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BACKGROUND: The BioBrick construction as an approach in synthetic biology provides the ability to assemble various gene fragments. To date, different BioBrick strategies have been exploited for assembly and cloning of a variety of gene fragments. We present a new BioBrick strategy, here referred as Asis-Sal-Pac BioBrick, which we used for the assembly of NDV as a candidate for single-stranded non-segmented, negative-sense RNA genome viruses. RESULTS: In the present study, we isolated three NDVs from clinical samples which were classified into the VIId genotype based on their pathogenicity and phylogenetic analyses. Then, SalI, AsisI, and PacI enzymes were used to design and develop a novel BioBrick strategy, which enabled us to assemble the NDV genome, adopting the "rule of six". In this method, in each assembly step, the restriction sites in the newly formed destination plasmid are reproduced, which will be used for the next insertion. In this study using two overlapping PCRs, the cleavage site of the F gene was also modified from 112RRQKRF117to 112GRQGRL117 in order to generate the attenuated recombinant NDV. Finally, in order to construct the recombinant NDV viruses, the plasmids harboring the assembled full-length genome of the NDV and the helper plasmids were co-transfected into T7-BHK cells. The rescue of the recombinant NDVwas confirmed by RT-PCR and HA tests. CONCLUSIONS: These findings suggest that the combination of reverse genetic technology and BioBrick assembly have the potential to be applied for the development of novel vaccine candidates. This promising strategy provides an effective and reliable approach to make genotype-matched vaccines against specific NDV strains or any other virus.
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BACKGROUND: Designing a potent recombinant vaccine, using the appropriate subunits with the greatest effect on stimulating the immune system, especially in the case of intracellular pathogens such as gram negative Brucella Melitensis bacteria, is of great importance. In this study, three repeats of 27 amino acids of the immunogenic epitope derived from OMP31 antigen (3E) from the Brucella melitensis, in a protective manner against Brucellosis have been used. To fortify the delivery system of recombinant antigens, IL-2 cytokine as a molecular adjuvant was fused to recombinant constructs. Recombinant proteins were evaluated for immunological studies in a mouse model (BALB/c). RESULTS: The results showed that all recombinant proteins could stimulate the immune system to produce Th1 cytokines and antibodies in compare to the negative control treatments. 3E-IL2 and then OMP31-IL2 proteins stimulated higher levels of IFN-γ and IL-2 compared to the other treatments (p < 0.05). Also, the results indicated that experimental treatments produced a higher level of IgG2a isotype than IgG1 isotype. In addition, the findings of the experiment showed that the presence of chemical adjuvant (IFA) along with molecular adjuvant can play a significant role in stimulating the immune system. After determining the potency of recombinant structures, their efficacy in stimulating the immune system were also evaluated. B. melitensis M16 strain was used to challenge 30 days after last immunization. The microbial load of the splenocyte in the treatments receiving chimeric proteins were significantly lower. Also, Wright serological test confirmed that these treatments had the lowest agglutination rate, as well as the positive treatment, while in the negative treatments in excess of blood serum dilutions, agglutination rate were more than 2 + . CONCLUSIONS: 3E-IL2 treatment showed the best performance compared to other recombinant proteins and could be considered as the suitable candidate for further research on the production of recombinant vaccine against Brucella.
