RESUMO
Lectins are proteins that reversibly bind to carbohydrates and are commonly found across many species. The Banana Lectin (BanLec) is a member of the Jacalin-related Lectins, heavily studied for its immunomodulatory, antiproliferative, and antiviral activity. In this study, a novel sequence was generated in silico considering the native BanLec amino acid sequence and 9 other lectins belonging to JRL. Based on multiple alignment of these proteins, 11 amino acids of the BanLec sequence were modified because of their potential for interference in active binding site properties resulting in a new lectin named recombinant BanLec-type Lectin (rBTL). rBTL was expressed in E. coli and was able to keep biological activity in hemagglutination assay (rat erythrocytes), maintaining similar structure with the native lectin. Antiproliferative activity was demonstrated on human melanoma lineage (A375), evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). rBTL was able to inhibit cellular growth in a concentration-dependent manner, in an 8-h incubation, 12 µg/mL of rBTL led to a 28.94% of cell survival compared to cell control with 100%. Through a nonlinear fit out log-concentration versus biological response, an IC50% of 3.649 µg/mL of rBTL was determined. In conclusion, it is possible to state that the changes made to the rBTL sequence maintained the structure of the carbohydrate-binding site without changing specificity. The new lectin is biologically active, with an improved carbohydrate recognition spectrum compared to nBanLec, and can also be considered cytotoxic for A375 cells.
Assuntos
Escherichia coli , Lectinas , Humanos , Animais , Ratos , Lectinas/genética , Lectinas/farmacologia , Escherichia coli/genética , Lectinas de Plantas/genética , Lectinas de Plantas/farmacologia , Lectinas de Plantas/química , Sequência de Aminoácidos , CarboidratosRESUMO
This study investigated the ability of Staphylococcus aureus isolates from milk to form biofilm, through detection of adhesion genes, investigating exopolysaccharide (EPS) production and biofilm formation on polystyrene (PS) and stainless steel (SS) surfaces, and by quantifying the expression of ebpS and cna genes under different temperatures and culture media. Among the 31 isolates, the adhesion genes ebpS and cna were found in 81% and 61% of the isolates, respectively. The screening tests for phenotype revealed that 58% of the isolates were EPS producers, and 45% showed the ability to produce biofilm on PS. Nine of the 31 isolates were selected to verify their ability to form biofilm on SS, of which 3 were non-biofilm producers, 3 were poor biofilm producers, and 3 were moderate biofilm producers. However, all nine isolates produced biofilm on SS, regardless of their phenotypic profile on PS. Reverse-transcriptase quantitative PCR (RT-qPCR) revealed no variation in the expression levels of ebpS and cna genes at different temperatures, except for isolate S24 at 10 °C, for both genes tested. Moreover, RT-qPCR assays revealed that the expression levels of the adhesion genes ebpS and cna are isolate- and temperature-dependent; however, they are independent of the phenotypic biofilm-formation profile.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Biofilmes , Humanos , Leite , Staphylococcus aureus/genética , TemperaturaRESUMO
Leptospirosis is a zoonotic disease worldwide and caused by the pathogenic spirochetes of the genus Leptospira. Bacterins make up the vaccines used against leptospirosis, but they only succeed in providing short-term and serovar-specific protection. The use of Mycobacterium bovis BCG as a live vaccine vector expressing leptospiral antigens is a promising alternative, particularly due to its adjuvant properties. Four distinct portions P1 (lipL32), P2 (ligAni), P3 (lemA:ligAni) and P4 (lipL32:lemA) of a recombinant chimera composed of the lipL32, lemA and ligANI genes from Leptospira interrogans were cloned individually according to the BioBricks® strategy in the plasmid pUP500/PpAN. These constructs were individually transformed into a BCG Pasteur strain, and protein expression was detected by Western blot. For vaccination, 5 groups of 10 Golden Syrian hamsters were used, aged 4-6 weeks - group 1, rBCG (LipL32); group 2, rBCG (LigAni); group 3, rBCG (LemA:LigAni); group 4, (LipL32:LemA); group 5, wild-type BCG Pasteur (negative control). Two doses containing 106 CFU of rBCG were administered subcutaneously, the challenge was performed with 5 × LD50 of Leptospira interrogans serovar Copenhageni L1-130, and the animals were observed for a 30-day period until the endpoint was reached. Humoral immunity was assessed via indirect ELISA, while renal colonisation was assessed by culture and quantitative real-time PCR. All vaccinated groups were protected against lethal challenge and renal colonisation, in comparison with negative control group (P < 0.05). Recombinant vaccines were not effective at inducing significant humoral immunity, which suggests the induction of cellular immunity - a characteristic of M. bovis BCG. In conclusion, all formulations provide 100% significant protection against leptospirosis in hamsters with no renal colonisation. The use of rBCG as a vaccine vector represents a promising alternative for the control of animal leptospirosis, allowing for protection against clinical signs of leptospirosis and renal colonisation.
