RESUMO
Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) are two of the most devastating and economically significant pathogens affecting pig populations worldwide. Administration of a combination of vaccines against swine pathogens has been demonstrated to be as efficacious as the administration of single vaccines. In this study, we developed and tested a novel bivalent subunit vaccine against CSFV and PCV2. The safety and efficacy of this vaccine were demonstrated in mice and specific pathogen-free (SPF) piglets. In addition to investigating the serological responses after immunization, challenge studies with both viruses were also conducted. The results showed that this CSFV/PCV2 bivalent vaccine elicited a high level of neutralizing antibodies against both viruses and provided protection in challenge studies. In conclusion, the CSFV/PCV2 bivalent vaccine is safe and effective against CSFV or PCV2 challenge.
Assuntos
Infecções por Circoviridae , Circovirus , Vírus da Febre Suína Clássica , Doenças dos Suínos , Vacinas Virais , Animais , Suínos , Camundongos , Anticorpos Antivirais , Vacinas Combinadas , Vacinas de Subunidades Antigênicas , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/veterináriaRESUMO
Pfu DNA polymerase is a vital enzyme in PCR-related experiments. However, it is not easy to achieve high-level expression and high purity through one-step purification. This paper illustrates the method to acquire the full-length open reading frame of Pfu DNA polymerase. Without altering its amino acids, we have modified the codon usage, based on that of the enhanced green fluorescence protein (eGFP), and named it rPfu. The synthesized rPfu gene has been subcloned into the pET28a plasmid and expressed in four Escherichia coli strains without the pLysS plasmid. Three strains have expressed a high level of soluble Pfu DNA polymerase. With the aid of Ni-NTA Hisâ¢Bind® resin, we could obtain high purity (>95%) soluble recombinant protein. Compared with the commercial, proofreading DNA polymerase, rPfu's bioactivity was 12,987 U/mg; that is, 88,311 U of rPfu could be obtained from 50 mL cultured E. coli. The purified rPfu was able to amplify the length of DNA fragments at least 5.5 kb. The method of increasing soluble protein's yield using the eGFP codon usage may introduce a new possibility to the expression of other soluble recombinant proteins.
Assuntos
Uso do Códon , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismoRESUMO
Intramuscular vaccines have greatly reduced hospitalization and death due to severe COVID-19. However, most countries are experiencing a resurgence of infection driven predominantly by the Delta and Omicron variants of SARS-CoV-2. In response, booster dosing of COVID-19 vaccines has been implemented in many countries to address waning immunity and reduced protection against the variants. However, intramuscular boosting fails to elicit mucosal immunity and therefore does not solve the problem of persistent viral carriage and transmission, even in patients protected from severe disease. In this study, two doses of stabilized prefusion SARS-CoV-2 spike (S-2P)-based intramuscular vaccine adjuvanted with Alum/CpG1018, MVC-COV1901, were used as a primary vaccination series, followed by an intranasal booster vaccination with nanoemulsion (NE01)-adjuvanted S-2P vaccine in a hamster model to demonstrate immunogenicity and protection from viral challenge. Here we report that this vaccination regimen resulted not only in the induction of robust immunity and protection against weight loss and lung pathology following challenge with SARS-CoV-2, but also led to increased viral clearance from both upper and lower respiratory tracts. Our findings showed that intramuscular MVC-COV1901 vaccine followed by a booster with intranasal NE01-adjuvanted vaccine promotes protective immunity against both viral infection and disease, suggesting that this immunization protocol may offer a solution in addressing a significant, unmet medical need for both the COVID-19 and future pandemics.