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Proteínas da Membrana Bacteriana Externa/imunologia , Vacina contra Brucelose/genética , Brucella melitensis/imunologia , Brucelose/prevenção & controle , Animais , Proteínas da Membrana Bacteriana Externa/genética , Vacina contra Brucelose/imunologia , Brucelose/imunologia , Feminino , Imunoglobulina G , Interleucina-2 , Camundongos Endogâmicos BALB C , Baço/microbiologia , Vacinas Sintéticas/imunologiaRESUMO
Influenza H9N2 virus mostly infects avian species but poses a potential health risk to humans. Little is known about the mammalian host immune responses to H9N2 virus. To obtain insight into the innate immune responses of human lung epithelial cells to the avian H9N2 virus, the expressions of pro-inflammatory cytokines and chemokine in the human airway epithelial cells infected with avian H9N2 virus were examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). H9N2 virus was able to cultivate in the human lung epithelial cell line (A549) and stimulate production of pro-inflammatory cytokines (IL-1ß, IL-6) and chemokine (IL-8). Expressions of cytokine genes were up-regulated to a significantly higher level for IL-1ß (p < 0.01), IL-6 (p < 0.01 after 12 hours and p < 0.05 after 24 hours) and IL-8 (p < 0.01 after 12 hours and p < 0.001 after 24 hours) in virus-cultured A549 cells as compared with non-virus-cultured cells. The amount of IL-6 and IL-1ß proteins secreted into the culture medium was also increased after virus culture infection of A549 cell line compared to non-virus-cultured A549 cells and were significant in both IL-1ß (p < 0.05 in 18 hours and p < 0.001 in 24-48 hours harvested supernatant) and IL-6 (p < 0.001). Silencing the p65 component of NF-κB in A549 cells suppressed the stimulatory effects of influenza virus on secretion of pro-inflammatory cytokines and chemokine. The findings in this study will broaden our understanding of host innate immune mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.
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Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Vírus da Influenza A Subtipo H9N2/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Influenza Humana/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Regulação para Cima , Replicação ViralRESUMO
DNA vaccine represents a powerful approach for prevention of avian H5N1 influenza infection. Yet, DNA vaccine-induced immune responses might be limited by the short duration of antigen expression. As a strategy to enhance adaptive immune responses elicited by a hemagglutinin 5 (H5) DNA vaccine, we explored the effect of co-administration of a DNA encoding X-linked inhibitor of apoptosis protein (XIAP) as a modulator of apoptosis and a stimulator of inflammatory signaling. In cultured cells as early as 24 hours (h), we found that the DNA vaccine encoded H5 antigen was a potent stimulator of apoptosis, and the H5 pro-apoptotic activity was significantly suppressed by the co-expression of full-length XIAP or mutant XIAP (ΔRING). However, full-length XIAP showed a higher potency than mutant XIAP (ΔRING) in the inhibition of H5-induced apoptosis. We also compared the immunizing ability of transmembrane and secretory forms of H5. Mice vaccinated (twice with 3-week intervals) with the secretory form of H5 showed higher hemagglutination inhibition (HI) antibody titers than mice vaccinated with the transmembrane form of H5. Furthermore, co-administration of XIAP with the secretory form of H5 resulted into a stronger antibody response than the transmembrane form of H5. Our findings suggest that in the design of DNA vaccines for a given pro-apoptotic antigen, using an anti-apoptotic molecular adjuvant and the secretory form of antigen may be a greater stimulus to induce immune responses.
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Adjuvantes Imunológicos/administração & dosagem , Virus da Influenza A Subtipo H5N1/fisiologia , Vacinas contra Influenza/administração & dosagem , Influenza Aviária/prevenção & controle , Vacinas de DNA/administração & dosagem , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Apoptose , Aves , Células CHO , Cricetulus , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Imunidade Humoral , Imunização , Vacinas contra Influenza/genética , Influenza Aviária/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Seminal plasma and follicular fluid (FF) cytokine analysis are valuable tools for diagnoses and validation of therapeutic approaches for improving the chance of conception. Despite the initial discovery over a decade ago, the IL-17 family has not received much attention in the case of infertility. In this study, we analyzed the level of IL-17A in seminal plasma, follicular fluid and blood serum of infertile patients with different clinical diagnoses by Enzyme Linked Immunosorbent Assay (ELISA). The results showed that the level of IL-17A was higher in seminal plasma and blood serum of varicocele patients than the control group. The level of this cytokine was higher in follicular fluid of endometriosis, polycystic ovary syndrome (PCOS) and tubal factor patients than the control group. A similar elevation in IL-17A level was observed in blood serum of these patients. Furthermore, there was a correlation between the numbers of meiosis I (MI) oocytes and the level of blood serum and follicular fluid IL-17A in PCOS patients. Our data suggest a putative role of IL-17A in mediating these conditions and may have possible applications in the development of more effective diagnostic tools and therapeutic treatments for human reproductive disorders.
