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Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.
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Arabidopsis , Proteínas de Fluorescência Verde , Arabidopsis/virologia , Arabidopsis/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas de Transferência de Genes , Plasmídeos/genética , Poliaminas/química , Protoplastos/metabolismo , Nanoestruturas/química , DNA/química , DNA/administração & dosagemRESUMO
Plants, essential for food, oxygen, and economic stability, are under threat from human activities, biotic threats, and climate change, requiring rapid technological advancements for protection. Biohybrid systems, merging synthetic macromolecules with biological components, have provided improvement to biological systems in the past, namely, in the biomedical arena, motivating an opportunity to enhance plant well-being. Nevertheless, strategies for plant biohybrid systems remain limited. In this study, we present a method using grafting-from ring-opening metathesis polymerization (ROMP) under physiological conditions to integrate norbornene-derived polymers into live plants by spray coating. The approach involves creating biological macroinitiators on leaf surfaces, which enable subsequent polymerization of norbornene-derived monomers. Characterization techniques, including FTIR spectroscopy, SEM EDS imaging, ICP-MS, nanoindentation, and XPS, confirmed the presence and characterized the properties of the polymeric layers on leaves. The demonstrated modifiability and biocompatibility could offer the potential to maintain plant health in various applications, including the development of thermal barriers, biosensors, and crop protection layers.
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Norbornanos , Folhas de Planta , Norbornanos/química , Folhas de Planta/química , Polimerização , Polímeros/química , PlásticosRESUMO
Ulcerative Colitis (UC) is an inflammatory disease characterized by colonic mucosal lesions associated with an increased risk of carcinogenesis. UC pathogenesis involves environmental and genetic factors. Genetic studies have indicated the association of gene variants coding for the divalent metal ion transporter SLC11A1 protein (formerly NRAMP1) with UC susceptibility in several animal species. Two mouse lines were genetically selected for high (AIRmax) or low (AIRmin) acute inflammatory responses (AIR). AIRmax is susceptible, and AIRmin is resistant to DSS-induced colitis and colon carcinogenesis. Furthermore, AIRmin mice present polymorphism of the Slc11a1 gene. Here we investigated the possible modulating effect of the Slc11a1 R and S variants in DSS-induced colitis by using AIRmin mice homozygous for Slc11a1 R (AIRminRR) or S (AIRminSS) alleles. We evaluated UC by the disease activity index (DAI), considering weight loss, diarrhea, blood in the anus or feces, cytokines, histopathology, and cell populations in the distal colon epithelium. AIRminSS mice have become susceptible to DSS effects, with higher DAI, IL6, G-CSF, and MCP-1 production and morphological and colon histopathological alterations than AIRminRR mice. The results point to a role of the Slc11a1 S allele in DSS colitis induction in the genetic background of AIRmin mice.
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Colite Ulcerativa , Colite , Animais , Camundongos , Carcinogênese , Colite/induzido quimicamente , Colite/genética , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Inflamação/genética , Camundongos Endogâmicos C57BL , Polimorfismo GenéticoRESUMO
INTRODUCTION: Rapid antigen tests (RAgTs) for SARS-CoV-2 are considered adequate for diagnosis at the point of care. Our objective was to establish the agreement between reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RAgTs in the pediatric population. POPULATION AND METHODS: All patients aged 1 month to 17 years and 11 months seen at the Emergency Fever Unit of a children's hospital between 6-11-2021 and 10-3-2021 were recruited. The Panbio COVID-19 Ag® test (Abbott Diagnostic) was compared to the reference method RT-qPCR (as per the protocol suggested by the United States Centers for Disease Control and Prevention). RESULTS: A total of 6491 patients were included. The prevalence of COVID-19 was 2.8%. Symptoms were observed in 92.1%. Sensitivity, specificity, and the kappa index of agreement for the RAgT were 71.0%, 99.9%, and 0.813, respectively. The kappa index and the RAgT sensitivity were significantly higher in the group aged 13-17 years (0.89 and 82.4%, respectively) compared to the groups aged 0-5 and 6-12 years. This may be due to the lower viral load observed in patients younger than 12 years. CONCLUSIONS: Although RAgTs shorten the time to result and improve the isolation strategy for COVID-19 patients, their sensitivity in children younger than 12 years or asymptomatic children is not within the recommended ranges, especially during periods of low disease prevalence.
