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Bacillus thuringiensis is the most widely used biopesticide, targets a diversity of insect pests belonging to several orders. However, information regarding the B. thuringiensis strains and toxins targeting Zeugodacus cucurbitae is very limited. Therefore, in the present study, we isolated and identified five indigenous B. thuringiensisstrains toxic to larvae of Z. cucurbitae. However, of five strains NBAIR BtPl displayed the highest mortality (LC50 = 37.3 µg/mL) than reference strain B. thuringiensis var. israelensis (4Q1) (LC50 = 45.41 µg/mL). Therefore, the NBAIR BtPl was considered for whole genome sequencing to identify the cry genes present in it. Whole genome sequencing of our strain revealed genome size of 6.87 Mb with 34.95% GC content. Homology search through the BLAST algorithm revealed that NBAIR BtPl is 99.8% similar to B. thuringiensis serovar tolworthi, and gene prediction through Prokka revealed 7406 genes, 7168 proteins, 5 rRNAs, and 66 tRNAs. BtToxin_Digger analysis of NBAIR BtPl genome revealed four cry gene families: cry1, cry2, cry8Aa1, and cry70Aa1. When tested for the presence of these four cry genes in other indigenous strains, results showed that cry70Aa1 was absent. Thus, the study provided a basis for predicting cry70Aa1 be the possible reason for toxicity. In this study apart from novel genes, we also identified other virulent genes encoding zwittermicin, chitinase, fengycin, and bacillibactin. Thus, the current study aids in predicting potential toxin-encoding genes responsible for toxicity to Z. cucurbitae and thus paves the way for the development of B. thuringiensis-based formulations and transgenic crops for management of dipteran pests.
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Bacillus thuringiensis , Proteínas Bacterianas , Genoma Bacteriano , Secuenciación Completa del Genoma , Bacillus thuringiensis/genética , Animales , Proteínas Bacterianas/genética , Toxinas de Bacillus thuringiensis/genética , Endotoxinas/genética , Control Biológico de Vectores , Tephritidae/genética , Tephritidae/microbiología , Proteínas Hemolisinas/genética , Larva/genética , FilogeniaRESUMEN
Bacillus thuringiensis Berliner is recognized as a predominant bioinsecticide but its antifungal potential has been relatively underexplored. A novel B. thuringiensis strain NBAIR BtAr was isolated and morphologically characterized using light and scanning electron microscopy, revealing presence of bipyramidal, cuboidal, and spherical parasporal crystals. The crude form of lipopeptides was extracted from NBAIR BtAr and assessed for its antagonistic activity in vitro, and demonstrated 100 % inhibition of Sclerotium rolfsii Sacc. at a minimum inhibitory concentration of 50 µL of the crude lipopeptide extract per mL of potato dextrose agar. To identify the antagonistic genes responsible, we performed whole genome sequencing of NBAIR BtAr, revealing the presence of circular chromosome of 5,379,913 bp and 175,362 bp plasmid with 36.06 % guanine-cytosine content and 5814 protein-coding sequences. Average nucleotide identity and whole genome phylogenetic analysis delineated the NBAIR BtAr strain as konkukian serovar. Gene ontology analysis revealed associations of 1474, 1323, and 1833 genes with biological processes, molecular function, and cellular components, respectively. Antibiotics & secondary metabolite analysis shell analysis of the whole genome yielded secondary metabolites biosynthetic gene clusters with 100 %, 85 %, 40 %, and 35 % similarity for petrobactin, bacillibactin, fengycin, and paenilamicin, respectively. Also, novel biosynthetic gene clusters, along with antimicrobial genes, including zwittermicin A, chitinase, and phenazines, were identified. Moreover, the presence of eight bacteriophage sequences, 18 genomic islands, insertion sequences, and one CRISPR region indicated prior occurrences of genetic exchange and thus improved competitive fitness of the strain. Overall, the whole genome sequence of NBAIR BtAr is presented, with its taxonomic classification and critical genetic attributes that contribute to its strong antagonistic activity against S. rolfsii.