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Líquido Folicular/metabolismo , Infertilidade Feminina/metabolismo , Infertilidade Masculina/metabolismo , Interleucina-17/metabolismo , Sêmen/metabolismo , Adulto , Endometriose/metabolismo , Feminino , Humanos , Interleucina-17/sangue , Masculino , Síndrome do Ovário Policístico/metabolismo , Varicocele/metabolismo , Adulto JovemRESUMO
BACKGROUND: The hygiene hypothesis suggests that early life exposure to helminth infections can reduce hypersensitivity in the immune system. OBJECTIVE: The present study aims to evaluate the effects of Toxocara cati (T. cati) somatic products on allergic airway inflammation. METHODS: Between 2018 and 2020, T. cati adult worms were collected from stray cats in Mashhad, Iran (31 out of 186 cats), and their somatic extract was collected. Thirty BALB/c mice were equally divided into three groups, including the OVA group (sensitized and challenged with ovalbumin), the somatic administered group (received somatic extract along with ovalbumin sensitization), and the PBS group (sensitized and challenged with phosphate buffer saline). Bronchoalveolar lavage (BAL) fluid was collected to assess the number of cells, and lung homogenates were prepared for cytokine analysis. Histopathological analysis of the lungs was performed, and inflammatory cells and mucus were detected. Cytokine levels (IL-4, IL-5, IL-10) were measured using enzyme-linked immunosorbent assay (ELISA), and ovalbumin-specific immunoglobulin E (IgE) levels were determined using a capture ELISA. RESULTS: The somatic group significantly decreased regarding the lung pathological changes, including peribronchiolitis, perivasculitis, and eosinophil influx, compared to the group treated with ovalbumin alone. These changes were accompanied by a decrease in proinflammatory cytokines IL-4 and IL-5 and an increase in the anti-inflammatory cytokine IL-10, indicating a shift toward a more balanced immune response. The number of inflammatory cells in the BAL fluid was also significantly reduced in the somatic group, indicating a decrease in inflammation. CONCLUSION: These preclinical findings suggest that in experimental models, T. cati somatic extract exhibits promising potential as a therapeutic agent for mitigating allergic airway inflammation. Its observed effects on immune response modulation and reduction of inflammatory cell infiltration warrant further investigation in clinical studies to assess its efficacy and safety in human patients.
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Citocinas , Camundongos Endogâmicos BALB C , Toxocara , Animais , Camundongos , Toxocara/imunologia , Toxocara/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/imunologia , Imunoglobulina E/imunologia , Imunoglobulina E/sangue , Ovalbumina/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/parasitologia , Pulmão/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/imunologia , Asma/imunologia , Asma/tratamento farmacológico , Modelos Animais de Doenças , Gatos , Feminino , Toxocaríase/tratamento farmacológico , Toxocaríase/imunologia , Toxocaríase/parasitologiaRESUMO
The innate immune sensing of nucleic acids using effective immunoadjuvants is critical for increasing protective immune responses against cancer. Stimulators of interferon genes (STING) and toll-like receptor 9 (TLR9) agonists are considered promising candidates in several preclinical tumor models with the potential to be used in clinical settings. However, the effects of such treatment on tumor stroma are currently unknown. In this study, we investigated the immunotherapeutic effects of ADU-S100 as a STING agonist and CpG ODN1826 as a TLR9 agonist in a preclinical model of colon carcinoma. Tumor-bearing mice were treated intratumorally on days 10 and 16 post-tumor inoculation with ADU-S100 and CpG ODN1826. Cytokine profiles in the tumor and spleen, tumor cell apoptosis, the infiltration of immune cells, and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) were evaluated to identify the immunological mechanisms after treatment. The powerful antitumor activity of single and combination treatments, the upregulation of the expression of pro-inflammatory cytokines in the tumor and spleen, and the recruitment and infiltration of the TME by immune cells revealed the synergism of immunoadjuvants in the eradication of the colon carcinoma model. Remarkably, the significant downregulation of CAFs in the TME indicated that suppression of tumorigenesis occurred after immunoadjuvant therapy. The results illustrate the potential of targeting the STING and TLR9 pathways as powerful immunoadjuvants in the treatment of preclinical colon carcinoma and the possibility of harnessing these pathways in future therapeutic approaches.
