Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 15.472
Filtrar
1.
BMC Vet Res ; 15(1): 274, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370852

RESUMO

BACKGROUND: In Poland, the leader in goose production in Europe, goose parovirus infection, or Derzsy's disease (DD), must be reported to the veterinary administration due to the serious economic and epizootic threat to waterfowl production. Prophylactic treatment for DD includes attenuated live or inactivated vaccines. Moreover, the control of DD includes the monitoring of maternal derived antibody (MDA) levels in the offspring and antibody titers in the parent flock after vaccination. The aim of this study was to develop an ELISA for the detection of goose parvovirus (GPV) antibodies. RESULTS: Two recombinant protein fragments derived from VP3 (viral protein 3) GPV, namely VP3ep6 and VP3ep4-6 with a mass of 20.9 and 32.3 kDa, respectively, were produced using an Escherichia coli expression system. These proteins were purified by one-step nickel-affinity chromatography, which yielded protein preparations with a purity above 95%. These recombinant proteins were useful in the detection of serum anti-GPV antibodies, and this was confirmed by Western blotting. However, recombinant VP3ep4-6 protein showed a greater ability to correctly identify sera from infected geese. In the next stage of the project, a pool of 166 goose sera samples, previously examined by a virus neutralization test (VN), was tested. For further studies, one recombinant protein (VP3ep4-6) was selected for optimization of the test conditions. After optimization, the newly developed ELISA was compared to other serological tests, and demonstrated high sensitivity and specificity. CONCLUSION: In conclusion, the VP3ep4-6 ELISA method described here can be used for the detection of antibodies to GPV in serum.


Assuntos
Anticorpos Antivirais/sangue , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Doenças das Aves Domésticas/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática/normas , Infecções por Parvoviridae/sangue , Infecções por Parvoviridae/diagnóstico , Doenças das Aves Domésticas/sangue , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade
2.
Vet Immunol Immunopathol ; 212: 9-14, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31213252

RESUMO

Targeting antigens to endocytic receptors on the surface of dendritic cells is a new strategy for increasing the adaptive immune response. The objective of the current study was the construction and bacterial expression of a recombinant antibody single-chain fragment variable (ScFv) directed against chicken DEC 205, an endocytic receptor, for use in the genetic fusion of antigens. In particular, we use as antigen the hemagglutinin-neuraminidase (HN) of Newcastle disease virus. Our results show that inoculation of chickens with HN genetically fused to the ScFv anti-DEC 205 induced an evidently higher immune response against HN, in contrast to inoculation with unconjugated HN. In addition, neutralizing antibodies against Newcastle disease virus were detected only in the serum from chickens immunized with HN fused to ScFv anti-DEC 205. Inoculated fused antigens to ScFv against endocytic receptor DEC 205 resulted in a greater antibody-specific anti-HN production compared with antigens applied alone. The results of this study show that the strategy described here has the potential to be used in the development of more effective vaccines against infectious diseases in chickens.


Assuntos
Antígenos CD/imunologia , Lectinas Tipo C/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Vírus da Doença de Newcastle/enzimologia , Doenças das Aves Domésticas/prevenção & controle , Receptores de Superfície Celular/imunologia , Anticorpos de Cadeia Única/biossíntese , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Galinhas/imunologia , Escherichia coli/genética , Hemaglutininas Virais/imunologia , Neuraminidase/imunologia , Doenças das Aves Domésticas/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/imunologia , Vacinas Virais/imunologia
3.
Cancer Immunol Immunother ; 68(7): 1143-1155, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31177328

RESUMO

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Neoplasias Mamárias Experimentais/terapia , Receptor ErbB-2/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral/transplante , Células Dendríticas/imunologia , Evolução Molecular , Feminino , Imunogenicidade da Vacina/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptor ErbB-2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Tolerância a Antígenos Próprios/genética , Microambiente Tumoral/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico
4.
Parasitol Int ; 72: 101938, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201923

RESUMO

Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60-70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Malária Falciparum/diagnóstico , Proteínas de Protozoários/imunologia , Animais , Antígenos de Protozoários/genética , Bombyx/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Malária Falciparum/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Merozoítos/imunologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Testes Sorológicos
5.
Nat Commun ; 10(1): 2261, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113940