Assuntos
COVID-19 , Vacinas Virais , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Cricetinae , Humanos , SARS-CoV-2RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern negatively impact the effectiveness of vaccines. In this study, we challenge hamsters with the delta variant after 2- or 3-dose inoculations with SARS-CoV-2 vaccines constructed from stabilized prefusion spike proteins (S-2P) of Wuhan (W) and beta (B) variants. Compared to 3 doses of W S-2P, 2 doses of W S-2P followed by a third dose of B S-2P induced the highest neutralizing antibody titer against live SARS-CoV-2 virus and enhanced neutralization of omicron variant pseudovirus. Reduced lung live virus titer and pathology suggested that all vaccination regimens protect hamsters from SARS-CoV-2 delta variant challenge.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Cricetinae , Adjuvantes Imunológicos , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
BACKGROUND: Variants of concern (VoCs) have the potential to diminish the neutralizing capacity of antibodies elicited by vaccines. MVC-COV1901 is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine consisting of recombinant prefusion stabilized spike protein S-2P adjuvanted with CpG 1018 and aluminum hydroxide. We explored the effectiveness of MVC-COV1901 against the VoCs. METHODS: Serum samples were taken from rats and phase 1 clinical trial human subjects immunized with a low, medium, or high dose of MVC-COV1901. The neutralizing titers of serum antibodies were assayed with pseudoviruses coated with the SARS-CoV-2 spike protein of the wild-type (WT), D614G, Alpha, or Beta variants. RESULTS: Rats vaccinated twice with vaccine containing high doses of antigen retained high levels of neutralization activity against the Beta variant, albeit with a slight reduction compared to WT. After the third dose, neutralizing titers against the Beta variant were noticeably enhanced regardless of the amount of antigen used for immunization. In humans, vaccinated phase 1 subjects still showed appreciable neutralization abilities against the D614G, Alpha, and Beta variants, although neutralizing titers were significantly reduced against the Beta variant. CONCLUSIONS: Two doses of MVC-COV1901 were able to elicit neutralizing antibodies against SARS-CoV-2 variants with an overall tendency of inducing higher immune response at a higher dose level. The neutralizing titers to the Beta variant in rats and humans were lower than those for WT and the Alpha variant. An additional third dose in rats was able to partially compensate for the reduction in neutralization against the Beta variant. We have demonstrated that immunization with MVC-COV1901 was effective against VoCs.
Assuntos
COVID-19 , SARS-CoV-2 , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Humanos , Ratos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus , Vacinas de Subunidades Antigênicas , Proteínas do Envelope ViralRESUMO
The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 µg or 5 µg of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 µg or 5 µg of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , COVID-19/prevenção & controle , Oligodesoxirribonucleotídeos/administração & dosagem , Glicoproteína da Espícula de Coronavírus/imunologia , Hidróxido de Alumínio/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Linhagem Celular , Cricetinae , Feminino , Humanos , Imunização , Injeções Intramusculares , Oligodesoxirribonucleotídeos/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Carga Viral/efeitos dos fármacosRESUMO
The COVID-19 pandemic is a worldwide health emergency which calls for an unprecedented race for vaccines and treatment. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein, S-2P. To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of neutralizing antibodies in sera of immunized mice against pseudotyped lentivirus reporter or live wild-type SARS-CoV-2. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018 and alum as a candidate vaccine to prevent COVID-19 disease.