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Ascomicetos , Bacillus thuringiensis , Genoma Bacteriano , Lipopéptidos , Pruebas de Sensibilidad Microbiana , Filogenia , Secuenciación Completa del Genoma , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Lipopéptidos/farmacología , Lipopéptidos/metabolismo , Lipopéptidos/genética , Lipopéptidos/biosíntesis , Ascomicetos/genética , Ascomicetos/metabolismo , Ascomicetos/efectos de los fármacos , Antifúngicos/farmacología , Antifúngicos/metabolismo , Plásmidos/genética , Antibiosis , Agentes de Control Biológico/metabolismo , Composición de BaseRESUMEN
Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for P. manihoti based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of P. manihoti, and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of P. manihoti only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in P. manihoti samples. The assay successfully amplified P. manihoti DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring P. manihoti in quarantine stations and facilitating in development of a portable diagnostic kit.
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Indian sandalwood (Santalum album), valued for its medicinal properties, is an indigenous species of India. Circular or irregular pale yellow lesions surrounded by a purple halo with prominent pinhead sized black fruiting bodies at the centre of the lesion were observed on leaves of sandalwood seedlings in a nursery located in Karnataka, with a disease incidence of 75% (n = 100 investigated plants) during July 2020. The disease prevailed in monsoon followed by winter season (July 2020 - January 2021); summer was less supportive for the disease incidence. As the disease progressed, lesions expanded and merged, causing necrosis of the whole leaf. Isolation of the pathogen involved excision of small sections of diseased tissues from the lesions followed by surface sterilization with 70% ethanol for 30 s and in 1% NaClO for 1 min. Sections were rinsed in sterile distilled water, placed on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Ten isolates of Colletotrichum ssp. were obtained with an isolation frequency = 10/12×100 = 83%. One representative single-spore isolate (CSSA-1) was used for further study. Initially, pure cultures exhibited a white mycelium which later turned gray with time, and had orange conidial masses in a concentric ring pattern with the aggregation of black acervuli at the center of the culture Conidia were single celled, hyaline, and cylindrical having smooth rounded ends and the size ranged from 12.6 to 18.5 µm in length, and 3.5 to 5.6 µm in width (n = 100). The morphological characteristics were in agreement with the species description of fungi in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm the species designation of the isolate CSSA-1, a multilocus phylogenetic analysis was performed using six genomic loci (Weir et al. 2012; Marin-Felix et al. 2017). The internal transcribed spacer (ITS) region of rDNA and a partial sequence of the beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and glutamine synthetase (GS) genes were amplified using ITS1/ITS4, BT2F/BT4R, GAPDHF/R, 79F/354R, 512F/783R and GSF/GSR primers, respectively. The ITS (OK254122), TUB2 (OL462863), GAPDH (OL462859), CHS-1 (OL462860), actin (OL462861), and glutamine synthetase (OL961822) sequences of representative isolate CSSA-1 showed 99 to 100% identity with sequences MZ148628, MK967339, MN525882, MW192791, MT263504, MF111030, MH370542 and KX578767, respectively to the holotype isolate of Colletotrichum siamense (Prihastuti et al. 2009). The sequences were analysed with representative sequences of Colletotrichum and a multilocus Bayesian inference phylogenetic tree with ITS-GAPDH-ACT-CHS1-GS-TUB2 concatenated data sets (concatenated with Sequence Matrix v.1.8 (Vaidya et al. 2011)) was constructed using Beast version 1.8.4 to confirm the isolate identification (Drummond et al. 2012; Hyde et al. 2014; Weir et al. 2012). Isolate CSSA-1 clustered with C. siamense isolates. To complete Koch's postulates, for the characterized isolate CSSA-1, a pathogenicity test was conducted on 3-month-old sandalwood seedlings by spore spray inoculation. Ten plants were inoculated with a conidial suspension (106 conidia/ml) and control plants inoculated with sterilized water then kept in a glass house at 25°C and >85% relative humidity with a 12-h photoperiod. Humidity was maintained by spraying the plants with water in the morning and evening to enhance the infection. Typical symptoms of anthracnose disease similar to naturally infected leaves were observed, which included circular pale yellow lesions surrounded by a purple halo with prominent pinhead sized black fruiting bodies at the center of the lesion 7 days after inoculation, while the control plants remained unaffected. The pathogen was reisolated from infected leaves and its identity was confirmed as C. siamense based on morphological characteristics. Previously, C. siamense was identified causing disease on chili in India (Gunjan and Shenoy, 2014), but to our knowledge this is the first report of leaf anthracnose caused by C. siamense on Indian sandalwood in India or globally. This study documents crucial information, paving way for epidemiologic studies and design of control strategies to combat this newly emerging disease.