RESUMO

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Células Cultivadas , Cristalografia por Raios X , DNA/imunologia , DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferons/imunologia , Interferons/metabolismo , Macrófagos , Modelos Moleculares , Nucleotídeos Cíclicos/imunologia , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/isolamento & purificação , Nucleotidiltransferases/metabolismo , Cultura Primária de Células , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
Cancer Immunol Immunother ; 68(7): 1211-1222, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31069460

RESUMO

Human tumor cells express antigens that serve as targets for the host cellular immune system. This phase 1 dose-escalating study was conducted to assess safety and tolerability of G305, a recombinant NY-ESO-1 protein vaccine mixed with glucopyranosyl lipid A (GLA), a synthetic TLR4 agonist adjuvant, in a stable emulsion (SE). Twelve patients with solid tumors expressing NY-ESO-1 were treated using a 3 + 3 design. The NY-ESO-1 dose was fixed at 250 µg, while GLA-SE was increased from 2 to 10 µg. Safety, immunogenicity, and clinical responses were assessed prior to, during, and at the end of therapy. G305 was safe and immunogenic at all doses. All related AEs were Grade 1 or 2, with injection site soreness as the most commonly reported event (100%). Overall, 75% of patients developed antibody response to NY-ESO-1, including six patients with increased antibody titer ( ≥ 4-fold rise) and three patients with seroconversion from negative (titer < 100) to positive (titer ≥ 100). CD4 T-cell responses were observed in 44.4% of patients; 33.3% were new responses and 1 was boosted ( ≥ 2-fold rise). Following treatment, 8 of 12 patients had stable disease for 3 months or more; at the end of 1 year, three patients had stable disease and nine patients were alive. G305 is a potent immunotherapeutic agent that can stimulate NY-ESO-1-specific antibody and T-cell responses. The vaccine was safe at all doses of GLA-SE (2-10 µg) and showed potential clinical benefit in this population of patients.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos de Neoplasias/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Glucosídeos/administração & dosagem , Lipídeo A/administração & dosagem , Proteínas de Membrana/administração & dosagem , Neoplasias/terapia , Adjuvantes Imunológicos/efeitos adversos , Adulto , Idoso , Antígenos de Neoplasias/efeitos adversos , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Drogas em Investigação/administração & dosagem , Drogas em Investigação/efeitos adversos , Feminino , Glucosídeos/efeitos adversos , Glucosídeos/imunologia , Humanos , Imunogenicidade da Vacina , Injeções Intramusculares , Lipídeo A/efeitos adversos , Lipídeo A/imunologia , Masculino , Proteínas de Membrana/efeitos adversos , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/imunologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Adulto Jovem
7.
Ticks Tick Borne Dis ; 10(4): 935-941, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31072731

RESUMO

Tick-borne encephalitis virus (TBEV) is a member of the Flavivirus genus and is the main pathogenic arbovirus circulating in Europe, Russia and China. The envelope (E) protein is exposed on the viral surface and is the main antigen that is employed in diagnostic tests based on the detection of protein-specific antibodies from serum samples of infected individuals. The high degree of similarity among the E proteins of flaviviruses can, in some cases, lead to cross-reactivity and false-positive results in serological tests. Increased specificity in the detection of positive sera for different Flavivirus infections is often obtained by using a portion of the E protein, namely, the DIII domain. Different strategies and expression systems have been described for E and DIII protein production. Here, we present the optimization of an easy and fast method for TBEV E and DIII antigen production and partial purification from E. coli inclusion bodies. The antigenic properties of the produced antigens are retained, as validated by ELISAs with anti-TBEV murine sera as well as sera from infected human patients. The potential applications of both proteins as diagnostic reagents were confirmed.