Assuntos
Vacinas contra COVID-19/imunologia , Imunogenicidade da Vacina , Glicoproteína da Espícula de Coronavírus/imunologia , Adjuvantes Imunológicos/uso terapêutico , Hidróxido de Alumínio/uso terapêutico , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Células CHO , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/uso terapêutico , Cricetinae , Cricetulus , Citocinas/sangue , Citocinas/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/uso terapêutico , Ratos , Ratos Sprague-Dawley , Baço/imunologia , Células Th1/imunologiaRESUMO
BACKGROUND: Canine mammary gland tumors (cMGTs) are the most common neoplasms in intact female canines and viewed as a suitable model for studying human breast cancers. Euphorbia royleana has been reported to have a variety of antitumor efficacies. We have prepared the crude extracts of E. royleana in ethanol and hexane solvents to evaluate the anti-tumor effects for cMGT in vitro and in vivo. RESULTS: The results showed that E. royleana could inhibit cell proliferation and colony formation in cMGT cells. The suppression of tumor cell growth resulted from necrosis and cell cycle arrest. Moreover, autophagy appears to play a critical role in E. royleana-mediated cell death by triggering cell apoptosis. The in vivo results also revealed that E. royleana treatment could reduce the size of solid tumors while exhibiting low toxicity in cMGT-bearing nude mice. CONCLUSIONS: The anti-tumor mechanisms of E. royleana were firstly verified to show it would cause autophagic cell death, apoptosis, and cell cycle arrest in canine mammary tumor cells. The in vitro and in vivo findings in the present study revealed E. royleana has potential anticancer effects for the treatment of canine mammary gland tumors.
Assuntos
Autofagia/efeitos dos fármacos , Euphorbia/química , Neoplasias Mamárias Animais/tratamento farmacológico , Extratos Vegetais/farmacologia , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Doenças do Cão/tratamento farmacológico , Cães , Feminino , Camundongos Nus , Extratos Vegetais/toxicidade , Células VeroRESUMO
Porcine circovirus type 2 (PCV2) is a pathogenic virus that causes high rates of porcine death, resulting in severe economic losses to the swine industry. In recent years, the prevalence of PCV2d genotype infection in pigs has increased, but most commercially available vaccines were developed against the PCV2a strain and do not ensure complete protection from PCV2d. Here, we first constructed an expression vector for the antigenic ORF2-encoded capsid protein of PCV2d (pLp3050-His6-tag-capsid). We then utilized Lactobacillus plantarum to express the protein at mucosal sites in orally vaccinated mice. After transducing L. plantarum with pLp3050-His6-tag-capsid, the expressed protein could be found in cell wall and cell-free supernatant fractions by Western blotting. Using flow cytometry, we found that L. plantarum cells with surface-displayed capsid protein increased with time after SppIP induction. Finally, mice that were orally immunized 18 times with capsid-expressing L. plantarum showed increased levels of capsid-specific sIgA and virus neutralizing activity at mucosal sites, suggesting mucosal immunity had been stimulated by the vaccine. Overall, our findings demonstrate the feasibility and utility of a PCV2d-based vaccine, which may be of great value in porcine agriculture.
RESUMO
Since 2016, Apis cerana sacbrood virus (AcSBV) has been recorded in Taiwan. It is epizootic in Apis cerana (Hymenoptera: Apidae) and causing serious loss of A. cerana. Herein, we performed a long-term survey of AcSBV prevalence in the populations of A. cerana in Northern Taiwan from January 2017 to July 2018. The surveillance of AcSBV prevalence in A. mellifera (Hymenoptera: Apidae) populations was starting and further confirmed by sequencing since April 2017; thus, these data were also included in this survey. In our survey, the average prevalence rates of AcSBV were 72 and 53% in A. cerana and A. mellifera, respectively, in 2017, which decreased to 45 and 27% in 2018. For the spatial analysis of AcSBV in two honey bee populations, Hsinchu showed the highest prevalence, followed by New Taipei, Yilan, Taipei, and Keelung, suggesting that AcSBV might have come from the southern part of Taiwan. Interestingly, the AcSBV prevalence rates from A. cerana and A. mellifera cocultured apiaries gradually synchronized. The result of phylogenetic analysis and comparison of the annual AcSBV prevalence in A. cerana-only, A. mellifera-only, and A. cerana/A. mellifera cocultured sample sites indicate cross-infection between A. cerana and A. mellifera; however, AcSBV may lose the advantage of virulence in A. mellifera. The evidence suggested that the transmission of AcSBV might occur among these two honey bee species in the field. Therefore, A. mellifera may serve as a guard species to monitor AcSBV in A. cerana, but the cross-infection still needs to be surveyed.