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Adjuvants can be used to enhance the immunogenicity of antigens and improve the efficacy of vaccines. Potent adjuvant action is known to often correlate with the activation of the transcription factor, nuclear factor-κB (NF-κB). Specific plant polysaccharides and a variety of phytochemicals from foods and traditional medicinal herbs have been shown to modulate NF-κB activation. In the present study, selected plant polysaccharides and phytochemicals were evaluated for use as a DNA vaccine adjuvant in a murine melanoma model. We observed that a specific ethanol extract fraction (DsCE-I) from the tuber of a key Traditional Chinese Medicine plant, Dioscorea ( Shan Yào), enhanced the protection against melanoma after immunization with a gene-based vaccine. A number of anti-inflammatory phytochemicals tested were able to partially diminish the inflammation-associated tumorigenesis elicited by LPS. Among the several phytochemical combinations investigated, the use of an adjuvant containing LPS in combination with emodin resulted in smaller tumors and higher survival rate in test mice than the use of other adjuvant treatments and the control sets in this DNA cancer vaccine model. A Dioscorea polysaccharide fraction (DsCE-I) and several specific phytochemicals warrant further exploration as useful adjuvants for anticancer vaccines.
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A nonviral method for gene transfer into mammalian cells has been developed using physical force which accelerates plasmid DNA-coated gold particles to high -speed and penetrate the mammalian cells. This technology of gene transfer via a biolistic transfection method has been shown to have multiple applications to mammalian gene transfer systems. This method has also been adapted for delivery of other macromolecules like RNA, microRNA, and proteins. A broad range of somatic cell types, including primary cell cultures and established cell lines, have been successfully transfected ex vivo or in vitro by using the gene gun technology, either as suspension or adherent cells in cultures. This chapter describes the general procedures for in vitro DNA transfection by particle-mediated delivery to nonadherent and adherent cells. These procedures can be readily employed by using the Helios gene gun system (Bio-Rad, Hercules, CA) based on the Accell design.
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Biolística/instrumentación , Melanoma Experimental/genética , Melanoma Experimental/patología , Transfección/instrumentación , Animales , Adhesión Celular , ADN/administración & dosificación , ADN/química , ADN/genética , Citometría de Flujo , Oro/química , Humanos , Ratones , Microesferas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , Piel/metabolismo , Transgenes/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Shikonin, a phytochemical purified from Lithospermum erythrorhizon, has been shown to confer diverse pharmacological activities, including accelerating granuloma formation, wound healing, anti-inflammation and others, and is explored for immune-modifier activities for vaccination in this study. Transdermal gene-based vaccine is an attractive approach for delivery of DNA transgenes encoding specific tumor antigens to host skin tissues. Skin dendritic cells (DCs), a potent antigen-presenting cell type, is known to play a critical role in transmitting and orchestrating tumor antigen-specific immunities against cancers. The present study hence employs these various components for experimentation. METHOD: The mRNA and protein expression of RANTES were detected by RT-PCR and ELISA, respectively. The regional expression of RANTES and tissue damage in test skin were evaluated via immunohistochemistry assay. Fluorescein isothiocyanate sensitization assay was performed to trace the trafficking of DCs from the skin vaccination site to draining lymph nodes. Adjuvantic effect of shikonin on gene gun-delivered human gp100 (hgp100) DNA cancer vaccine was studied in a human gp100-transfected B16 (B16/hgp100) tumor model. RESULTS: Among various phytochemicals tested, shikonin induced the highest level of expression of RANTES in normal skin tissues. In comparison, mouse RANTES cDNA gene transfection induced a higher level of mRANTES expression for a longer period, but caused more extensive skin damage. Topical application of shikonin onto the immunization site before gene gun-mediated vaccination augmented the population of skin DCs migrating into the draining lymph nodes. A hgp100 cDNA gene vaccination regimen with shikonin pretreatment as an adjuvant in a B16/hgp100 tumor model increased cytotoxic T lymphocyte activities in splenocytes and lymph node cells on target tumor cells. CONCLUSION: Together, our findings suggest that shikonin can effectively enhance anti-tumor potency of a gene-based cancer vaccine via the induction of RANTES expression at the skin immunization site.