Assuntos
Antígenos Virais/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/biossíntese , Antígenos Virais/genética , Clonagem Molecular , Encefalite Transmitida por Carrapatos/diagnóstico , Encefalite Transmitida por Carrapatos/imunologia , Ensaio de Imunoadsorção Enzimática , Infecções por Flavivirus/diagnóstico , Infecções por Flavivirus/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética
8.
Microb Pathog ; 133: 103552, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31121269

RESUMO

Aeromonas veronii is an opportunistic pathogen that is capable of infecting both aquatic livestock and mammals. Natural infection in fishes results in irreparable damage to the aquaculture industry. In this study, we sought to investigate whether recombinant Lactobacillus casei expressing the outer membrane protein W (OmpW) of A.veronii could elicit protective immunity against A.veronii infections. We generated two recombinant Lactobacillus casei (L.casei) strains expressing the OmpW of A.veronii (surface-displayed or secreted) and evaluated the effect on immune responses in a fish model. A 600-bp gene fragment was subcloned into the L.casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secreted). Expression of the recombinant OmpW protein was also confirmed by Western blot and immunofluorescence assays. Common carp immunized with Lc-pPG-1- OmpW and Lc-pPG-2- OmpW via oral administration elicited high serum specific antibody titers and high LZM, ACP, and SOD activities. High levels of the IL-10, IL-ß, IFN-γ, and TNF-α genes in different organs indicated that the inflammatory response and cell immune response were triggered. Additionally, when immunized fish were challenged with A.veronii, Lc-pPG1-OmpW and Lc-pPG2-OmpW demonstrated 40% and 50% protective efficacy. These data indicate that the combination of OmpW delivery and the lactic acid bacteria (LAB) approach may be a promising mucosal therapeutic strategy for treatment of A.veronii.


Assuntos
Aeromonas veronii/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Carpas/imunologia , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Imunização/veterinária , Lactobacillus casei/metabolismo , Administração Oral , Aeromonas veronii/imunologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas , Sequência de Bases , Carpas/microbiologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Doenças dos Peixes/imunologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Infecções por Bactérias Gram-Negativas/imunologia , Imunidade Celular , Imunidade Humoral , Interferon gama/genética , Interleucina-10/genética , Intestinos/microbiologia , Lactobacillus casei/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/genética
9.
Microb Pathog ; 133: 103560, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145981

RESUMO

Toxoplasma gondii is an intracellular zoonotic parasite that causes toxoplasmosis, which can cause economic losses and serious public health problems worldwide. A member of the T. gondii calcium-dependent protein kinases family, TgCDPK1 was recently identified as an essential regulator of exocytosis in T. gondii, and participated in direct parasite motility, host-cell invasion and egress. In the present study, the protective immunity of recombinant TgCDPK1 protein (rTgCDPK1) was evaluated against acute toxoplasmosis in mice. rTgCDPK1 were expressed and purified, BABL/c mice were intraperitoneally immunized with rTgCDPK1 and challenged with the highly virulent RH strain of T. gondii. The specific immune responses were analyzed by measuring the cytokine and serum antibody, and lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival curve were employed to evaluate the protective efficacy. From the results we found that special humoral and cellular responses could be elicited in vaccine mice, and higher level of IgG antibody, and the significant increased levels of Th1-type cytokines IFN-γ, IL-12 (p70), IL10 and CD3+CD4+CD8- and CD3+CD8+CD4- T cells could also be detected comparing to control mice (P < 0.05). All vaccinated mice prolonged survival time (14.90 ±â€¯2.89 days) challenge with 1000 tachyzoites of RH, while the control mice died within 8 days. These results indicated that TgCDPK1 protein was a potential vaccine candidate against acute toxoplasmosis.


Assuntos
Imunização , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/prevenção & controle , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Clonagem Molecular , Citocinas/metabolismo , Feminino , Genes de Protozoários/genética , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Linfócitos/imunologia , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Baço/imunologia , Análise de Sobrevida , Toxoplasma/genética , Toxoplasmose Animal/imunologia , Vacinas de DNA/imunologia
10.
Fish Shellfish Immunol ; 89: 448-457, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974220