Assuntos
Leptospira interrogans , Leptospira , Leptospirose , Mycobacterium bovis , Animais , Antígenos de Bactérias/genética , Vacina BCG , Vacinas Bacterianas , Cricetinae , Leptospira interrogans/genética , Leptospirose/prevenção & controle , Mycobacterium bovis/genéticaRESUMO
Abstract DNA vaccines have been evaluated as an option to prevent several diseases. In this study, the capacity of the xanthan biopolymer to improve the DNA vaccines immune response, administered intramuscularly, was evaluated. The experimental vaccines consisted of genes encoding fragments of the proteins LigA and LigB of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. The humoral immune response was evaluated by indirect ELISA. Cytokine expression levels were determined by RT-qPCR. Compared to the control group, the IgG antibody levels of animals immunized with pTARGET/ligAni and pTARGET/ligBrep plasmids associated with xanthan biopolymer were significantly higher than the control group. Additionally, there was a significant increase in IL-17 expression in animals vaccinated with pTARGET/ligBrep and xanthan.
Assuntos
Animais , Feminino , Camundongos , Polissacarídeos Bacterianos , DNA Recombinante/farmacologia , Adjuvantes Imunológicos/farmacologia , Xanthomonas campestris , Vacinas de DNA/farmacologia , Biopolímeros/farmacologia , Ensaio de Imunoadsorção Enzimática , Leptospira interrogans serovar icterohaemorrhagiae , AnticorposRESUMO
For more than 25 years, recombinant Mycobacterium bovis BCG has been genetically engineered for use as a vehicle for antigen expression and immunomodulation, typically through introducing or deleting a gene from BCG genome. However, BCG transformation efficacy is still unpredictable, and cloning and expression of sequences from mycobacteria is difficult to predict due to the lack of standardization. To overcome such limitations, we have employed the BioBrick format to construct a toolbox of several mycobacterial parts, including coding sequences, reporter genes, selective markers, promoters, and other regulatory sequences. Additionally, we have developed and characterized BioBrick-compatible episomal vectors that are able to replicate in M. bovis BCG to enable expression of heterologous antigens. The availability of a BCG Biobrick toolbox will enable any coding sequence to be optimally expressed in BCG. We believe that this mycobacterial toolbox represents a standardized and useful kit to enhance the efficacy and use of recombinant BCG.