Introducción. Los inmunoensayos de detección rápida de antígenos (TRA) del SARS-CoV-2, son considerados adecuados para el diagnóstico en el punto de atención. El objetivo fue conocer la concordancia entre la reacción en cadena de la polimerasa en tiempo real con transcriptasa inversa (RT-qPCR, por su sigla en inglés) y los TRA en población pediátrica. Población y métodos. Se reclutaron todos los pacientes entre 1 mes y 17 años 11 meses de edad atendidos en la Unidad Febril de Urgencia de un hospital pediátrico entre el 11 de junio y el 3 de octubre de 2021. Se utilizó el TRA Panbio COVID-19 Ag® (Abbott Diagnostic) y, como método de referencia, la RT-qPCR (según el protocolo de los Centros para el Control y la Prevención de Enfermedades). Resultados. Se incluyeron 6491 pacientes. La prevalencia de COVID-19 fue del 2,8 %. El 92,1 % de los sujetos presentaron síntomas. La sensibilidad, la especificidad y el índice kappa de concordancia para el TRA fueron del 71,0 %, 99,9 % y 0,813, respectivamente. El índice kappa y la sensibilidad del TRA fueron significativamente mayores en el grupo de 13 a 17 años (0,89 y 82,4 %, respectivamente) cuando se los comparó con los grupos de 0 a 5 y de 6 a 12 años. Esto podría deberse a la menor carga viral observada en los pacientes menores de 12 años. Conclusión. Si bien los TRA permiten acortar el tiempo de obtención de los resultados y mejorar la estrategia de aislamiento de pacientes con COVID-19, la sensibilidad en niños menores de 12 años o asintomáticos no se encontraría dentro de los rangos recomendados, sobre todo en períodos de baja prevalencia de la enfermedad.
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COVID-19 , COVID-19/diagnóstico , Teste para COVID-19 , Criança , Serviço Hospitalar de Emergência , Hospitais Pediátricos , Humanos , SARS-CoV-2 , Sensibilidade e Especificidade , Estados UnidosRESUMO
Introducción. Los inmunoensayos de detección rápida de antígenos (TRA) del SARS-CoV-2, son considerados adecuados para el diagnóstico en el punto de atención. El objetivo fue conocer la concordancia entre la reacción en cadena de la polimerasa en tiempo real con transcriptasa inversa (RT-qPCR, por su sigla en inglés) y los TRA en población pediátrica. Población y métodos. Se reclutaron todos los pacientes entre 1 mes y 17 años 11 meses de edad atendidos en la Unidad Febril de Urgencia de un hospital pediátrico entre el 11 de junio y el 3 de octubre de 2021. Se utilizó el TRA Panbio COVID-19 Ag® (Abbott Diagnostic) y, comométodo de referencia, la RT-qPCR (según el protocolo de los Centros para el Control y la Prevención de Enfermedades). Resultados. Se incluyeron 6491 pacientes. La prevalencia de COVID-19 fue del 2,8 %. El92,1 % de los sujetos presentaron síntomas. La sensibilidad, la especificidad y el índice kappa de concordancia para el TRA fueron del 71,0 %, 99,9 % y 0,813, respectivamente. El índice kappa yla sensibilidad del TRA fueron significativamentemayores en el grupo de 13 a 17 años (0,89 y 82,4 %,respectivamente) cuando se los comparó con los grupos de 0 a 5 y de 6 a 12 años. Esto podría deberse a la menor carga viral observada en los pacientes menores de 12 años. Conclusión. Si bien los TRA permiten acortar el tiempo de obtención de los resultados y mejorar la estrategia de aislamiento de pacientes con COVID-19, la sensibilidad en niños menores de 12 años o asintomáticos no se encontraría dentro de los rangos recomendados, sobre todo enperíodos de baja prevalencia de la enfermedad.