Assuntos
Himenópteros , Infecções , Animais , Abelhas , Filogenia , Prevalência , Vírus de RNA , TaiwanRESUMO
BACKGROUND: To fight against enterovirus A71 (EV-A71)-associated diseases, vaccine development was initiated in Taiwan focusing on two-month-old infants. METHODS: We conducted a phase II, double-blind, randomised, placebo-controlled study on infants and children aged two months to 11â¯years. This study was conducted in 4 parts (2a, 2b, 2c, and 2d) with age de-escalation sequentially. Two doses were administered with a 28-day or 56-day interval. Participants aged two months to Assuntos
Reações Cruzadas/imunologia
, Enterovirus Humano A/imunologia
, Infecções por Enterovirus/imunologia
, Infecções por Enterovirus/prevenção & controle
, Imunogenicidade da Vacina
, Vacinas de Produtos Inativados/imunologia
, Vacinas Virais/imunologia
, Fatores Etários
, Anticorpos Neutralizantes/imunologia
, Anticorpos Antivirais/imunologia
, Criança
, Pré-Escolar
, Feminino
, Humanos
, Esquemas de Imunização
, Lactente
, Masculino
, Avaliação de Resultados da Assistência ao Paciente
, Taiwan
, Vacinação
, Potência de Vacina
, Vacinas de Produtos Inativados/administração & dosagem
, Vacinas de Produtos Inativados/efeitos adversos
, Vacinas Virais/administração & dosagem
, Vacinas Virais/efeitos adversos
RESUMO
Proteomic approaches were applied to investigate whether Photobacterium damselae subsp. piscicida (Phdp) can directly sense and respond to growth conditions under different salinities, 0.85% and 3.5% NaCl concentrations, mimicking the osmotic conditions in host and marine water bodies, respectively. Proteins significantly altered were analyzed by two-dimensional gel electrophoresis (2-DE), liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-Q-TOF MS/MS) and bioinformatics analysis, thus resulting in 16 outer membrane proteins (OMPs), 12 inner membrane proteins (IMPs), and 20 cytoplasmic proteins (CPs). Quantitative real-time PCR was also applied to monitor the mRNA expression level of these target proteins. Cluster of orthologous groups of protein (COG) analysis revealed that when shifting from 3.5% to 0.85% salinity, the majority of the up-regulated proteins were involved in posttranslational modification, protein turnover, and chaperones, while the down-regulated proteins were mainly related to energy production and conversion, compatible solutes (carbohydrates, amino acids and their derivatives) biogenesis and transport. Differentially expressed proteins identified in the current study could be used to elucidate the salt adaptation mechanisms of Phdp during their transition between host cells and the marine habitats.
Assuntos
Photobacterium/genética , Photobacterium/fisiologia , Proteoma , Tolerância ao Sal , Animais , Eletroforese em Gel Bidimensional , Doenças dos Peixes/microbiologia , Osmorregulação/genética , Photobacterium/crescimento & desenvolvimento , Photobacterium/ultraestrutura , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Espectrometria de Massas em TandemRESUMO
Pigs co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) have been shown to develop more severe diseases than pigs infected with PRRSV or PCV2 only. The underlying interaction mechanisms between the two viruses in developing the disease are unclear. The present study investigates the mRNA expression of Toll-like receptor (TLR) signaling-related molecules in peripheral blood mononuclear cells from pigs infected with PRRSV or PCV2 or both. The mRNA expression levels were determined by quantitative real-time RT-PCR. Co-infection of pigs with PRRSV and PCV2 resulted in a negatively synergistic effect on the mRNA expression of the negative regulators of TLR, including A20, Bcl-3, IRAK-M, MKP-1, SARM1 and SIGIRR, as well as the TLR downstream transcription factors IRF-1 and IRF-3. A positively synergistic effect of a combined infection of PRRSV and PCV2 on the CD14 mRNA expression was also observed.