RESUMO

Mannose-binding lectin (MBL) is a pattern recognition receptor (PRR) that plays an important role in the innate immune response. In this study, a novel mannose-binding lectin was cloned from the swimmimg crab Portunus trituberculatus (designated as PtMBL). The complete cDNA of PtMBL gene was 1208 bp in length with an open reading frame (ORF) of 732 bp that encoded 244 amino acid proteins. PtMBL shared lower amino acid similarity with other MBLs, yet it contained the conserved carbohydrate-recognition domain (CRD) with QPD motif and was clearly member of the collectin family. PtMBL transcripts were mainly detected in eyestalk and gill with sexually dimorphic expression. The temporal expression of PtMBL in hemocytes showed different activation times after challenged with Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtMBL protein revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria. It could also bind and agglutinate (Ca2+-dependent) both bacteria and yeast. Furthermore, the agglutinating activity could be inhibited by both d-galactose and d-mannose, suggesting the broader pathogen-associated molecular patterns (PAMPs) recognition spectrum of PtMBL. These results together indicate that PtMBL could serve as not only a PRR in immune recognition but also a potential antibacterial protein in the innate immune response of crab.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Masculino , Lectina de Ligação a Manose/química , Micrococcus luteus/fisiologia , Filogenia , Pichia/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Vibrio alginolyticus/fisiologia
11.
Fish Shellfish Immunol ; 89: 403-410, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30978447

RESUMO

The p40 subunit is known as a component of Interleukin (IL)-12 and IL-23. In mammals, p40 can be secreted as a monomer or homodimer and acts independently to mediate cellular responses. Recently, three p40 paralogues were isolated and identified from grass carp and other fish species, but whether they exist independently as well as their functional consequences and significance remain unclear. In the present study, using grass carp as the model, we for the first time demonstrated the existence of natural fish p40a, p40b and p40c (gcp40a, gcp40b and gcp40c) mainly as a monomer in culture supernatant of head kidney leukocytes (HKLs). Particularly, their excessive secretion induced by various immune stimuli suggests possible involvement of free p40s in fish immune responses. To define their functions, recombinant grass carp p40a/b/c (rgcp40a, rgcp40b and rgcp40c) were prepared by Pichia pastoris expression system, and they possessed the activities to enhance the secretion of pro-inflammatory cytokines including Il-1ß and tumor necrosis factor-α (Tnf-α) in grass carp HKLs. These pro-inflammatory properties of p40 isoforms prompted us to investigate their roles during the inflammatory process. In line with this, in vivo study revealed the pathogenic effect of rgcp40a on intestinal inflammation, whereas gcp40a polyclonal antibodies remarkably ameliorated Aeromonas hydrophila-induced intestinal histopathological changes. Taken together, our results uncover the biological significance of free p40s in teleost, and provide new clue for targeting fish intestinal inflammation.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Subunidade p40 da Interleucina-12/imunologia , Aeromonas hydrophila/fisiologia , Animais , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Inflamação/imunologia , Inflamação/veterinária , Isoformas de Proteínas/imunologia , Proteínas Recombinantes/imunologia
12.
Vet Microbiol ; 231: 154-159, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955803

RESUMO

Pigs suffer enteritis induced by pathogenic bacteria infection and toxins in the moldy feed, which cause intestinal epithelial damage and diarrhea through the whole breeding cycle. Interleukin-22 (IL-22) plays a critical role in maintaining intestinal mucosal barrier function through repairing intestinal epithelial damage. However, little was known about the effects of IL-22 against apoptosis caused by toxins and infection of intestinal pathogens in the intestinal epithelium, especially in pigs. In this study, we had successfully used prokaryotic expression system to produce recombinant porcine interleukin-22. Meanwhile, purified rIL-22 could activate STAT3 signal pathway and have been demonstrated to be safe to IPEC-J2 cells by increasing E-cadherin expression, without proinflammatory cytokines changes. Furthermore, rIL-22 reversed apoptosis induced by deoxynivalenol (DON) and played a vital part in repairing the intestinal injury. We also found that rIL-22 stimulated epithelial cells to secrete pBD-1 against enterotoxigenic E. coli (ETEC) K88 infection, as well as alleviating apoptosis ratio. This study provided a theoretical basis for curing intestinal inflammation caused by ETEC infection and epithelial apoptosis induced by DON with rIL-22 in pigs.