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Fibroblastos Associados a Câncer , Carcinoma , Neoplasias do Colo , Animais , Camundongos , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Fibroblastos Associados a Câncer/metabolismo , Neoplasias do Colo/terapia , Imunoterapia , Receptor Toll-Like 9/agonistas , Microambiente TumoralRESUMO
Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in a large percentage of breast cancers. Monoclonal antibodies targeting HER2 are vastly used for both diagnostic and therapeutic aims. However, identifying a new molecular probe against HER2 with improved diagnostic and therapeutic features is of great importance. In this report, we have applied the cell systematic evolution of ligands by exponential enrichment (SELEX) strategy for 16 selection rounds to generate an enriched pool of aptamers that specifically recognize the HER2 positive cell line. During the Cell SELEX procedure, a human HER2-overexpressing breast cancer cell line and a human HER2 negative breast cancer cell line were used. Our results reveal that polymerase chain reaction (PCR) amplification of random DNA libraries and the selected single-stranded DNA pool in different Cell SELEX rounds are different from what we expect from PCR amplification of homologous DNA. Our results also confirmed previous studies describing positive HER2 status of SK-BR3 and the absence of the HER2 expression in the MDA-MB468. We also developed a new method, Cell enzyme-linked assay, to monitor the enrichment of aptamers in a given round of Cell SELEX. This method would also be useful in other experiments using live cell enzyme-linked immunosorbent assay on adherent cells.
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Aptâmeros de Nucleotídeos/biossíntese , Neoplasias da Mama/diagnóstico , Sondas de DNA/biossíntese , Receptor ErbB-2/análise , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , Sondas de DNA/química , Feminino , Humanos , Ligantes , Receptor ErbB-2/química , Receptor ErbB-2/genéticaRESUMO
The present study investigated the effects of overripe pulp and green peel extract and powder of banana fruit (Musa. cavendish) on haematological, biochemical, immunological, health, and performance of Holstein dairy calves. In all, 40 newborn calves were randomly divided into four groups of 10 animals. In the control group, animals received no banana meal. In group 1, calves were supplemented with 2 g (dry matter)/kg body weight/day of overripe banana pulp extract. The calves in group 2 were supplemented with 1 g (dry matter) of overripe banana pulp extract/kg body weight/day and 1 g (dry matter) of green banana peel extract/kg body weight/day. The animals in group 3 were supplemented with 2 g/kg body weight/day of green banana peel powder. The feeding period of calves on the tested supplements was 5 days. Blood samples and other evaluations were taken on day 0 (at birth, before supplementation) and on days 7, 15 and 30. Just a trend towards better average daily weight gain was seen in groups 2 and 3 than others (p = 0.073). Significant group and sampling time interactions were seen for the quantities of RBC (group 1 was lower than other groups at day 30), MCV (group 3 was lower than other groups at day 30) and MCH (group 1 was higher than other groups at day 30) (p < 0.05). A trend towards significance in values of IgG (group 1 was lower than other groups at days 15 and 30) and bilirubin (higher values at day 7 in groups 1 and 2 than control, higher amounts at days 15 and 30 in groups 3 and 2 than control, respectively) was also observed. In conclusion, banana supplementation in neonatal calves had beneficial effects on the values of RBC, MCV, MCH, bilirubin, IgG and average daily weight gain in dairy calves.