Assuntos
DNA Bacteriano/genética , Genes Bacterianos/genética , Mycobacterium bovis/genética , Plasmídeos/genética , Linhagem Celular , Genes Reporter , Vetores Genéticos , Humanos , Regiões Promotoras GenéticasRESUMO
Leptospirosis is a zoonosis that is responsible for one million human cases per year. Fusing multiple immunogenic antigens represents a promising approach to delivering an effective vaccine against leptospirosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a potential vaccine vector due to its adjuvant properties and safety. Two chimeric genes based on genic sequences of ligANI, ligBrep, lipL32, and lemA, were individually cloned into five BioBrick vectors with different promoters (pAN, Hsp60, 18â¯kDa, Ag85B and Ag85B plus signal sequence) for antigen expression in BCG. Groups of ten hamsters were vaccinated with recombinant BCG (rBCG) strains in two doses of 106 CFU and challenged with 5â¯×â¯LD50 of L. interrogans serovar Copenhageni. All rBCG vaccines expressing chimera 1, based on antigens LipL32, LigANI, and LemA, under the control of any promoter, protected 80-100% of the hamsters from challenge (Pâ¯<â¯0.05) and four of them also protected from renal carrier status; for chimera 2, based on LigANI and LigBrep antigens, the only vaccine that afforded survival rates statistically different from the control was the vaccine that incorporated the pAN promoter (60% of survival). A single vaccine dose was sufficient to induce significant IgG levels by all vaccine compositions evaluated; however, humoral response was not related to protection. These findings suggest that the combination of potential vaccine candidates in chimeric antigens and the use of BCG as a live vector are promising strategies by which it is possible to obtain an effective and sterilizing vaccine against leptospirosis.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Leptospira/imunologia , Leptospirose/prevenção & controle , Mycobacterium bovis , Animais , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Cricetinae , Feminino , Imunoglobulina G/sangue , Leptospira/genética , Masculino , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/imunologiaRESUMO
The aims of this study were to evaluate the presence of genes associated with adhesion (cadF), invasion (ciaB), and cytotoxin production (cdtA, cdtB, and cdtC) among Campylobacter jejuni isolates from a poultry slaughterhouse and to investigate the effect of different temperatures on the expression of these virulence-associated genes. A total of 88 C. jejuni isolates from cecum, liver, chicken carcasses, chilled water, and scalding water were submitted to PCR assay for detection of virulence genes. Representative isolates were selected for gene expression evaluation at 37 and 42 °C, according to their virulence gene profile and genotypic typing. All C. jejuni isolates carried the five virulence-associated genes, which play an important role in the infectious process. Differential gene expression by RT-qPCR was observed among C. jejuni isolates at 37 and 42 °C. The expression levels at 37 °C showed upregulation of the ciaB, cdtA, cdtB, and cdtC genes in five isolates, with the exception of ciaB for isolate 4. At 42 °C, upregulation was observed for ciaB and cdtC, cdtA and cdtB, and cadF in four, three, and two isolates, respectively. The C. jejuni isolates expressed the virulence genes evaluated, and the expression is gene- and isolate-dependent and varied according the temperature.
Assuntos
Antígenos de Bactérias/genética , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/genética , Toxinas Bacterianas/genética , Campylobacter jejuni/genética , Proteínas de Transporte/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Virulência/genética , Animais , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/veterinária , Campylobacter jejuni/isolamento & purificação , Campylobacter jejuni/patogenicidade , Galinhas , DNA Bacteriano/genética , Genes Bacterianos , Reação em Cadeia da Polimerase , Doenças das Aves Domésticas/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temperatura , Fatores de VirulênciaRESUMO
Recombinant Mycobacterium bovis BCG vaccines (rBCG) were first developed in the 1990s as a means of expressing antigens from multiple pathogens. This review examines the key structural factors of recombinant M. bovis that influence the expression of the heterologous antigens and the generation of genetic and functional stability in rBCG, which are crucial for inducing strong and lasting immune responses. The fundamental aim of this paper is to provide an overview of factors that affect the expression of recombinant proteins in BCG and the generation of the immune response against the target antigens, including mycobacterial promoters, location of foreign antigens, and stability of the vectors. The reporter systems that have been employed for evaluation of these molecular features in BCG are also reviewed here.