Assuntos
Escherichia coli Enterotoxigênica/patogenicidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Interleucinas/farmacologia , Tricotecenos/efeitos adversos , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Apoptose , Linhagem Celular , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Inflamação , Interleucinas/imunologia , Mucosa Intestinal/citologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Suínos
13.
Vet Microbiol ; 231: 177-182, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955806

RESUMO

Canine parvovirus (CPV) is one of the most important cause of mortality in young dogs and no specific treatment exists. Since prolonged leukopenia greatly increases the risk of death in infected pups, strategies to counteract this decline were investigated. The outcomes of CPV naturally infected pups treated with the recombinant canine granulocyte-colony stimulating factor (rcG-CSF), in combination with the routine therapy, were compared with similarly-managed infected pups not treated with rcG-CSF. A non-randomized prospective clinical trial was performed on 62 CPV infected pups with WBC counts <3000 cells/µL and two different groups were selected based on a non-randomized approach. Group A dogs (31/62) received 5 µg/Kg of rcG-CSF daily from the hospitalization day until WBC reached the reference range (3-5 days) and group B (31/62) received 1 ml of placebo injection. All dogs in group A recovered, while five dogs in group B died. The rcG-CSF treatment demonstrated a statistically significant effect on WBC counts (p < 0.0001) and, surprisingly, also on lymphocytes and monocytes counts (p < 0.0001). There was no significant effect of treatment on neutrophil count (p = 0.5502). Although lymphocytes and monocytes are not a specific target for rcG-CSF, our study highlights that rcG-CSF is able to improve haematological parameters compared to untreated dogs and a clear increase in their number was detected, as previously described for humans treated with the homologous molecule.


Assuntos
Doenças do Cão/prevenção & controle , Fator Estimulador de Colônias de Granulócitos/imunologia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Contagem de Leucócitos/veterinária , Infecções por Parvoviridae/veterinária , Animais , Doenças do Cão/imunologia , Doenças do Cão/virologia , Cães , Feminino , Masculino , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/prevenção & controle , Parvovirus Canino , Estudos Prospectivos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico
14.
Fish Shellfish Immunol ; 89: 170-178, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928663

RESUMO

Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48 h compared with the PBS control. However, the expression level significantly increased after 36 h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24 h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii, since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Peroxirredoxina VI/genética , Peroxirredoxina VI/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Cromatografia de Afinidade , Dano ao DNA , DNA Super-Helicoidal/fisiologia , Perfilação da Expressão Gênica , Estresse Oxidativo , Peptidoglicano/farmacologia , Peroxirredoxina VI/química , Filogenia , Poli I-C/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência
15.
Microb Pathog ; 131: 9-14, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30930220

RESUMO

Acinetobacter baumannii is considered as a major cause of nosocomial infection worldwide. Various vaccine formulations have been mostly studied based on secreted or surface-exposed proteins of A. baumannii in murine models. Serum resistance proteins are critical virulence factors in bloodstream infections. AbOmpA and PKF are two major factors involved in serum resistance and could be considered as promising vaccine targets. In this study IgG1, IgG2c, Total-IgG concentrations, survival rates and spleen bacterial loads were studied in C57/BL mice model according to PKF, AbOmpA and AbOmpA + PKF vaccine formulations. The findings showed significant raises of IgG2c and Total-IgG in all three vaccinated groups in comparison with the control group. Whereas, there were low concentrations of IgG1 in all immunization plans. Colony counts of mice spleen showed the bacterial load of PKF plan had the most decrease of bacterial load (DBL = 5 log10 CFU/g). Taken together, this evaluation indicated that PKF vaccination plan induced a polarized Th1 response and rendered an effective protection against bloodstream infection caused by A. baumannii.


Assuntos
Infecções por Acinetobacter/imunologia , Infecções por Acinetobacter/prevenção & controle , Acinetobacter baumannii/patogenicidade , Formação de Anticorpos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Fatores R/sangue , Sepse/microbiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Animais , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Clonagem Molecular , Modelos Animais de Doenças , Genes Bacterianos/genética , Imunização , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Fatores R/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Baço/imunologia , Taxa de Sobrevida , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologia
16.
Fish Shellfish Immunol ; 90: 363-375, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30974219