Introduction. Rapid antigen tests (RAgTs) for SARS-CoV-2 are considered adequate for diagnosis at the point of care. Our objective was to establish the agreement between reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RAgTs in the pediatric population. Population and methods. All patients aged 1 month to 17 years and 11 months seen at the Emergency Fever Unit of a children's hospital between 6-11-2021 and 10-3-2021 were recruited. The Panbio COVID-19 Ag® test (Abbott Diagnostic) was compared to the reference method RT-qPCR (as per the protocol suggested by the United States Centers for Disease Control and Prevention). Results. A total of 6491 patients were included. The prevalence of COVID-19 was 2.8%. Symptoms were observed in 92.1%. Sensitivity, specificity, and the kappa index of agreement for the RAgT were 71.0%, 99.9%, and 0.813, respectively. The kappa index and the RAgT sensitivity were significantly higher in the group aged 1317 years (0.89 and 82.4%, respectively) compared to the groups aged 05 and 612 years. This may be due to the lower viral load observed in patients younger than 12 years. Conclusion. Although RAgTs shorten the time to result and improve the isolation strategy for COVID-19 patients, their sensitivity in children younger than 12 years or asymptomatic children is not within the recommended ranges, especially during periods of low disease prevalence.
Assuntos
Humanos , Lactente , Pré-Escolar , Criança , Adolescente , COVID-19/diagnóstico , Sensibilidade e Especificidade , Serviço Hospitalar de Emergência , Teste para COVID-19 , SARS-CoV-2 , Hospitais PediátricosRESUMO
Current tissue engineering techniques frequently rely on hydrogels to support cell growth, as these materials strongly mimic the extracellular matrix. However, hydrogels often need ad hoc customization to generate specific tissue constructs. One popular strategy for hydrogel functionalization is to add nanoparticles to them. Here, we present a plant viral nanoparticle the turnip mosaic virus (TuMV), as a promising additive for gelatin methacryloyl (GelMA) hydrogels for the engineering of mammalian tissues. TuMV is a flexuous, elongated, tubular protein nanoparticle (700-750 nm long and 12-15 nm wide) and is incapable of infecting mammalian cells. These flexuous nanoparticles spontaneously form entangled nanomeshes in aqueous environments, and we hypothesized that this nanomesh structure could serve as a nanoscaffold for cells. Human fibroblasts loaded into GelMA-TuMV hydrogels exhibited similar metabolic activity to that of cells loaded in pristine GelMA hydrogels. However, cells cultured in GelMA-TuMV formed clusters and assumed an elongated morphology in contrast to the homogeneous and confluent cultures seen on GelMA surfaces, suggesting that the nanoscaffold material per se did not favor cell adhesion. We also covalently conjugated TuMV particles with epidermal growth factor (EGF) using a straightforward reaction scheme based on a Staudinger reaction. BJ cells cultured on the functionalized scaffolds increased their confluency by approximately 30% compared to growth with unconjugated EGF. We also provide examples of the use of GelMA-TuMV hydrogels in different biofabrication scenarios, include casting, flow-based-manufacture of filaments, and bioprinting. We envision TuMV as a versatile nanobiomaterial that can be useful for tissue engineering.