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Bovinos/fisiologia , Imunidade/efeitos dos fármacos , Musa/química , Extratos Vegetais/metabolismo , Ração Animal/análise , Animais , Análise Química do Sangue/veterinária , Bovinos/sangue , Bovinos/crescimento & desenvolvimento , Bovinos/imunologia , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Frutas/química , Testes Hematológicos/veterinária , Extratos Vegetais/administração & dosagem , Pós , Distribuição AleatóriaRESUMO
BACKGROUND: Cystic echinococcosis (CE) is a zoonotic disease caused by infection with Echinococcus granulosus. Toll-like receptors (TLRs) as the first line of defense against various parasites play a critical role in sensing and triggering anti-parasite responses. METHODS: The study was conducted at the Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Iran in 2019. Ovine peripheral blood mononuclear cells (PBMCs) were stimulated with hydatid cyst-derived antigens including hydatid cyst fluid (HCF), germinal layer antigens (GL), somatic and excretory/secretory (ES) products of protoscoleces (PSC). To investigate whether the expression of TLR2 and TLR4 was altered during exposure to these antigens, PBMCs were stimulated with two different concentrations at different time points. RESULTS: After exposure of PBMCs to ES and somatic antigens of protoscoleces (PSC) the expression of TLR2 and TLR4 was down-regulated in comparison with control group. Similarly, HCF markedly down-regulated TLR2 and TLR4 transcripts independent of dose and time. GL antigens significantly down-regulated TLR2, while TLR4 expression was up-regulated as compared with control group. CONCLUSION: Hydatid cyst-derived antigens could dysregulate the expression of TLR2 and TLR4 in ovine PBMCs, suggesting a possible mechanism to suppress host immunity to establish chronic infection.
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BACKGROUND: The apply of aptamers as a new generation's way to probe diagnostic for the detection of target molecules has gained ground. Aptamers can be used as alternatives to diagnostic antibodies for detection of blood groups due to their unique features. This study was aimed to produce DNA diagnostic aptamer detecting the antigen of A1 blood group using the Cell-Selex method. MATERIALS AND METHODS: DNA aptamer was isolated against A1 RBC antigen after ten stages of Cell-Selex and amplification by an asymmetric polymerase chain reaction. The progress of the stages of selection was evaluated using flow cytometry analysis, which the DNA aptamer isolated from the tenth cycle with an affinity of 70% fluorescent intensity, was selected from four positive colonies followed by determination of the sequences and secondary structures. RESULTS: The aptameric sequence obtained from C4 cloning was calculated with the highest binding affinity to A1 antigen having an apparent dissociation constant (Kd value) of at least 29.5 ± 4.3 Pmol, which was introduced as the selected aptamer-based on ΔG obtained from a colony of C4 equal to -13.13. CONCLUSION: The aptamer obtained from using Cell-Selex method could be used as an example for the development of diagnostic tools such as biosensors for detecting A1 blood group antigens.
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Toxocariasis is considered a neglected disease despite the importance of Toxocara spp. infections for human health and is little recognized as a significant problem by public health institutions in developing countries. Epidemiological studies suggest that infection with Toxocara cati contributes to the development of allergic asthma.In the present study, we investigated the effect of T. cati infection on experimental allergic airway inflammation using murine model. BALB/c mice were infected by oral administration with 500 embryonated T. cati eggs followed by ovalbumin (OVA) sensitization and challenge to induce allergic airway inflammation. Infection with T. cati in combination with OVA treatment leads to exacerbation of pulmonary inflammation, eosinophilia, airway hyperresponsiveness, OVA specific IgE. Cytokines measurement in bronchoalveolar lavage indicated that the levels of IL-4 and IL-5 in BAL fluid significantly increased after T. cati infected, OVA treated or a combination of both. Increased level of IL-5 was measured in the lungs of T. cati-infected or OVA-treated mice compared with controls. Moreover, combining infection and OVA treatment significantly increase the level of these cytokines. A direct association between T. cati infection and asthma was found in murine model. Although a wide range of helminth species have been demonstrated to modulate allergic responses, most notably the intestinal nematode T. cati, increases airway hyperresponsiveness, lung histopathology, eosinophil recruitment, and Th2 cytokines in alum-sensitized models of airway allergy.