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Antiinflamatorios no Esteroideos/farmacología , Vacunas contra el Cáncer/inmunología , Quimiocina CCL5/inmunología , Naftoquinonas/farmacología , Vacunas de ADN/inmunología , Antígeno gp100 del Melanoma/inmunología , Animales , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/metabolismo , Línea Celular Tumoral , Quimiocina CCL5/biosíntesis , Quimiocina CCL5/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Masculino , Ratones , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/terapia , Vacunas de ADN/genética , Vacunas de ADN/metabolismo , Antígeno gp100 del Melanoma/biosíntesis , Antígeno gp100 del Melanoma/genéticaRESUMEN
Matrix metalloproteinases MMP-2 and -9 are known to be overexpressed in ultraviolet B (UVB)-irradiated skin tissues and contribute to the acceleration of photoaging and the development of skin cancer. But the specific molecular mechanisms that can control or interfere with the expression and regulation of these MMP-2 and -9 activities in skin are not clearly understood. The aim of the present study was to analyze the suppressive effects of ferulic acid (FA), an abundant phenolic compound present in various dietary and medicinal plants, on UVB radiation-induced MMP-2 and -9 activities in mouse skin. For attenuation of chronic UVB irradiation damage to skin, inhibition of MMP-2 and -9 protein expression was detected using immunohistochemistry analysis. However, the in situ suppressive effects of FA did not interfere with the transcription or translation of MMP-2 and -9, suggesting that its action could be mediated via the proteasome pathway. Histological analyses showed that FA attenuates the degradation of collagen fibers, abnormal accumulation of elastic fibers and epidermal hyperplasia induced by UVB, demonstrating the functional and physiological relevance of FA effects in UVB-irradiated skin tissues. Together, our findings provide a novel and increased insight into the in vivo action of FA and suggest a possible clinical application in skin pathophysiological conditions associated with overexpression of MMP-2 and -9.
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Ácidos Cumáricos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloproteinasas de la Matriz/metabolismo , Piel/metabolismo , Rayos Ultravioleta/efectos adversos , Animales , Células Cultivadas , Femenino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Procesamiento Proteico-Postraduccional , Piel/efectos de los fármacos , Piel/patología , Piel/efectos de la radiaciónRESUMEN
Specific cytokines have been tested clinically for immunotherapy of cancers; however, cytotoxicity has often impaired their usefulness. Consequently, alternative approaches are increasingly desirable. Dioscorea spp. tuber is a widely used traditional Chinese medicinal herb claimed to confer immunostimulatory activity. In this study, we evaluated Dioscorea as an adjuvant therapy for use alongside chemotherapy for cancer. Phytocompounds from Dioscorea tubers were ethanol fractioned and used for ex vivo splenocyte proliferation assay or in vivo force-feeding of mice pre-treated with the chemotherapy agent 5-fluorouracil. Co-treatment with a 50-75% ethanol-partitioned fraction of the tuber extract of D. batatas (DsCE-II) and interleukin (IL)-2 resulted in a significantly higher rate of murine splenocyte cell proliferation ex vivo than treatment with DsCE-II or IL-2 alone. This DsCE-II fraction, which contains a polysaccharide with a high proportion of ß-1,4-linkage mannose (≥64%), also promoted the regeneration of specific progenitor cell populations in damaged bone marrow tissues of 5-fluorouracil-treated mice. Colony-forming unit (CFU) analyses demonstrated that the population of CFU-GM cells, but not CFU-GEMM or BFU-E cells, preferentially recovered to ~67% in the bone marrow of immune-suppressed mice fed with DsCE-II. DsCE-II efficacy level was ~85% of that obtained by subcutaneous administration of recombinant G-CSF proteins (5 µg kg(-1)) in mice tested in parallel. This study suggests that the DsCE-II fraction of D. batatas extract may be considered for further development as a dietary supplement for use alongside chemotherapy during cancer treatment.