Assuntos
Adjuvantes Imunológicos/genética , Vacina BCG/genética , Mycobacterium bovis/imunologia , Tuberculose/prevenção & controle , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Genes Reporter , Vetores Genéticos/genética , Humanos , Mycobacterium bovis/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Leptospirosis is an emerging zoonosis attributed to multiple reservoirs. Climatic conditions influence the transmission of pathogenic leptospires, which require warm and humid conditions for survival. The influence of seasonality in human and animal leptospirosis in the subtropical region of Brazil remains poorly understood. METHODS: We performed a retrospective study to describe the patterns of human and animal exposure to leptospirosis and their association with precipitation events in Southern Brazil. Rainfall data were obtained from satellite images. Serum samples were tested using the microscopic agglutination test (MAT); samples with titer ≥ 100 were defined as seroreactive. Linear regression and Pearson's correlation were performed to assess whether there is a relationship between these variables. RESULTS: We found that precipitation events were not significantly associated with the exposure to leptospirosis in humans or animal species, except for dogs. The interspecies analysis revealed an association between canine and human exposure to leptospirosis. Leptospira kirschneri serovar Butembo (serogroup Autumnalis) presented the highest seroreactivity in humans. CONCLUSION: This study provides valuable insights in human and animal leptospirosis in Southern Brazil. These insights will be essential to design intervention measures directed to reduce disease dissemination.
Assuntos
Leptospirose/epidemiologia , Zoonoses/epidemiologia , Animais , Anticorpos Antibacterianos/sangue , Brasil/epidemiologia , Cães , Humanos , Leptospirose/veterinária , Estudos Retrospectivos , Fatores de RiscoRESUMO
Enzootic Pneumonia (EP) is caused by the Mycoplasma hyopneumoniae pathogenic bacteria, and it represents a significant respiratory disease that is responsible for major economic losses within the pig industry throughout the world. The bacterins that are currently commercially available have been proven to offer only partial protection against M. hyopneumoniae, and the development of more efficient vaccines is required. Several recombinant antigens have been evaluated via different immunization strategies and have been found to be highly immunogenic. This work describes the construction and immunological characterization of a multi-antigen chimera composed of four M. hyopneumoniae antigens: P97R1, P46, P95, and P42. Immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in Escherichia coli, and the chimeric protein was recognized by specific antibodies against each subunit, as well as by convalescent pig sera. The immunogenic properties of the chimera were then evaluated in a mice model through two recombinant vaccines that were formulated as follows: (1) purified chimeric protein plus adjuvant or (2) recombinant Escherichia coli bacterin. The immune response induced in BALB/c mice immunized with each formulation was characterized in terms of total IgG levels, IgG1, and IgG2a isotypes against each antigen present in the chimera. The results of the study indicated that novel chimeric protein is a potential candidate for the future development of a more effective vaccine against EP.
Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Imunoglobulina G/sangue , Mycoplasma hyopneumoniae/imunologia , Pneumonia Suína Micoplasmática/prevenção & controle , Adjuvantes Imunológicos , Animais , Antígenos de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Imunização/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mycoplasma hyopneumoniae/genética , Pneumonia Suína Micoplasmática/microbiologia , Proteínas Recombinantes , Suínos , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES: The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS: Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS: The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION: The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.
Assuntos
Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Mycobacterium bovis/imunologia , Animais , Antígenos de Bactérias/metabolismo , Vacina BCG/genética , Escherichia coli/genética , Feminino , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/genética , Plasmídeos/genética , Plasmídeos/imunologiaRESUMO
BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.