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Coccidiosis is considered to be one of the most important challenge in the poultry industry causes economic losses due to the destruction in the digestive tract of chicken. It disturbs amino acids profile and their digestibility, leading to weight lost and economic burden. Using dietary arginine may decrease the adverse effects of coccidiosis on chicken digestive tract. This study aimed to evaluate the effects of dietary inclusion of arginine on intestine histological parameters, serum amino acid concentration and ileal amino acid digestibility of broiler chicks infected with coccidiosis. A total number of 384 one-d-old broiler chicks (Ross 308) of mixed sex with initial weight of 42⯱â¯2â¯g was allocated into 8 groups with 8 birds/pen from grower period. At 21 days of age, broiler chicks were infected with a mixture of Eimeria spp. Broiler chicks were divided into infected and un-infected groups and received arginine at recommended levels of 85, 100, 125 and 150 %. Intestinal morphology and lesions, serum amino acid concentration and ileal amino acid digestibility were evaluated. Broiler chicks infected with Eimeria spp. showed lower villus height and villus height: crypt depth ratio and also higher intestinal lesions (Pâ¯<â¯0.05). Coccidia infection decreased the ileal amino acid digestibility for all studied amino acids and also reduced serum concentrations of amino acids, except lysine and isoleucine (Pâ¯<â¯0.05). Dietary supplementation of arginine especially in higher levels significantly increased villus height and villus height:crypt depth ratio and decreased lesions (Pâ¯<â¯0.05). Moreover, dietary supplementing of arginine increased the serum concentration of arginine (Pâ¯<â¯0.05), but it did not have any significant effect on its digestibility (Pâ¯>â¯0.05). In sum, coccidiosis decreases amino acid digestibility and serum amino acid concentration, but dietary inclusion of higher levels of arginine significantly improved histological parameters of broiler chicks infected with coccidiosis.
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Arginina/metabolismo , Galinhas , Coccidiose/veterinária , Doenças das Aves Domésticas/parasitologia , Ração Animal/análise , Animais , Arginina/administração & dosagem , Coccidiose/sangue , Coccidiose/parasitologia , Coccidiose/patologia , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Eimeria/fisiologia , Feminino , Masculino , Doenças das Aves Domésticas/sangue , Doenças das Aves Domésticas/patologia , Distribuição AleatóriaRESUMO
The objective of this study was to investigate the presence of Toxocara eggs on the hair of stray cats. The total number of stray cats trapped and included in the trial was 167 that were collected weekly from different residential areas of Mashhad, in northeastern Iran, from November 2016 to December 2017. Among the 167 cats, 18 (10.8%) of them were positive to T. cati eggs in their hair. In the positive cats, 7 (39%) were adult, 1 (6%) was juvenile and 10 (55%) were kittens. Overall, the mean number of eggs from positive cats was 3.9 ± 1.7 eggs per gram (epg) of hair per cat with an average of 3.1 ± 1.4 in adults, 4.9 in juveniles and 4.3 ± 1.6 in kittens. In total, 39.9% of the eggs recovered were non-viable 35.5% were viable, 22.2% were embryonating and 2.3% were embryonated which embryonated eggs were found only in juveniles. Based on our data, kittens were responsible for 61.7% of the total number of eggs. The age of the cat was found to be an important risk factor associated with parasitic infection.. This study showed that cat hair contaminated by T. cati eggs in different developmental stages represents of potential source for human toxocariasis.