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BACKGROUND: Several Echinacea species have been used as nutraceuticals or botanical drugs for "immunostimulation", but scientific evidence supporting their therapeutic use is still controversial. In this study, a phytocompound mixture extracted from the butanol fraction (BF) of a stem and leaf (S+L) extract of E. purpurea ([BF/S+L/Ep]) containing stringently defined bioactive phytocompounds was obtained using standardized and published procedures. The transcriptomic and proteomic effects of this phytoextract on mouse bone marrow-derived dendritic cells (BMDCs) were analyzed using primary cultures. RESULTS: Treatment of BMDCs with [BF/S+L/Ep] did not significantly influence the phenotypic maturation activity of dendritic cells (DCs). Affymetrix DNA microarray and bioinformatics analyses of genes differentially expressed in DCs treated with [BF/S+L/Ep] for 4 or 12 h revealed that the majority of responsive genes were related to cell adhesion or motility (Cdh10, Itga6, Cdh1, Gja1 and Mmp8), or were chemokines (Cxcl2, Cxcl7) or signaling molecules (Nrxn1, Pkce and Acss1). TRANSPATH database analyses of gene expression and related signaling pathways in treated-DCs predicted the JNK, PP2C-α, AKT, ERK1/2 or MAPKAPK pathways as the putative targets of [BF/S+L/Ep]. In parallel, proteomic analysis showed that the expressions of metabolic-, cytoskeleton- or NF-κB signaling-related proteins were regulated by treatment with [BF/S+L/Ep]. In vitro flow cytometry analysis of chemotaxis-related receptors and in vivo cell trafficking assay further showed that DCs treated with [BF/S+L/Ep] were able to migrate more effectively to peripheral lymph node and spleen tissues than DCs treated as control groups. CONCLUSION: Results from this study suggest that [BF/S+L/Ep] modulates DC mobility and related cellular physiology in the mouse immune system. Moreover, the signaling networks and molecules highlighted here are potential targets for nutritional or clinical application of Echinacea or other candidate medicinal plants.
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Movimiento Celular/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Echinacea/química , Genoma/genética , Extractos Vegetales/farmacología , Proteómica/métodos , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Dendríticas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Femenino , Perfilación de la Expresión Génica , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Receptores Inmunológicos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Extractos de Tejidos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Much of the current research in cancer therapeutics is aimed at developing drugs or vaccines to target key molecules for combating tumor cell growth, metastasis, proliferation, or changes in the associated stromal microenvironment. Studies on a wide spectrum of plant secondary metabolites extractable as natural products from fruits, vegetables, teas, spices, and traditional medicinal herbs show that these plant natural products can act as potent anti-inflammatory, antioxidant or anticancer agents. The recent advances in genomics and metabolomics have enabled biologists to better investigate the potential use of immunomodulatory natural products for treatment or control of various cancerous diseases. The cancer preventive or protective activities of the various immunomodulatory natural products lie in their effects on cellular defenses including detoxifying and antioxidant enzyme systems, and the induction of anti-inflammatory and antitumor or antimetastasis responses, often by targeting specific key transcription factors like nuclear factor kappa B (NF-kappaB), activator protein (AP-1), signal transducers and activators of transcription (STAT) and others. This review presents recent findings and hypotheses on the molecular mechanisms through which various inflammatory activities are linked to tumorigenic processes and the specific immunomodulatory natural products that may suppress inflammation and the associated tumor progression and metastasis both IN VITRO and IN VIVO. In addition to tumor cells PER SE, the various associated roles of myeloid-derived suppressor cells, stromal fibroblasts, myofibroblasts, and inflammatory immune cells, and the possible effects of phytomedicines on these cells in the tumor microenvironment will be discussed.