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Agriculture is facing new challenges, with global warming modifying the survival chances for crops, and new pests on the horizon. To keep up with these challenges, gene delivery provides tools to increase crop yields. On the other hand, gene delivery also opens the door for molecular farming of pharmaceuticals in plants. However, towards increased food production and scalable molecular farming, there remain technical difficulties and regulatory hurdles to overcome. The industry-standard is transformation of plants via Agrobacterium tumefaciens, but this method is limited to certain plants, requires set up of plant growth facilities and fermentation of bacteria, and introduces lipopolysaccharides contaminants into the system. Therefore, alternate methods are needed. Mechanical inoculation and spray methods have already been discussed in the literature - and here, we compare these methods with a newly introduced petiole injection technique. Because our interest lies in the development of plant viruses as immunotherapies targeting human health as well as gene delivery vectors for agriculture applications, we turned toward tobacco mosaic virus as a model system. We studied the effectiveness of three inoculation techniques: mechanical inoculation, Silwet-77 foliar spray and petiole injections. The foliar spray method was optimized, and we used 0.03% Silwet L-77 to induce infection using either TMV or a lysine-added mutant TMV-Lys. We developed a method using a needle-laden syringe to target and inject the plant virus directly into the vasculature of the plant - we tested injection into the stem and petiole. Stem inoculation resulted in toxicity, but the petiole injection technique was established as a viable strategy. TMV and TMV-Lys were purified from single plants and pooled leaf samples - overall there was little variation between the techniques, as measured by TMV or TMV-Lys yields, highlighting the feasibility of the syringe injection technique to produce virus nanoparticles. There was variation between yields from preparation to preparation with mechanical, spray and syringe inoculation yielding 40-141 mg, 36-56 mg, 18-56 mg TMV per 100 grams of leaves. Similar yields were obtained using TMV-Lys, with 24-38 mg, 17-28, 7-36 mg TMV-Lys per 100 grams of leaves for mechanical, spray and syringe inoculation, respectively. Each method has its advantages: spray inoculation is highly scalable and therefore may find application for farming, the syringe inoculation could provide a clean, aseptic, and controlled approach for molecular farming of pharmaceuticals under good manufacturing protocols (GMP) and would even be applicable for gene delivery to plants in space.
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We identified Pycard and BC017158 genes as putative effectors of the Quantitative Trait locus (QTL) that we mapped at distal chromosome 7 named Irm1 for Inflammatory response modulator 1, controlling acute inflammatory response (AIR) and the production of IL-1ß, dependent on the activation of the NLRP3 inflammasome. We obtained the mapping through genome-wide linkage analysis of Single Nucleotide Polymorphisms (SNPs) in a cross between High (AIRmax) and Low (AIRmin) responder mouse lines that we produced by several generations of bidirectional selection for Acute Inflammatory Response. A highly significant linkage signal (LOD score peak of 72) for ex vivo IL-1ß production limited a 4 Mbp interval to chromosome 7. Sequencing of the locus region revealed 14 SNPs between "High" and "Low" responders that narrowed the locus to a 420 Kb interval. Variants were detected in non-coding regions of Itgam, Rgs10 and BC017158 genes and at the first exon of Pycard gene, resulting in an E19K substitution in the protein ASC (apoptosis associated speck-like protein containing a CARD) an adaptor molecule in the inflammasome complex. Silencing of BC017158 inhibited IL1-ß production by stimulated macrophages and the E19K ASC mutation carried by AIRmin mice impaired the ex vivo IL-1ß response and the formation of ASC specks in stimulated cells. IL-1ß and ASC specks play major roles in inflammatory reactions and in inflammation-related diseases. Our results delineate a novel genetic factor and a molecular mechanism affecting the acute inflammatory response.
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Proteínas Adaptadoras de Sinalização CARD , Inflamassomos , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ligação Genética , Inflamassomos/genética , Inflamassomos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos , Locos de Características QuantitativasRESUMO
Two non-inbred mouse lines, phenotypically selected for maximal (AIRmin) and minimal (AIRmax) acute inflammatory response, show differential susceptibility/resistance to the development of several chemically-induced tumor types. An intercross pedigree of these mice was generated and treated with the chemical carcinogen dimethylhydrazine, which induces lung and intestinal tumors. Genome wide high-density genotyping with the Restriction Site-Associated DNA genotyping (2B-RAD) technique was used to map genetic loci modulating individual genetic susceptibility to both lung and intestinal cancer. Our results evidence new common quantitative trait loci (QTL) for those phenotypes and provide an improved understanding of the relationship between genomic variation and individual genetic predisposition to tumorigenesis in different organs.
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Neoplasias do Colo , Locos de Características Quantitativas , Animais , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Predisposição Genética para Doença , Pulmão , Camundongos , Camundongos EndogâmicosRESUMO
A laboratory exercise for undergraduate advanced students of enzymology and biocatalysis is presented. Since enzyme assays can be quenched or continuous, this experiment compares the performance of two quenching agents for lactase, in a continuous setup. Enzymatic activity of ß-galactosidase (Aspergillus oryzae) was determined based on the release of 4-nitrophenol from 4-nitrophenyl ß-D-galactopyranoside using a microplate reader. Sodium carbonate and borate buffer were tested as quenching agents, and experimental control was the unstopped assay. Based on released 4-nitrophenol, enzyme activity, and rate constant k, the students could assess the performance of each termination agent. The experiment promotes disciplinary and transversal competencies, including research-based learning, critical thinking, and introduce the students to high-throughput techniques that are common in the research and development environment.