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Pelo Animal/parasitologia , Animais Selvagens/parasitologia , Doenças do Gato/epidemiologia , Óvulo , Toxocara/isolamento & purificação , Toxocaríase/transmissão , Animais , Gatos , Estudos Transversais , Fezes/parasitologia , Feminino , Irã (Geográfico)/epidemiologia , Masculino , Contagem de Ovos de Parasitas , Toxocaríase/epidemiologiaRESUMO
BACKGROUND: Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane. RESULTS: In this study we first isolated and purified schizonts of Theileria annulata from infected leukocytes in Theileria annulata vaccine cell line (S15) by aerolysin-percoll technique. Then, the free GPIs of schizont stage and isolated GPI from cell membrane glycoproteins were purified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Furthermore, enzyme linked immunosorbent assay (ELISA) on the serum samples obtained from naturally infected, as well as Theileria annulata-vaccinated animals, confirmed a significant (P < 0.01) high level of anti-GPI antibody in their serum. CONCLUSIONS: The results presented in this study show, to our knowledge for the first time, the isolation of GPI from the schizont stage of Theileria annulata and demonstrate the presence of anti-GPI antibody in the serum of naturally infected as well as vaccinated animals. This finding is likely to be valuable in studies aimed at the evaluation of chemically structures of GPIs in the schizont stage of Theileria annulata and also for pathogenicity and immunogenicity studies with the aim to develop GPI-based therapies or vaccines.
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Anticorpos Antiprotozoários/sangue , Formação de Anticorpos , Doenças dos Bovinos/prevenção & controle , Glicosilfosfatidilinositóis/imunologia , Vacinas Protozoárias/imunologia , Theileria annulata/imunologia , Theileriose/prevenção & controle , Animais , Bovinos , Doenças dos Bovinos/imunologia , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Cromatografia Gasosa-Espectrometria de Massas , Glicosilfosfatidilinositóis/análise , Leucócitos/parasitologia , Vacinas Protozoárias/administração & dosagem , Esquizontes/química , Esquizontes/imunologia , Theileria annulata/química , Theileriose/imunologiaRESUMO
Strategies to design delivery vehicles are critical in modern vaccine-adjuvant development. Nanoparticles (NPs) encapsulating antigen(s) and adjuvant(s) are promising vehicles to deliver antigen(s) and adjuvant(s) to antigen-presenting cells (APCs), allowing optimal immune responses against a specific pathogen. In this study, we developed a novel adjuvant delivery approach for induction of efficient in vivo immune responses. Polyethylenimine (PEI) was physically conjugated to poly(lactic-co-glycolic) acid (PLGA) to form PLGA/PEI NPs. This complex was encapsulated with resiquimod (R848) as toll-like receptor (TLR) 7/8 agonist, or monophosphoryl lipid A (MPLA) as TLR4 agonist and co-assembled with cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN) as TLR9 agonist to form a tripartite formulation [two TLR agonists (inside and outside NPs) and PLGA/PEI NPs as delivery system]. The physicochemical characteristics, cytotoxicity and cellular uptake of these synthesized delivery vehicles were investigated. Cellular viability test revealed no pronounced cytotoxicity as well as increased cellular uptake compared to control groups in murine macrophage cells (J774 cell line). In the next step, PLGA (MPLA or R848)/PEI (CpG ODN) were co-delivered with ovalbumin (OVA) encapsulated into PLGA NPs to enhance the induction of immune responses. The immunogenicity properties of these co-delivery formulations were examined in vivo by evaluating the cytokine (IFN-γ, IL-4, and IL-1ß) secretion and antibody (IgG1, IgG2a) production. Robust and efficient immune responses were achieved after in vivo administration of PLGA (MPLA or R848)/PEI (CpG ODN) co-delivered with OVA encapsulated in PLGA NPs in BALB/c mice. Our results demonstrate a rational design of using dual TLR agonists in a context-dependent manner for efficient nanoparticulate adjuvant-vaccine development.