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Antiinflamatorios/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Fitoterapia , Preparaciones de Plantas/uso terapéutico , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayos Clínicos como Asunto , Humanos , Inflamación/tratamiento farmacológico , Ratones , Metástasis de la Neoplasia/tratamiento farmacológico , Neoplasias/prevención & control , Neovascularización Patológica/tratamiento farmacológico , Preparaciones de Plantas/química , Preparaciones de Plantas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Gene-based immunization with transgenic DNA vectors expressing tumor-associated antigens (TAA), cytokines, or chemokines, alone or in combination, provides an attractive approach to increase the cytotoxic T cell immunity against various cancer diseases. With this consideration, particle-mediated or gene gun technology has been developed as a nonviral method for gene transfer into various mammalian tissues. It has been shown to induce both humoral and cell-mediated immune responses in both small and large experimental animals. A broad range of somatic cell types, including primary cultures and established cell lines, has been successfully transfected ex vivo or in vitro by gene gun technology, either as suspension or adherent cultures. Here, we show that protocols and techniques for use in gene gun-mediated transgene delivery system for skin vaccination against melanoma using tumor-associated antigen (TAA) human gpl00 and reporter gene assays as experimental systems.
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Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Técnicas de Transferencia de Gen , Animales , ADN/genética , Dermis/inmunología , Epidermis/inmunología , Regulación de la Expresión Génica , Genes Reporteros , Oro , Humanos , Neoplasias Pulmonares/secundario , Melanoma/inmunología , Melanoma/patología , RatonesRESUMEN
BACKGROUND: Porcine circovirus type 2 (PCV2) is the primary cause of an emerging swine disease, postweaning multisystemic wasting syndrome, that is responsible for economic losses. To develop an effective vaccine for PCV2, we evaluated a heterologous prime-boost vaccine approach, using a gene gun-mediated naked DNA vector as a priming and modified vaccinia virus ankara (MVA) as a booster, in Balb/c mice. METHODS: Three open reading frames (ORF) of PCV2 viral samples from infected pigs were amplified, and gene gun-mediated DNA priming vaccination was performed followed by boosts with MVA vectors expressing the same ORFs of PCV2. After vaccination, mice were challenged with PCV2 virus, and virus titers in the lungs and lymph nodes were measured. RESULTS: The combination of ORF-2 and -3 in this gene-based vaccine strategy resulted in high antibody titers and virus neutralization activity in serum, reduced PCV2 virus load, and reduced levels of apoptosis in the lungs. No cross-reaction was observed between ORF-1 and -2, but weak cross-reaction was observed between ORF-1 and -3, and between ORF-2 and -3. Following vaccination, expression of chemokines, macrophage inflammatory protein-1beta and regulated upon activation, normal T cell expressed and secreted, increased significantly. The expression of T helper 1-type cytokine (interferon-gamma) and specific lysis of PCV2-infected cells increased; concomitantly, the level of T helper 2-type cytokine (interleukin-10) decreased in test mice. The expression of tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor increased significantly in mice vaccinated with ORF-2/-3, and with ORF-1/-2/-3. CONCLUSIONS: This prime-boost vaccination strategy, using a gene gun for DNA priming and recombinant MVA for boosts, may be an attractive vaccine strategy against PCV2 infection in swine.