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Boratos , Lactase , Carbonatos , Humanos , Laboratórios , beta-GalactosidaseRESUMO
Bothrops jararaca venom (BjV) can induce mast cell degranulation. In order to investigate the role of mast cells and the interference of the host genetic background in the inflammation induced by BjV, we have used mouse strains selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory response (AIR). Mice were pretreated with an inhibitor of mast cell degranulation, cromolyn (CROM), and injected in footpads or intraperitoneally (i.p.) with BjV. Pain was measured with von Frey hairs, cell migration in the peritoneum by flow cytometry, and reactive oxygen species (ROS) production by chemiluminescence assays. The nociceptive response to BjV was higher in AIRmax than AIRmin mice; however, this difference was abolished by pretreatment with CROM. BjV induced peritoneal neutrophil (CD11b+ GR-1+) infiltration and ROS secretion in AIRmax mice only, which were partially inhibited by CROM. Our findings evidence a role for mast cells in pain, neutrophil migration, and ROS production triggered by BjV in AIRmax mice that are more susceptible to the action of BjV.
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Bothrops , Venenos de Crotalídeos , Animais , Movimento Celular , Venenos de Crotalídeos/efeitos adversos , Inflamação/induzido quimicamente , Mastócitos , Camundongos , Dor , Espécies Reativas de OxigênioRESUMO
Crotoxin (CTX), the main neurotoxin from Crotalus durissus terrificus snake venom, has anti-inflammatory, immunomodulatory and antinociceptive activities. However, the CTX-induced toxicity may compromise its use. Under this scenario, the use of nanoparticle such as nanostructured mesoporous silica (SBA-15) as a carrier might become a feasible approach to improve CTX safety. Here, we determined the benefits of SBA-15 on CTX-related neuroinflammatory and immunomodulatory properties during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis that replicates several histopathological and immunological features observed in humans. We showed that a single administration of CTX:SBA-15 (54 µg/kg) was more effective in reducing pain and ameliorated the clinical score (motor impairment) in EAE animals compared to the CTX-treated EAE group; therefore, improving the disease outcome. Of interest, CTX:SBA-15, but not unconjugated CTX, prevented EAE-induced atrophy and loss of muscle function. Further supporting an immune mechanism, CTX:SBA-15 treatment reduced both recruitment and proliferation of peripheral Th17 cells as well as diminished IL-17 expression and glial cells activation in the spinal cord in EAE animals when compared with CTX-treated EAE group. Finally, CTX:SBA-15, but not unconjugated CTX, prevented the EAE-induced cell infiltration in the CNS. These results provide evidence that SBA-15 maximizes the immunomodulatory and anti-inflammatory effects of CTX in an EAE model; therefore, suggesting that SBA-15 has the potential to improve CTX effectiveness in the treatment of MS.
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Crotoxina/administração & dosagem , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Imunomodulação/efeitos dos fármacos , Dióxido de Silício , Nanomedicina Teranóstica , Animais , Biomarcadores , Biópsia , Crotoxina/efeitos adversos , Crotoxina/química , Citocinas/metabolismo , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/diagnóstico , Feminino , Camundongos , Músculo Esquelético/imunologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Índice de Gravidade de Doença , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Avaliação de SintomasRESUMO
AIRmax and AIRmin mouse strains phenotypically selected for high and low acute inflammatory responsiveness (AIR) are, respectively, susceptible or resistant to developing hepatocellular carcinoma (HCC) induced by the chemical carcinogens urethane and diethylnitrosamine (DEN). Early production of TNF-α, IL-1ß, and IL-6 in the liver after DEN treatment correlated with tumor development in AIRmax mice. Transcriptome analysis of livers from untreated AIRmax and AIRmin mice showed specific gene expression profiles in each line, which might play a role in their differential susceptibility to HCC. Linkage analysis with SNP markers in F2 (AIRmax×AIRmin) intercross mice revealed two quantitative trait loci (QTL) in chromosomes 2 and 9, which are significantly associated with the number and progression of urethane-induced liver tumors. An independent linkage analysis with an intercross population from A/J and C57BL/6J inbred mice mapped regions in chromosomes 1 and 7 associated with the progression of urethane-induced liver tumors, evidencing the heterogeneity of HCC genetic control.