Assuntos
Animais , Feminino , Camundongos , Plasmídeos/genética , Plasmídeos/imunologia , Vacina BCG/genética , Vacina BCG/imunologia , Vetores Genéticos/imunologia , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Escherichia coli/genética , Vetores Genéticos , Camundongos Endogâmicos BALB CRESUMO
Leptospirosis is a major public health problem with an incidence of over one million human cases each year. It is a globally distributed, zoonotic disease and is associated with significant economic losses in farm animals. Leptospirosis is caused by pathogenic Leptospira spp. that can infect a wide range of domestic and wild animals. Given the inability to control the cycle of transmission among animals and humans, there is an urgent demand for a new vaccine. Inactivated whole-cell vaccines (bacterins) are routinely used in livestock and domestic animals, however, protection is serovar-restricted and short-term only. To overcome these limitations, efforts have focused on the development of recombinant vaccines, with partial success. Reverse vaccinology (RV) has been successfully applied to many infectious diseases. A growing number of leptospiral genome sequences are now available in public databases, providing an opportunity to search for prospective vaccine antigens using RV. Several promising leptospiral antigens were identified using this approach, although only a few have been characterized and evaluated in animal models. In this review, we summarize the use of RV for leptospirosis and discuss the need for potential improvements for the successful development of a new vaccine towards reducing the burden of human and animal leptospirosis.
Assuntos
Vacinas Bacterianas/imunologia , Leptospira/imunologia , Animais , Antígenos de Bactérias/imunologia , Humanos , Leptospira/genética , Leptospirose/imunologia , Leptospirose/microbiologia , Vacinas Sintéticas/imunologiaRESUMO
Peripheral inflammatory stimuli may activate a brain neuroinflammatory processes with consequences in brain function. The present study investigated if anthocyanins (ANT) consumption was able to prevent the memory loss, the neuronal damage, and the neuroinflammatory processes triggered by the intraperitoneal lipopolysaccharide (LPS) administration. C57BL6 male mice were treated with ANT (30-100 mg/kg by gavage). With a single dose or during 10 days, before be challenged with LPS (250 µg/kg intraperitoneally single administration), a classical inductor of inflammation. The data obtained showed that ANT was able to confer protection against the memory impairment after 10 days of ANT treatment (100 mg/kg). This phytonutrient also prevented the hypothermia episode induced by LPS. Moreover, ANT prevented the increase in protein carbonyl, NOx, and MDA levels in the hippocampus and cerebral cortex (4 and 24 h) in animal challenged with LPS. ANT showed a protective effect on the increase in the pro-inflammatory cytokines content, especially Interleukin (IL)-1ß, tumoral necrosis factor-α and on the reduction of IL-10 induced by LPS. ANT 100 mg/kg prevented the infiltration of peripheral immune cells in the hippocampus at 24 h post-LPS administration. In parallel, LPS increased the activity of myeloperoxidase in cortex and hippocampus, and ANT prevented this effect, also reducing microglia (Iba-1) and astrocyte (GFAP) immunoreactivity. Thus, our data support that ANT are a promising therapeutic component against brain disorders associated with process of neuroinflammation. Graphical Abstract á .
Assuntos
Antocianinas/uso terapêutico , Inflamação/tratamento farmacológico , Transtornos da Memória/tratamento farmacológico , Animais , Antocianinas/farmacologia , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Hipocampo/enzimologia , Hipocampo/patologia , Hipotermia Induzida , Inflamação/complicações , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/administração & dosagem , Masculino , Transtornos da Memória/complicações , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Lectins are proteins able to interact specifically and reversibly with carbohydrates. They are present in all living beings, particularly in legume seeds, which have many biological functions. The aim of this study was to isolate, characterize and verify antioxidant, anti-hemolytic, antitumor and gastroprotective activities in a lectin present in seeds of Phaseolus lunatus L. var. cascavel (PLUN). The isolation of lectin was performed by size exclusion chromatography on Sephadex G-100, which was isolated from a protein capable of agglutinating only human erythrocytes type A, being this the only inhibited haemagglutination n-acetyl-d-galactosamine. Its weight was estimated by PAGE is 128kDa. The lectin is thermostable up to 80°C and is active between pH 2-11. As 8M urea was able to denature the lectin. PLUN is a glycoprotein consisting of 2% carbohydrate and has antioxidant action with ascorbic acid equivalent antioxidant capacity (µMAA/g) of 418.20, 326 and 82.9 for total antioxidant activity, ABTS radical capture and capture of DPPH radical, respectively. The lectin has antitumor activity against melanoma derived cells at doses of 100 and 50mg/ml, reducing up to 83% tumor cells, and gastroprotective action, reducing up to 63% damaged area of ââthe stomach induced by ethanol.