Assuntos
Circovirus/genética , Regulação da Expressão Gênica/genética , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Transdução de Sinais/genética , Doenças dos Suínos/virologia , Receptores Toll-Like/metabolismo , Animais , Anticorpos Antivirais/sangue , Coinfecção/virologia , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Sus scrofa , Suínos , Doenças dos Suínos/metabolismo , Receptores Toll-Like/genéticaRESUMO
Field and experimental studies have shown that co-infection of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) increases the severity of the disease. The present study investigates the mRNA expression profile of Toll-like receptors (TLRs) in pigs co-infected with PRRSV and PCV2. SPF pigs were infected with PRRSV, PCV2 or in a combination of both. The mRNA expression levels of TLRs and related cytokines in peripheral blood mononuclear cells (PBMCs) of pigs were determined by quantitative real-time RT-PCR. The mRNA expression profiles of TLRs by PBMCs from pigs co-infected with PRRSV and PCV2 displayed two distinct patterns: an increased expression profile for TLRs2, 4 and 8, and a decreased expression profile for TLRs3, 7 and 9. An up-regulated expression of IL-1ß and IL-10 mRNA and a down-regulated expression of INF-α and TNF-α mRNA in PBMCs of co-infected pigs were also observed.
Assuntos
Infecções por Circoviridae/veterinária , Coinfecção/veterinária , Regulação da Expressão Gênica/imunologia , Leucócitos Mononucleares/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Doenças dos Suínos/imunologia , Receptores Toll-Like , Animais , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/virologia , Circovirus/imunologia , Coinfecção/imunologia , Coinfecção/virologia , Citocinas/genética , Citocinas/imunologia , Perfilação da Expressão Gênica , Leucócitos Mononucleares/virologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Suínos , Doenças dos Suínos/virologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
The objective of the present study was to assess the effect of alginate-encapsulated infectious pancreatic necrosis virus antigens in inducing the immune response of Atlantic salmon as booster vaccines. One year after intraperitoneal injection with an oil-adjuvanted vaccine, post-smolts were orally boosted either by 1) alginate-encapsulated IPNV antigens (ENCAP); 2) soluble antigens (UNENCAP) or 3) untreated feed (control). This was done twice, seven weeks apart. Sampling was done twice, firstly at 7 weeks post 1st oral boost and the 2nd, at 4 weeks after the 2nd oral boost. Samples included serum, head kidney, spleen and hindgut. Serum antibodies were analyzed by ELISA while tissues were used to assess the expression of IgM, IgT, CD4, GATA3, FOXP3, TGF-ß and IL-10 genes by quantitative PCR. Compared to controls, fish fed with ENCAP had a significant increase (p<0.04) in serum antibodies following the 1st boost but not after the 2nd boost. This coincided with significant up-regulation of CD4 and GATA3 genes. In contrast, serum antibodies in the UNENCAP group decreased both after the 1st and 2nd oral boosts. This was associated with significant up-regulation of FOXP3, TGF-ß and IL-10 genes. The expression of IgT was not induced in the hindgut after the 1st oral boost but was significantly up-regulated following the 2nd one. CD4 and GATA3 mRNA expressions exhibited a similar pattern to IgT in the hindgut. IgM mRNA expression on the other hand was not differentially regulated at any of the times examined. Our findings suggest that 1) Parenteral prime with oil-adjuvanted vaccines followed by oral boost with ENCAP results in augmentation of the systemic immune response; 2) Symmetrical prime and boost (mucosal) with ENCAP results in augmentation of mucosal immune response and 3) Symmetrical priming and boosting (mucosal) with soluble antigens results in the induction of systemic immune tolerance.
Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/prevenção & controle , Vírus da Necrose Pancreática Infecciosa/imunologia , Salmo salar/imunologia , Vacinação , Vacinas , Alginatos , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/prevenção & controle , Doenças dos Peixes/imunologia , Ácido Glucurônico , Ácidos Hexurônicos , Imunidade Ativa/imunologia , Imunidade nas Mucosas/imunologiaRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), derived from the outer-membrane protein (OMP) fraction, has been used as a potential candidate for vaccine development. The gene-encoding 37 kDa GAPDH outer membrane protein (OMP) from Edwardsiella ictaluri was amplified using polymerase chain reaction (PCR) and was cloned and expressed in Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and nucleotide and amino acid sequencing were used to analyze the expressed antigenic protein and gene encoding this protein. Comparative DNA and protein sequence analysis of GAPDH from E. ictaluri GAPDHs from several Gram-negative bacterial species within the Enterobacteriaceae family revealed that the GAPDHs within this group are highly conserved and share a sequence similarity of 75-100% with E. ictaluri GDPDH. Rabbit antiserum raised against the E. ictaluri recombinant GAPDH (rGAPDH) protein recognized purified GADPH, indicating that it has a strong immunogenicity. Tilapia fish were intraperitoneally immunized with formalin-killed E. ictaluri whole cells, and rGAPDH (30 µg fish(-1)) from E. ictaluri, both of which were emulsified in ISA 763A adjuvant. At 3 months after immunization, fish were challenged with the E. tarda strain to assess vaccine efficacy; the relative percent survival (RPS) values were found to exceed 71.4%. The specific mean antibody titer log2 level of groups vaccinated with rGAPDH at 3 months was significantly higher than that of non-vaccinated fish (control group). Therefore, this recombinant protein can be considered a multi-purpose candidate vaccine against several pathogenic bacteria.
Assuntos
Vacinas Bacterianas/imunologia , Edwardsiella ictaluri/enzimologia , Edwardsiella tarda/imunologia , Infecções por Enterobacteriaceae/prevenção & controle , Doenças dos Peixes/prevenção & controle , Gliceraldeído 3-Fosfato/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Clonagem Molecular , Infecções por Enterobacteriaceae/imunologia , Escherichia coli/genética , Doenças dos Peixes/imunologia , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Análise de Sobrevida , Tilápia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
NBM-T-L-BMX-OS01 (BMX) was derived from the semisynthesis of osthole, isolated from Cnidium monnieri (L.) Cuss., and was identified to be a potent inhibitor of HDAC8. This study shows that HDAC8 is highly expressed in the pancreas and the brain. The function of HDAC8 in the brain has not been adequately studied. Because BMX enhances neurite outgrowth and cAMP response element-binding protein (CREB) activation, the effect of BMX on neural plasticity such as learning and memory is examined. To examine declarative and nondeclarative memory, a water maze, a passive one-way avoidance task, and a novel object recognition task were performed. Results from the water maze revealed that BMX and suberoylanilide-hydroxamic-acid-(SAHA-) treated rats showed shorter escape latency in finding the hidden platform. The BMX-treated animals spent more time in the target quadrant in the probe trial performance. An analysis of the passive one-way avoidance results showed that the BMX-treated animals stayed longer in the illuminated chamber by 1 day and 7 days after footshock. The novel object recognition task revealed that the BMX-treated animals showed a marked increase in the time spent exploring novel objects. Furthermore, BMX ameliorates scopolamine-(Sco-) induced learning and memory impairment in animals, indicating a novel role of BMX in learning and memory.
RESUMO
This study established a highly permissive and decontaminated cell line for growing porcine circovirus type 2 (PCV2). A porcine kidney-15 cell line (PK-15) contaminated with porcine circovirus type 1 (PCV1) was decontaminated by neutralizing with rabbit anti-PCV1 hyperimmune serum. Subsequently, by limiting dilution and cell subcloning, four PCV1-free monoclonal cells were grown to monolayers. Each cell clone and PK-15 cell were infected with PCV2. The PKKC cell clone yielded up to 10(6.8)TCID(50)/ml at 6 days post-infection. In addition, PKKC was free of extraneous viral contamination and exhibited a cytopathic effect (CPE) to PCV2 at 6 days post-infection. The advantages of the PKKC cell are that it can grow a high PCV2 titer and exhibit CPE; therefore, it can be used for PCV2 cultivation, vaccine production, and diagnostic purposes.