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Animais não Endogâmicos , Carcinoma Hepatocelular/genética , Predisposição Genética para Doença , Inflamação/imunologia , Neoplasias Hepáticas/genética , Alelos , Animais , Carcinogênese/genética , Carcinoma Hepatocelular/imunologia , Modelos Animais de Doenças , Ligação Genética , Endogamia , Inflamação/genética , Interleucina-1beta/genética , Interleucina-6/genética , Neoplasias Hepáticas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Locos de Características Quantitativas , Transcriptoma , Fatores de Necrose Tumoral/genéticaRESUMO
The inflammatory and autoimmune events preceding clinical symptoms in rheumatoid arthritis (RA) and other autoimmune diseases are difficult to study in human patients. Therefore, animal models that share immunologic and clinical features with human RA, such as pristane-induced arthritis (PIA), are valuable tools for assessing the primordial events related to arthritis susceptibility. PIA-resistant HIII and susceptible LIII mice were injected i.p. with pristane, and peritoneal lavage fluid was harvested in the early (7 days) and late (35 days) preclinical phases of PIA. Chemokine and cytokine levels were measured in lavage supernatant with ELISA, peritoneal inflammatory leukocytes were immunophenotyped by flow cytometry, and gene expression was determined by qRT-PCR. Leukocyte recruitment was quantitatively and qualitatively divergent in the peritoneum of HIII and LIII mice, with an early increase of CC chemokines (CCL2/CCL3/CCL5/CCL12/CCL22) in the susceptible LIII strain. Also, cytokines such as IL-12p40, IL-23, and IL-18 were elevated in LIII mice while IL-6 was increased in HIII animals. The results show that an early peritoneal CC chemokine response is an important feature of arthritis susceptibility and defines potential biomarkers in this model.
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Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Quimiocinas CC/imunologia , Inflamação , Peritônio/imunologia , Animais , Artrite Experimental/induzido quimicamente , Biomarcadores , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Interleucina-6/imunologia , Masculino , Camundongos , Fenótipo , Terpenos/administração & dosagemRESUMO
The exact role of inflammatory immune response in bone healing process is still unclear, but the success of the alveolar bone healing process seems to be associated with a moderate and transitory inflammatory response, while insufficient or exacerbated responses seems to have a detrimental influence in the healing outcome. In this context, we performed a comparative analysis of mice strains genetically selected for maximum (AIRmax) or minimum (AIRmin) acute inflammatory response to address the influence of inflammation genes in alveolar bone healing outcome. Experimental groups comprised 8-week-old male or female AIRmax and AIRmin submitted to extraction of upper right incisor, and evaluated at 0, 3, 7, 14 and 21â¯days after upper incision extraction by micro-computed tomography (µCT), histomorphometry, birefringence, immunohistochemistry and molecular (PCRArray) analysis. Overall, the results demonstrate a similar successful bone healing outcome at the endpoint was evidenced in both AIRmin and AIRmax strains. The histormophometric analysis reveal a slight but significant decrease in blood clot and inflammatory cells density, as well a delay in the bone formation in AIRmax strain in the early times, associated with a decreased expression of BMP2, BMP4, BMP7, TGFb1, RUNX2, and ALP. The evaluation of inflammatory cells nature reveals increased GR1+ cells counts in AIRmax strain at 3d, associated with increased levels of neutrophil chemoattractants such as CXCL1 and CXCL2, and its receptor CXCR1, while F4/80+ cell prevails in AIRmin strain at 7d. Also, our results demonstrate a relative predominance of M2 macrophages in AIRmin strain, associated with an increased expression of ARG1, IL10, TGFb, while M1 macrophages prevail in AIRmax, which parallel with increased IL-1B, IL-6 and TNF expression. At late repair stage, AIRmax presents evidences of increased bone remodeling, characterized by increased density of blood vessels and osteoclasts in parallel with decreased bone matrix density, as well increased levels of MMPs, osteoclastogenic and osteocyte markers. In the view of contrasting inflammatory and healing phenotypes of AIRmin and AIRmax strains in other models, the unpredicted phenotype observed suggests the existence of specific QTLs (Quantitative trait loci) responsible for the regulation 'sterile' inflammation and bone healing events. Despite the similar endpoint healing, AIRmax strain delayed repair was associated with increased presence of neutrophils and M1 macrophages, supporting the association of M2 cells with faster bone healing. Further studies are required to clarify the elements responsible for the regulation of inflammatory events at bone healing sites, as well the determinants of bone healing outcome.