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Fármacos Gastrointestinais/farmacologia , Phaseolus/química , Lectinas de Plantas/farmacologia , Úlcera Gástrica/tratamento farmacológico , Acetilgalactosamina/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Benzotiazóis/antagonistas & inibidores , Benzotiazóis/química , Compostos de Bifenilo/antagonistas & inibidores , Compostos de Bifenilo/química , Linhagem Celular Tumoral , Eritrócitos/efeitos dos fármacos , Etanol , Fármacos Gastrointestinais/química , Fármacos Gastrointestinais/isolamento & purificação , Testes de Hemaglutinação , Humanos , Masculino , Camundongos , Peso Molecular , Picratos/antagonistas & inibidores , Picratos/química , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Desnaturação Proteica , Sementes/química , Extração em Fase Sólida/métodos , Estômago/efeitos dos fármacos , Estômago/patologia , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/patologia , Ácidos Sulfônicos/antagonistas & inibidores , Ácidos Sulfônicos/química , Ureia/químicaRESUMO
Neosporosis is a disease caused by the apicomplexan parasite Neospora caninum, which is closely related to Toxoplasma gondii. N. caninum infection represents an important cause of reproductive failure in sheep, goats, horses, and cattle worldwide. The diagnosis of neosporosis is based on the detection of pathogen-specific antibodies in animal sera or the presence of tissue cysts. However, morphological similarities and serological cross-reactivity between N. caninum and T. gondii can result in the misdiagnosis. In this study, the N. caninum tachyzoite surface protein Ncp-43 was expressed in a recombinant form to elicit polyclonal antibodies (pAb) response. The pAb was purified and conjugated to horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC) to detect the recombinant and native Ncp-43 proteins, respectively. The pAb and pAb/HRP were able to recognize rNcp-43 by dot blot and ELISA, and pAb/FITC immunolabeled the apical complex of tachyzoites. A blocking enzyme-linked immunosorbent assay (b-ELISA) was performed to evaluate pAb/HRP as a diagnostic tool. The mean percent inhibition for the positive and negative serum samples from cattle with neosporosis was significantly different (P < 0.0001). These results suggest that the pAb may bind to the same epitopes of Ncp-43 as anti-N. caninum antibodies in the positive samples tested. The b-ELISA using the pAb/HRP can facilitate diagnostic testing for neosporosis, since fewer steps are involved, and cross-reactivity with secondary antibodies is avoided. In summary, this report describes the production of antibodies against N. caninum, and evaluates the potential of these tools for the development of new diagnostic tests for neosporosis.
Assuntos
Anticorpos/química , Antígenos de Protozoários/química , Neospora/isolamento & purificação , Proteínas de Protozoários/química , Animais , Anticorpos/sangue , Anticorpos/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/parasitologia , Coccidiose/veterinária , Ensaio de Imunoadsorção Enzimática , Masculino , Neospora/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ∆leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ∆leuD strain. The inhibitory effect of BCG ∆leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ∆leuD/Ag85B treatment was evaluated by Western blotting. BCG ∆leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ∆leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ∆leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ∆leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer.
Assuntos
Antígenos de Bactérias/imunologia , Citotoxicidade Imunológica , Expressão Gênica , Mycobacterium bovis/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapia , Antígenos de Bactérias/genética , Apoptose , Linhagem Celular Tumoral , Humanos , Imunoterapia , Modelos Biológicos , Mycobacterium bovis/genética , Regulação para Cima , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/fisiopatologiaRESUMO
Mycobacterium bovis is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with leuD. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with M. bovis, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur.