RESUMO

Superoxide dismutases (SODs) are important antioxidant enzymes that occur in virtually all oxygen-respiring organisms, and copper/zinc SOD (Cu/ZnSOD) is one of the most important SODs. In the present study, Macrobrachium rosenbergii Cu/Zn-SOD was expressed in a yeast eukaryotic system. The open reading frame (ORF) of MrCu/ZnSOD was cloned into the plasmid vector pHAC181, and the recombinant plasmid was integrated into the downstream region of the GAL1 promoter in Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination. The resulting recombinant MrCu/ZnSOD consisted of a 3 × HA-tag at its C-terminal. Via western blot, the molecular weight of the recombinant MrCu/ZnSOD was estimated at about 30 kDa. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of this recombinant MrCu/ZnSOD ranged from 0.556 to 0.840 µM, and from 0.967 to 2.015 µM, respectively. The recombinant MrCu/ZnSOD protein was able to agglutinate four Gram-negative bacterial strains, as well as two of three Gram-positive strains (except Staphylococcus aureus). This demonstrated that the recombinant protein possessed some antimicrobial activity against certain Gram-positive and Gram-negative bacteria. M. rosenbergii were fed with the recombinant yeast strain MrCu/ZnSOD for 4 weeks and then challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. This group of prawns presented lower mortality, higher enzymatic activity, and higher expression of the mRNA of immune-related genes than that in the control groups. Taken together, these results suggest that MrCu/ZnSOD is an antioxidant enzyme and antimicrobial peptide involved in the crustacean innate immune system and offers protection to the host against pathogenic bacteria.


Assuntos
Palaemonidae/genética , Palaemonidae/imunologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/imunologia , Vibrio parahaemolyticus/imunologia , Aglutinação , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/metabolismo , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Microrganismos Geneticamente Modificados/metabolismo , Palaemonidae/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/metabolismo
17.
Virus Res ; 266: 34-42, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30965063

RESUMO

The porcine epidemic diarrhea virus (PEDV) collagenase equivalent domain (COE, residues 499-638), a crucial antigenic region within the viral spike (S) glycoprotein, has been widely utilized for the development of subunit vaccines to prevent viral infection. In the current study, we immunized BALB/c mice with recombinant truncated PEDV COE protein and obtained 14 COE-specific monoclonal antibodies (mAbs). Based on the reactivity analysis of the mAbs with two prevalent PEDV strains in G2 type and the attenuated CV777 strain in G1 type, 6 mAbs were selected for subsequent identification of COE mAb-binding epitopes. Dot-blot hybridization and enzyme-linked immunosorbent assays (ELISAs) identified the peptide 592TSLLASACTIDLFGYP607 as a novel linear B-cell epitope involved in binding of mAbs 4D8F10 and 6F3E3. Subsequently, alanine (A)-scanning mutagenesis demonstrated that residues 606Y, 605G and 604F were core residues involved in recognition. Importantly, this novel COE epitope, including core residues, is conserved among G1 and G2 type PEDV strains. Further experiment indicates that the mAbs 4D8F10 and 6F3E3 were suitable for PEDV detection via mAb binding to the conserved epitope. The current work actually provides potential uses for the development of diagnostic methods to detect PEDV.


Assuntos
Epitopos de Linfócito B/imunologia , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Cercopithecus aethiops , Sequência Conservada , Infecções por Coronavirus/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Feminino , Células HEK293 , Humanos , Imunização , Camundongos Endogâmicos BALB C , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
18.
Fish Shellfish Immunol ; 90: 20-29, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31009809