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Processo Alveolar/patologia , Inflamação/patologia , Cicatrização , Processo Alveolar/diagnóstico por imagem , Animais , Antígenos CD/metabolismo , Birrefringência , Osso Esponjoso/diagnóstico por imagem , Osso Esponjoso/patologia , Inflamação/diagnóstico por imagem , Camundongos , Alvéolo Dental/diagnóstico por imagem , Alvéolo Dental/patologia , Microtomografia por Raio-XRESUMO
Mouse lines selected for maximal (AIRmax) or minimal acute inflammatory reaction (AIRmin) were used to characterize the immune response and the influence of genetic background during pristane-induced arthritis (PIA). Susceptible AIRmax mice demonstrated exacerbated cellular profiles during PIA, with intense infiltration of lymphocytes, as well as monocytes/macrophages and neutrophils, producing higher levels of IL-1ß, IFN-γ, TNF-α, IL-10, total IgG3, and chemokines. Resistant AIRmin mice controlled cell activation more efficiently than the AIRmax during arthritis progression. The weight alterations of the spleen and thymus in the course of PIA were observed. Our data suggest that selected AIRmax cellular and genetic immune mechanisms contribute to cartilage damage and arthritis severity, evidencing many targets for therapeutic actions.
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Artrite Experimental/imunologia , Citocinas/imunologia , Imunoglobulina G/imunologia , Terpenos/efeitos adversos , Doença Aguda , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/genética , Artrite Experimental/patologia , Citocinas/genética , Imunoglobulina G/genética , Inflamação , Camundongos , Baço/imunologia , Baço/patologia , Terpenos/farmacologia , Timo/imunologia , Timo/patologiaRESUMO
OBJECTIVE: To provide guidance to criminologists for conducting experiments in light of two common discouraging factors: the belief that they are overly time-consuming and the belief that they can compromise the ethical principles of human subjects' research. METHOD: A case study approach is used, based on a large-scale randomized controlled trial experiment in which we exposed participants to a 5-s TASER shock, to describe how the authors overcame ethical, methodological, and logistical difficulties. RESULTS: We derive four pieces of advice from our experiences carrying out this experimental trial: (1) know your limitations, (2) employ pilot testing, (3) remain flexible and patient, and (4) "hold the line" to maintain the integrity of the research and the safety of human subjects. CONCLUSIONS: Criminologists have an obligation to provide the best possible evidence regarding the impact and consequences of criminal justice practices and programs. Experiments, considered by many to be the gold standard of empirical research methodologies, should be used whenever possible in order to fulfill this obligation.
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Criminologia , Projetos de Pesquisa , Estimulação Elétrica Nervosa Transcutânea/instrumentação , Armas , Adolescente , Adulto , Idoso , Lesões por Armas de Eletrochoque , Ética em Pesquisa , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Somatic KRAS mutations are common in human lung adenocarcinomas and are associated with worse prognosis. In mice, Kras is frequently mutated in both spontaneous and experimentally induced lung tumors, although the pattern of mutation varies among strains, suggesting that such mutations are not random events. We tested if the occurrence of Kras mutations is under genetic control in two mouse intercrosses. Codon 61 mutations were prevalent, but the patterns of nucleotide changes differed between the intercrosses. Whole genome analysis with SNPs in (A/J x C57BL/6)F4 mice revealed a significant linkage between a locus on chromosome 19 and 2 particular codon 61 variants (CTA and CGA). In (AIRmax × AIRmin) F2 mice, there was a significant linkage between SNPs located on distal chromosome 6 (around 135 Mbp) and the frequency of codon 61 mutation. These results reveal the presence of two loci, on chromosomes 6 and 19, that modulate Kras mutation frequency in different mouse intercrosses. These findings indicate that somatic mutation frequency and type are not simple random events, but are under genetic control.