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BACKGROUND: Recently the role of gastrointestinal nematodes in modulating the immune responses in inflammatory and immune-mediated conditions such as allergy and autoimmune diseases has been introduced. This is mainly due to the suppressive effects of somatic and excretory secretory (ES) products of nematodes on the immune responses. In this study, we evaluated the immunomodulatory potentials of somatic products of Marshallagia marshalli, a gastrointestinal nematodes of sheep, to suppress the immune-mediated responses in a murine model of allergic airway inflammation. BALB/c mice were intraperitoneally (IP) sensitized with ovalbumin (OVA)/Alum and then challenged with 1% OVA. Somatic products of M. marshalli were administered during each sensitization. The effects of somatic products on development of allergic airway inflammation were evaluated by analyzing inflammatory cells recruitment, histopathological changes, cytokines production (IL-4, IL-13, IL-10, TGF-ß) and serum antibody titers (IgG1, IgG2a). RESULTS: Somatic products of M. marshalli were able to suppress the induction of allergic airway inflammation in mice. Modulation of Th2 type responses (IL-4, IL-13, IgG1) via upregulations of IL-10 and TGF-ß production was observed after injection of somatic products of M. marshalli. In addition, inflammatory cells infiltration and pathological disorders were significantly diminished following administration of somatic products. CONCLUSIONS: Our data raised the possibility that helminths could be a potential therapeutic candidate to alleviate the inflammatory conditions in allergic asthma. According to these results, we concluded that M. marshalli may contain immune-modulatory molecules that attenuate allergic airway inflammation via induction of regulatory cytokines. Further investigations are required to identify molecules that might have potentials for development of novel therapeutic targets.
Assuntos
Asma/tratamento farmacológico , Células Th2/imunologia , Extratos de Tecidos/uso terapêutico , Trichostrongyloidea/química , Compostos de Alúmen , Animais , Asma/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Modelos Animais de Doenças , Regulação para Baixo , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Imunização , Imunomodulação , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-4/biossíntese , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Ovinos , Extratos de Tecidos/administração & dosagem , Extratos de Tecidos/imunologiaRESUMO
The establishment of good experimental models for Theileria sp. infection is important for theileriosis research. Routinely, infection of ticks is accomplished by feeding on parasite-infected animals (sheep, cows and horses), which raises practical and ethical problems, driving the search for alternative methods of tick infection. Artificial tick feeding systems are based mainly on rearing ticks on host-derived or hand-made artificial membranes. We developed a modified feeding assay for infecting nymphal stages of Hyalomma anatolicum ticks with Theileria lestoquardi, a highly pathogenic parasite of sheep. We compared two different membranes: an artificial silicone membrane and a natural alternative using mouse skin. We observed high attachment rates with mouse skin, whereas in vitro feeding of H. anatolicum nymphs on silicone membranes was unsuccessful. We could infect H. anatolicum nymphs with T. lestoquardi and the emerging adult ticks transmitted infective parasites to sheep. In contrast, similar infections with Rhipicephalus bursa, a representative tick with short mouth-parts that was proposed as a vector for T. lestoquardi, appeared not to be a competent vector tick species. This is the first report of an experimentally controlled infection of H. anatolicum with T. lestoquardi and opens avenues to explore tick-parasite dynamics in detail.
Assuntos
Doenças dos Ovinos/transmissão , Ovinos/parasitologia , Pele/parasitologia , Theileria/patogenicidade , Theileriose/transmissão , Carrapatos/classificação , Carrapatos/parasitologia , Animais , Camundongos , Modelos Teóricos , Coelhos , Doenças dos Ovinos/parasitologia , Theileriose/parasitologiaRESUMO
Toll-like receptors (TLRs) are essential components of the innate immune system. They play an important role in the pathogenesis of allergic diseases, especially asthma. Since TLRs significantly orchestrate innate and adaptive immune response, their manipulation has widely been considered as a potential approach to control asthma symptoms. It is well established that helminths have immunoregulatory effects on host immune responses, especially innate immunity. They release bioactive molecules such as excretory-secretory (ES) products manipulating TLRs expression and signaling. Thus, given the promising results derived from preclinical studies, harnessing helminth-derived molecules affecting TLRs can be considered as a potential biological therapy for allergic diseases. Prospectively, the data that are available at present suggest that, in the near future, it is possible that helminth antigens will offer new therapeutic strategies and druggable targets for fighting allergic diseases. This review describes the interactions between helminths and TLRs and discusses the potential possibilities for asthma therapy. In this opinion paper, the authors aimed to review the updated literatures on the interplay between helminths, TLRs, and asthma with a view to proposing helminth-based asthma therapy.