Assuntos
Linhagem Celular , Circovirus/crescimento & desenvolvimento , Animais , Células Clonais/virologia , Rim/citologia , Suínos , Fatores de Tempo , Carga Viral , Cultura de Vírus/métodosRESUMO
It is well known that cytokinins are a class of phytohormones that promote cell division in plant roots and shoots. However, their targets, biological functions, and implications in mammalian systems have rarely been examined. In this study, we show that one cytokinin, zeatin riboside, can prevent pheochromocytoma (PC12) cells from serum deprivation-induced apoptosis by acting on the adenosine A(2A) receptor (A(2A)-R), which was blocked by an A(2A)-R antagonist and a protein kinase A (PKA) inhibitor, demonstrating the functional ability of zeatin riboside by mediating through A(2A)-R signaling event. Since the A(2A)-R was implicated as a therapeutic target in treating Huntington's disease (HD), a cellular model of HD was applied by transfecting mutant huntingtin in PC12 cells. By using filter retardation assay and confocal microscopy we found that zeatin riboside reversed mutant huntingtin (Htt)-induced protein aggregations and proteasome deactivation through A(2A)-R signaling. PKA inhibitor blocked zeatin riboside-induced suppression of mutant Htt aggregations. In addition, PKA activated proteasome activity and reduced mutant Htt protein aggregations. However, a proteasome inhibitor blocked both zeatin riboside-and PKA activator-mediated suppression of mutant Htt aggregations, confirming mediation of the A(2A)-R/PKA/proteasome pathway. Taken together, zeatin riboside might have therapeutic potential as a novel neuroprotectant and a lead for treating neurodegenerative disorders.
Assuntos
Citocininas/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Receptor A2A de Adenosina/fisiologia , Animais , Células PC12 , RatosRESUMO
Glycation, one of the post-translational modifications, is known to influence protein structure and biological function. Advanced glycation end products (AGEs) have been shown to cause pathologies of diabetes. Glycation levels in patients with Alzheimer's disease (AD) are higher than in normal people. However, whether the glycation of susceptible proteins is a triggering event for cell damage or simply a result remains to be elucidated. In this study, we demonstrated that ribose-conjugated BSA (Rib-BSA) directly induces PC12 cell death in a dose- and time-dependent manner. The IC(50) is 4.6 µM. Unlike glucose-incubated BSA, Rib-BSA rapidly forms cytotoxic AGEs. PC12 is vulnerable to Rib-BSA. However, fructose can induce AGE formation, although no effect on cell survival was observed. This effect of Rib-BSA is reversed by pretreatment of pioglitazone and rosiglitazone, which belongs to thiazolidinediones (TZDs) and are peroxisome proliferator-activated receptor (PPAR-γ) ligands. Moreover, Rib-BSA upregulates inducible nitric oxide synthase (iNOS), cycloxygenase 2 (COX-2) expression, and p-38 phosphorylation and leaves extracellular regulated protein1/2 (ERK1/2) phosphorylation unchanged. The Rib-BSA-induced signaling changes are blocked by rosiglitazone and confirmed by PPAR-γ small-interfering RNA transfection. The reduction of cell survival by Rib-BSA is blocked by the iNOS inhibitor and p38 inhibitor. No effect on cell survival was observed using the COX-2 inhibitor. Consequently, these results show that Rib-BSA directly inducing PC12 cell death is a triggering event and TZDs protect PC12 cell from Rib-BSA damage. Signaling molecules, such as PPAR-γ, P38, and iNOS, are involved in Rib-BSA-mediated cytotoxicity.