Assuntos
Adenocarcinoma/genética , Predisposição Genética para Doença/genética , Neoplasias Pulmonares/genética , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Códon/genética , Cruzamentos Genéticos , Feminino , Frequência do Gene , Masculino , Camundongos Endogâmicos C57BL , Camundongos EndogâmicosRESUMO
Pristane-induced arthritis (PIA) in mice is an experimental model that resembles human rheumatoid arthritis, a chronic autoimmune disease that affects joints and is characterized by synovial inflammation and articular cartilage and bone destruction. AIRmax and AIRmin mouse lines differ in their susceptibility to PIA, and linkage analysis in this model mapped arthritis severity QTLs in chromosomes 5 and 8. miRNAs are a class of small RNA molecules that have been extensively studied in the development of arthritis. We analyzed miRNA and gene expression profiles in peritoneal cells of AIRmax and AIRmin lines, in order to evaluate the genetic architecture in this model. Susceptible AIRmax mice showed higher gene (2025 vs 1043) and miRNA (240 vs 59) modulation than resistant AIRmin mice at the onset of disease symptoms. miR-132-3p/212-3p, miR-106-5p, miR-27b-3p, and miR-25-3p were among the miRNAs with the highest expression in susceptible animals, showing a negative correlation with the expression of predicted target genes (Il10, Cd69, and Sp1r1). Our study showed that global gene and miRNA expression profiles in peritoneal cells of susceptible AIRmax and resistant AIRmin lines during pristane-induced arthritis are distinct, evidencing interesting targets for further validation.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , MicroRNAs/genética , Peritônio/fisiologia , Animais , Artrite Experimental/induzido quimicamente , Células Cultivadas , Suscetibilidade a Doenças , Feminino , Humanos , Interleucina-10/genética , Masculino , Camundongos , Camundongos Mutantes , Peritônio/patologia , Locos de Características Quantitativas/genética , Terpenos/administração & dosagem , TranscriptomaRESUMO
The SM/J mouse strain is resistant to chemically-induced lung tumorigenesis despite having a haplotype, in the pulmonary adenoma susceptibility locus (Pas1) locus, that confers tumor susceptibility in other strains. To clarify this inconsistent genotype-phenotype correlation, we crossed SM/J mice with another resistant strain and conducted genome-wide linkage analysis in the (C57BL/6J × SM/J)F2 progeny exposed to urethane to induce lung tumors. Overall, >80% of F2 mice of both sexes developed from 1 to 20 lung tumors. Genotyping of 372 F2 mice for 744 informative non-redundant SNPs dispersed over all autosomal chromosomes revealed four quantitative trait loci (QTLs) affecting lung tumor multiplicity, on chromosomes 3 (near rs13477379), 15 (rs6285067), 17 (rs33373629) and 18 (rs3706601), all with logarithm of the odds (LOD) scores >5. Four QTLs modulated total lung tumor volume, on chromosome 3 (rs13477379), 10 (rs13480702), 15 (rs6285067) and 17 (rs3682923), all with LOD scores >4. No QTL modulating lung tumor multiplicity or total volume was detected in Pas1 on chromosome 6. The present study demonstrates that the SM/J strain carries, at the Pas1 locus, the resistance allele: a finding that will facilitate identification of the Pas1 causal element. More generally, it demonstrates that lung tumorigenesis is under complex polygenic control even in a pedigree with low susceptibility to this neoplasia, suggesting that the genetics of lung tumorigenesis is much more complex than evidenced by the pulmonary adenoma susceptibility and resistance loci that have, so far, been mapped in a small number of crosses between a few inbred strains.