Assuntos
Vacina BCG/administração & dosagem , Doenças dos Bovinos/prevenção & controle , Mycobacterium bovis/imunologia , Tuberculose Bovina/prevenção & controle , Animais , Bovinos , Doenças dos Bovinos/imunologia , Primers do DNA , Citometria de Fluxo , Interferon gama/biossíntese , Reação em Cadeia da Polimerase , Teste Tuberculínico , Tuberculose Bovina/imunologiaRESUMO
BACKGROUND: Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins. RESULTS: The vaccine candidates LigANI and LipL32 were cloned and expressed in P. pastoris as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis. CONCLUSIONS: The expression of LigANI and LipL32 in P. pastoris resulted in a significant increase in yield compared to expression in E. coli. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Leptospira/imunologia , Leptospirose/imunologia , Lipoproteínas/genética , Pichia/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Glicosilação , Humanos , Leptospira/enzimologia , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/metabolismoRESUMO
Introducao: Atualmente no Brasil, as doencas cardiovasculares (DCVs) sao as patologias de maior morbimortalidade. Os principais fatores de risco sao ma alimentacao, sedentarismo, tabagismo, etilismo, obesidade, dislipidemias, Diabetes mellitus e estresse. O objetivo do estudo foi avaliar os perfis lipidico e glicidico e identificar fatores de risco associados as doencas cardiovascularesem caminhoneiros. Materiais e Metodos: A amostra foi composta por 45 caminhoneiros que passaram pelo Centro Unificado de Fronteira, entre Brasil e Argentina, nos dias 23 e 24 de abril e 21 de maio de 2007. Foi avaliado o indice de massa corporal, a circunferencia abdominal, a pressao arterial, habitos alimentares, tabagismo e historia familiar de DCVs. O perfil lipidico foi definido pelas determinacoes do colesterol total, HDL e LDL colesterol, triglicerideos; e o perfil glicidico, atraves da glicemia de jejum. Resultados: Foram encontrados 33 casos de dislipidemias, classificados em hipercolesterolemia (33%), hiperlipidemia mista (15%) e hipertrigliceridemia (30%). Os fatores de risco presentes foram: obesidade (42%), obesidade visceral (78%), hipertensao (47%) e tabagismo (20%).Conclusao: Mesmo se referindo a um grupo especifico, o trabalho foi importante, pois identificou fatores de risco presentes na categoria profissional de motoristas de caminhao e com isso sua maior predisposicao ao desenvolvimento de DCVs.
Introduction: Nowadays in Brazil, the cardiovascular diseases (CVDs) are the major morbidities pathologies. The maincause of these pathologies is improper lifestyle that results in risk factors as: bad feeding, sedentary lifestyle, smoking, alcoholism, obesity, dyslipidemia, Diabetes mellitus and stress. This study had as objective the lipid and glucose profiles evaluation, and the identification of risk factors related to the truck drivers CVDs development. Materials and Methods: The sample was composed by 45 truck drivers that went by the Unified Center of Border, among Brazil and Argentina, at the days April 23, April 24 and May 21, 2007. Thestudy evaluated the CMI (Corporal mass index), the abdominal circumference and the arterial pressure and investigated the physical activity practice, alimentary behavor, tobacco consumption, coronary artery disease family history and Diabetes mellitus. The lipid profile was defined by the biochemical determinations of the total cholesterol, the HDL fraction, the LDL fraction, triglyceride, and the glucose profile, through the fast glycemia. Results: The study demonstrated that 33 of dislipidemias cases classified at hypercholesterolemia(33%), mingle hiperlipidemia (15%) and hypertriglyceridemia (30%). The risk factors showed were obesity (42%), visceral obesity (78%), hypertension (47%) and smoke (20%). Conclusion: Although the data presented in this study refers to only one placeand a specific group, the work was important, because it evaluated the risk factors exhibition extension that the truck drivers' professional category is submitted, and with that its largest predisposition to the development of CVDs.