RESUMO

Granulocyte colony-stimulating factor (GCSF) is a pleiotropic cytokine that plays a key role in regulation of hematopoiesis, innate and adaptive immune responses in mammals. However, bioactivity of GCSF in teleost fish remains largely unknown. In this study, a GCSFb homologue from large yellow croaker (Larimichthys crocea) (LcGCSFb) was cloned by RACE-PCR techniques. The open reading frame (ORF) of LcGCSFb is 603 bp long and encoded a protein precursor of 200 amino acids (aa), with a 19-aa signal peptide and a 181-aa mature peptide. In healthy fish, the LcGCSFb was constitutively expressed in all examined tissues, with the highest levels in mucous tissues, such as gills, intestine, and stomach. Its transcripts in head kidney, spleen, gills, intestine and stomach were significantly induced by Vibrio alginolyticus challenge. LcGCSFb transcripts were also detected in primary head kidney leukocytes (PKL), primary head kidney macrophages (PKM), primary head kidney granulocytes (PKG) and head kidney cell line (LYCK), and markedly upregulated by inactivated V. alginolyticus. These data suggested that LcGCSFb may play a role in immune response against bacterial infection. In vivo administration of recombinant LcGCSFb protein (rLcGCSFb) significantly upregulated the expression levels of the inflammatory cytokines (IL-6 and TNFα), and transcription factor C/EBPß, which is required for proliferation of neutrophils. Furthermore, rLcGCSFb showed an ability to strengthen the phagocytosis of PKL in vitro. Taken together, LcGCSFb may be involved in antibacterial immunity via promoting the inflammatory response and the phagocytic activity of leukocytes. To our knowledge, this is the first report on immunoregulatory roles of GCSF in teleost.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Fator Estimulador de Colônias de Granulócitos/química , Rim Cefálico/imunologia , Leucócitos/imunologia , Fagocitose/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência/veterinária , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio alginolyticus/fisiologia
19.
Monoclon Antib Immunodiagn Immunother ; 38(2): 70-74, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31009334

RESUMO

Zinc transporter ZIP6 (SLC39A6) or LIV-1 is a protein that belongs to a subfamily of proteins group that displays structural specifications of zinc transporters in the cell membrane. Overexpression of this protein is observed in breast, prostate, and kidney tumor cells. Lately, LIV-1 is a dependable marker for detection of estrogen receptor positive breast cancer, which can be used to detect luminal breast cancer type A. In this study, the gene construct containing extracellular domain of human LIV-1 gene was subcloned into pET22b expression vector, expressed and confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. It was shown for the first time that the extracellular domain of LIV-1 could be expressed in bacterial systems and can be used for rabbit immunization. The reactivity of the resulted antibody was evaluated in flow cytometry and enzyme-linked immunosorbent assay. In conclusion, this protein can be used for animal immunization toward preparation of a new monoclonal antibody that can be introduced as a drug in the treatment of breast cancer.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Neoplasias da Mama/metabolismo , Proteínas de Transporte de Cátions/imunologia , Proteínas de Neoplasias/imunologia , Proteínas Recombinantes/imunologia , Animais , Neoplasias da Mama/imunologia , Feminino , Humanos , Coelhos , Células Tumorais Cultivadas
20.
Virol Sin ; 34(3): 324-333, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989429

RESUMO

Interferon, a natural protein that is produced by a variety of cells during viral infection, activates the transcription of multiple functional genes in cells, regulates synergy among various signaling pathways, and mediates many biological functions such as antiviral activity, immune regulation, and cell growth. However, clinical research on interferon in livestock is lacking. In this study, recombinant porcine interferon (PoIFNα) was used as an adjuvant, in combination with inactivated influenza virus, to vaccinate 6-week-old pigs via nasal infusion. The transcription of target genes was then monitored and the functions of PoIFNα were determined with respect to the activation of mucosal immunity. We found that a combination of low-dose PoIFNα and inactivated influenza virus could significantly up-regulate the expression of immunoregulatory cytokines such as IL-2, IL-18, IFN-γ, IL-6, and IL-10 by real-time PCR, suggesting the induction of a strong mucosal innate immune response after administration. In addition, low-dose PoIFNα can significant enhancing the transcription of genes encoding homing factors including CCR9 and CCR10 (P < 0.001), thereby resulting in the induction of higher levels of HA-specific antibodies (P < 0.05), which can be determined by ELISA and IFA. Post-immunization challenges with H1N1 virus demonstrated that PoIFNα, combined with inactivated influenza virus, could alleviate clinical signs in pigs during the early stages of viral infection. These studies reveal low-dose PoIFNα as a potential mucosal adjuvant for influenza virus in pigs.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Imunidade nas Mucosas , Vacinas contra Influenza/imunologia , Interferon-alfa/imunologia , Infecções por Orthomyxoviridae/veterinária , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Citocinas/genética , Citocinas/imunologia , Imunidade Inata , Vacinas contra Influenza/administração & dosagem , Interferon-alfa/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Suínos , Vacinação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA