1): 3837, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29497069

RESUMO

The hepatitis B virus (HBV) infection is a critical health problem worldwide, and HBV preS1 is an important biomarker for monitoring HBV infection. Previously, we found that a murine monoclonal antibody, mAb-D8, targets the preS1 (aa91-107) fragment of HBV. To improve its performance, we prepared the single-chain variable region of mAb-D8 (scFvD8) and constructed the three-dimensional structure of the scFvD8-preS1 (aa91-107) complex by computer modelling. The affinity of scFvD8 was markedly increased by the introduction of mutations L96Tyr to Ser and H98Asp to Ser. Furthermore, a highly sensitive immunosensor was designed based on a proximity-dependent hybridization strategy in which the preS1 antigen competitively reacts with an antibody labelled with DNA, resulting in decreased proximity-dependent hybridization and increased electrochemical signal from the Fc fragment, which can be used for the quantisation of preS1. The results showed a wide detection range from 1 pM to 50 pM with a detection limit of 0.1 pM. The sensitivity and specificity of this immunosensor in clinical serum samples were 100% and 96%, respectively. This study provides a novel system based on proximity-dependent hybridization and the scFv antibody fragment for the rapid quantisation of antigens of interest with a high sensitivity.


Assuntos
Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/ultraestrutura , Precursores de Proteínas/imunologia , Precursores de Proteínas/ultraestrutura , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Humanos , Hibridomas/imunologia , Camundongos , Hibridização de Ácido Nucleico/métodos , Precursores de Proteínas/genética , Anticorpos de Cadeia Única/genética
11.
J Allergy Clin Immunol ; 142(2): 497-509.e9, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29361332

RESUMO

BACKGROUND: BM32 is a grass pollen allergy vaccine based on recombinant fusion proteins consisting of nonallergenic peptides from the IgE-binding sites of the 4 major grass pollen allergens and the hepatitis B preS protein. OBJECTIVE: We sought to study the safety and clinical efficacy of immunotherapy (allergen immunotherapy) with BM32 in patients with grass pollen-induced rhinitis and controlled asthma. METHODS: A double-blind, placebo-controlled, multicenter allergen immunotherapy field study was conducted for 2 grass pollen seasons. After a baseline season, subjects (n = 181) were randomized and received 3 preseasonal injections of either placebo (n = 58) or a low dose (80 µg, n = 60) or high dose (160 µg, n = 63) of BM32 in year 1, respectively, followed by a booster injection in autumn. In the second year, all actively treated subjects received 3 preseasonal injections of the BM32 low dose, and placebo-treated subjects continued with placebo. Clinical efficacy was assessed by using combined symptom medication scores, visual analog scales, Rhinoconjunctivitis Quality of Life Questionnaires, and asthma symptom scores. Adverse events were graded according to the European Academy of Allergy and Clinical Immunology. Allergen-specific antibodies were determined by using ELISA, ImmunoCAP, and ImmunoCAP ISAC. RESULTS: Although statistical significance regarding the primary end point was not reached, BM32-treated subjects, when compared with placebo-treated subjects, showed an improvement regarding symptom medication, visual analog scale, Rhinoconjunctivitis Quality of Life Questionnaire, and asthma symptom scores in both treatment years. This was accompanied by an induction of allergen-specific IgG without induction of allergen-specific IgE and a reduction in the seasonally induced increase in allergen-specific IgE levels in year 2. In the first year, more grade 2 reactions were observed in the active (n = 6) versus placebo (n = 1) groups, whereas there was almost no difference in the second year. CONCLUSIONS: Injections of BM32 induced allergen-specific IgG, improved clinical symptoms of seasonal grass pollen allergy, and were well tolerated.


Assuntos
Alérgenos/imunologia , Epitopos de Linfócito B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Pólen/imunologia , Precursores de Proteínas/imunologia , Rinite Alérgica Sazonal/imunologia , Vacinas/imunologia , Adolescente , Adulto , Alérgenos/genética , Dessensibilização Imunológica/métodos , Método Duplo-Cego , Epitopos de Linfócito B/genética , Feminino , Antígenos de Superfície da Hepatite B/genética , Humanos , Masculino , Pessoa de Meia-Idade , Efeito Placebo , Poaceae/imunologia , Pólen/genética , Precursores de Proteínas/genética , Resultado do Tratamento , Vacinação , Adulto Jovem
12.
Diabetes ; 67(4): 687-696, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29343547

RESUMO

The signal peptide region of preproinsulin (PPI) contains epitopes targeted by HLA-A-restricted (HLA-A0201, A2402) cytotoxic T cells as part of the pathogenesis of ß-cell destruction in type 1 diabetes. We extended the discovery of the PPI epitope to disease-associated HLA-B*1801 and HLA-B*3906 (risk) and HLA-A*1101 and HLA-B*3801 (protective) alleles, revealing that four of six alleles present epitopes derived from the signal peptide region. During cotranslational translocation of PPI, its signal peptide is cleaved and retained within the endoplasmic reticulum (ER) membrane, implying it is processed for immune recognition outside of the canonical proteasome-directed pathway. Using in vitro translocation assays with specific inhibitors and gene knockout in PPI-expressing target cells, we show that PPI signal peptide antigen processing requires signal peptide peptidase (SPP). The intramembrane protease SPP generates cytoplasm-proximal epitopes, which are transporter associated with antigen processing (TAP), ER-luminal epitopes, which are TAP independent, each presented by different HLA class I molecules and N-terminal trimmed by ER aminopeptidase 1 for optimal presentation. In vivo, TAP expression is significantly upregulated and correlated with HLA class I hyperexpression in insulin-containing islets of patients with type 1 diabetes. Thus, PPI signal peptide epitopes are processed by SPP and loaded for HLA-guided immune recognition via pathways that are enhanced during disease pathogenesis.


Assuntos
Ácido Aspártico Endopeptidases/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Insulina/imunologia , Precursores de Proteínas/imunologia , Linfócitos T Citotóxicos/imunologia , Aminopeptidases , Ácido Aspártico Endopeptidases/metabolismo , Linhagem Celular , Retículo Endoplasmático , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Antígeno HLA-A11/imunologia , Antígeno HLA-A2/imunologia , Antígeno HLA-A24/imunologia , Humanos , Técnicas In Vitro , Insulina/metabolismo , Antígenos de Histocompatibilidade Menor , Fatores de Proteção , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Fatores de Risco
13.
Virus Res ; 244: 213-217, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29196195

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals and is endemic in Africa, parts of South America and southern Asia. The causative agent, FMD virus (FMDV) is a member of the genus Aphthovirus, family Picornaviridae. Vaccines currently used against FMDV are chemically inactivated virus strains which are produced under high-level biocontainment facilities, thus raising their cost. The development of recombinant FMDV vaccines has focused predominantly on FMDV virus-like particle (VLP) subunit vaccines for which promising results have been achieved. These VLPs are attractive candidates because they avoid the use of live virus in production facilities, but conserve the complete repertoire of conformational epitopes of the virus. Recombinant FMDV VLPs are formed by the expression and assembly of the three structural proteins VP0, VP1 and VP3. This can be attained by co-expression of the three individual structural capsid proteins or by co-expression of the viral capsid precursor P1-2A together with the viral protease 3C. The latter proteolytically cleaves P1-2A into the respective structural proteins. These VLPS are produced in mammalian or insect cell culture systems, which are expensive and can be easily contaminated. Plants, such as Nicotiana benthamiana, potentially provide a more cost-effective and very highly scalable platform for recombinant protein and VLP production. In this study, P1-2A was transiently expressed in N. benthamiana alone, without the 3C protease. Surprisingly, there was efficient processing of the P1-2A polyprotein into its component structural proteins, and subsequent assembly into VLPs. The yield was ∼0.030µg per gram of fresh leaf material. Partially purified VLPs were preliminarily tested for immunogenicity in mice and shown to stimulate the production of FMDV-specific antibodies. This study, has important implications for simplifying the production and expression of potential vaccine candidates against FMDV in plants, in the absence of 3C expression.


Assuntos
Anticorpos Antivirais/biossíntese , Proteínas do Capsídeo/imunologia , Vírus da Febre Aftosa/efeitos dos fármacos , Febre Aftosa/prevenção & controle , Tabaco/genética , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Epitopos/genética , Epitopos/imunologia , Feminino , Febre Aftosa/imunologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/imunologia , Imunização , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Tabaco/química , Tabaco/metabolismo , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
14.
Am J Reprod Immunol ; 79(1)2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29048721

RESUMO

PROBLEM: Immunocastration or vaccination against the GnRH-I hormone is a promising alternative to reproductive control in different animal species. Given the low immunogenicity of this hormone, the use of adjuvants becomes necessary. METHOD OF STUDY: This study evaluated the effects of three adjuvants that induce different immune response profiles over gonadal function, fertility, and expression of GnRH-I. Female mice (n = 6) were vaccinated at days 1 and 30 with a recombinant antigen for immunocastration and different adjuvants that induced preferentially Th1/Th2, Th2, and Th1 immune profiles. RESULTS: Th1/Th2 response is the most efficient to block reproductive activity in vaccinated animals, reducing the number of luteal bodies and pre-ovulatory follicles. Th2 and Th1/Th2 responses induced an increase in GnRH-I at the hypothalamus. CONCLUSION: The immune profile induced by different adjuvants is essential on the effects over fertility, gonadal function, and hypothalamic GnRH-I expression in immunocastrated animals.


Assuntos
Hormônio Liberador de Gonadotropina/imunologia , Gônadas/fisiologia , Hipotálamo/metabolismo , Precursores de Proteínas/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Feminino , Fertilidade , Regulação da Expressão Gênica , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/patologia , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Precursores de Proteínas/genética , Receptores LHRH/metabolismo , Equilíbrio Th1-Th2 , Vacinação
15.
Pediatr Diabetes ; 19(1): 68-79, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28488272

RESUMO

AIMS/HYPOTHESIS: Among the beta-cell associated antigens, preproinsulin (PPI) has been shown to play a key role in the pathogenesis of type 1 diabetes (T1D). PPI-specific autoreactive CD8+ T cells emerge early during beta-cell destruction and persist in peripheral circulation during diabetes progression. However, the influence of insulin therapy on phenotype of autoreactive CD8+ T cells in T1D including, juvenile-onset T1D (JOT1D), and adult-onset T1D (AOT1D) is not yet known. METHODS: We followed the time course of PPI-specific CD8+ T cells in JOT1D and AOT1D subjects that achieved glycemic control after 1 year of insulin therapy, using major histocompatibility complex-I (MHC-I) dextramers by flow cytometry. RESULTS AND DISCUSSION: At follow-up, PPI-specific CD8+ T cells could be detected consistently in peripheral blood of all T1D subjects. Proportion of PPI-specific effector memory (TEM ) subsets decreased, while central memory T (TCM ) cells remained unchanged in both groups. Expression of granzyme-B and perforin in PPI-specific CD8+ T cells also remained unchanged. Further, on analysis of B-chain and signal peptide (SP) specific CD8+ T cell responses separately, we again observed decrease in TEM subset in both the groups, while increase in naive (TN ) subset was observed in B-chain specific CD8+ T cells only. CONCLUSION: Our study shows that PPI-specific CD8+ T cells can be detected in both JOT1D and AOT1D subjects over a period of time with reliable consistency in frequency but variable pathophysiological characteristics. Insulin therapy seems to reduce the PPI-specific TEM subsets; however, the PPI-specific TCM cells continue to persist as attractive targets for immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Diabetes Mellitus Tipo 1/imunologia , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Precursores de Proteínas/imunologia , Adulto , Idade de Início , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/tratamento farmacológico , Seguimentos , Granzimas/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Memória Imunológica , Insulina/imunologia , Insulina/farmacologia , Perforina/metabolismo
16.
J Control Release ; 268: 296-304, 2017 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-29080666

RESUMO

Loading of antigen on particles as well as the choice of skin as target organ for vaccination were independently described as effective dose-sparing strategies for vaccination. Combining these two strategies, sufficient antigen recognition may be achievable via the transcutaneous route even with minimal-invasive tools. Here, we investigated the skin penetration and cellular uptake of topically administered virus-like particles (VLPs), composed of the HIV-1 precursor protein Pr55gag, as well as the migratory activity of skin antigen-presenting cells (APCs). We compared VLP administration on ex vivo human skin pre-treated with cyanoacrylate tape stripping (CSSS, minimal-invasive) to administration by skin pricking and intradermal injection (invasive). CSSS as well as pricking treatments resulted in penetration of VLPs in the viable skin layers. Electron microscopy confirmed that at least part of VLPs remained intact during the penetration process. Flow cytometry of epidermal, dermal, and HLA-DR+ APCs harvested from culture media of skin explants cultivated at air-liquid interface revealed that a number of cells had taken-up VLPs. Similar results were found between invasive and minimal-invasive VLP application methods. CSSS pre-treatment was associated with significantly increased levels of IL-1α levels in cell culture media as compared to untreated and pricked skin. Our findings provide first evidence for effective cellular uptake of VLPs after dermal application and indicate that even mild physical barrier disruption, as induced by CSSS, provides stimulatory signals that enable the activation of APCs and uptake of large antigenic material.


Assuntos
Precursores de Proteínas/administração & dosagem , Pele/imunologia , Administração Cutânea , Animais , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular , Citocinas/imunologia , Humanos , Insetos , Plasmídeos , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia
17.
Int J Mol Sci ; 18(8)2017 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-28820494

RESUMO

Fungi are of increasing importance in sepsis. However, culture-based diagnostic procedures are associated with relevant weaknesses. Therefore, culture- and next-generation sequencing (NGS)-based fungal findings as well as corresponding plasma levels of ß-d-glucan, interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, -4, -6, -10, -17A, and mid-regional proadrenomedullin (MR-proADM) were evaluated in 50 septic patients at six consecutive time points within 28 days after sepsis onset. Furthermore, immune-response patterns during infections with Candida spp. were studied in a reconstituted human epithelium model. In total, 22% (n = 11) of patients suffered from a fungal infection. An NGS-based diagnostic approach appeared to be suitable for the identification of fungal pathogens in patients suffering from fungemia as well as in patients with negative blood cultures. Moreover, MR-proADM and IL-17A in plasma proved suitable for the identification of patients with a fungal infection. Using RNA-seq., adrenomedullin (ADM) was shown to be a target gene which is upregulated early after an epithelial infection with Candida spp. In summary, an NGS-based diagnostic approach was able to close the diagnostic gap of routinely used culture-based diagnostic procedures, which can be further facilitated by plasmatic measurements of MR-proADM and IL-17A. In addition, ADM was identified as an early target gene in response to epithelial infections with Candida spp.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sistema Imunitário/imunologia , Micoses/imunologia , Choque Séptico/imunologia , Adrenomedulina/sangue , Adrenomedulina/imunologia , Idoso , Biomarcadores/sangue , Candida/imunologia , Candida/fisiologia , Feminino , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-17/sangue , Interleucina-17/imunologia , Masculino , Pessoa de Meia-Idade , Micoses/diagnóstico , Micoses/microbiologia , Precursores de Proteínas/sangue , Precursores de Proteínas/imunologia , Choque Séptico/sangue , Choque Séptico/microbiologia , Fatores de Tempo
18.
Clin Cancer Res ; 23(17): 5267-5280, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28600477

RESUMO

Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct ß-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses.Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling.Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors.Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS-mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267-80. ©2017 AACR.


Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Gastrinas/antagonistas & inibidores , Precursores de Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Gastrinas/sangue , Gastrinas/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Camundongos , Mutação , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Precursores de Proteínas/sangue , Precursores de Proteínas/imunologia , Via de Sinalização Wnt/efeitos dos fármacos
19.
J Microbiol Biotechnol ; 27(7): 1336-1344, 2017 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-28478661

RESUMO

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma. With recent identification of HBV receptor, inhibition of virus entry has become a promising concept in the development of new antiviral drugs. To date, 10 HBV genotypes (A-J) have been defined. We previously generated two murine anti-preS1 monoclonal antibodies (mAbs), KR359 and KR127, that recognize amino acids (aa) 19-26 and 37-45, respectively, in the receptor binding site (aa 13-58, genotype C). Each mAb exhibited virus neutralizing activity in vitro, and a humanized version of KR127 effectively neutralized HBV infection in chimpanzees. In the present study, we constructed a humanized version (HzKR359-1) of KR359 whose antigen binding activity is 4.4-fold higher than that of KR359, as assessed by competitive ELISA, and produced recombinant preS1 antigens (aa 1-60) of different genotypes to investigate the binding capacities of HzKR359-1 and a humanized version (HzKR127-3.2) of KR127 to the 10 HBV genotypes. The results indicate that HzKR359-1 can bind to five genotypes (A, B, C, H, and J), and HzKR127-3.2 can also bind to five genotypes (A, C, D, G, and I). The combination of these two antibodies can bind to eight genotypes (A-D, G-J), and to genotype C additively. Considering that genotypes A-D are common, whereas genotypes E and F are occasionally represented in small patient population, the combination of these two antibodies might block the entry of most virus genotypes and thus broadly neutralize HBV infection.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Epitopos/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Precursores de Proteínas/imunologia , Receptores Virais/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/fisiologia , Anticorpos Monoclonais Humanizados/isolamento & purificação , Sítios de Ligação , Ensaio de Imunoadsorção Enzimática , Epitopos/fisiologia , Genótipo , Hepatite B/imunologia , Hepatite B/terapia , Hepatite B/virologia , Anticorpos Anti-Hepatite B/imunologia , Anticorpos Anti-Hepatite B/metabolismo , Humanos , Ligação Proteica , Internalização do Vírus
20.
Monoclon Antib Immunodiagn Immunother ; 36(3): 113-118, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28557609

RESUMO

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.


Assuntos
Anticorpos Monoclonais/química , Imunoensaio/normas , Imunoconjugados/química , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Proteínas do Mieloma/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Biomarcadores , Células CHO , Cricetulus , Expressão Gênica , Humanos , Hibridomas/imunologia , Imunoconjugados/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Mieloma Múltiplo/química , Mieloma Múltiplo/genética , Proteínas do Mieloma/genética , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Protrombina/genética , Protrombina/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Sensibilidade e Especificidade
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