RESUMO
BACKGROUND: COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. METHODS: Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers. RESULTS: DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. CONCLUSIONS: Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.
Assuntos
Lesão Pulmonar Aguda , COVID-19 , Armadilhas Extracelulares , Animais , Humanos , Camundongos , SARS-CoV-2 , Tratamento Farmacológico da COVID-19 , Modelos Animais de Doenças , Neutrófilos , Desoxirribonuclease I/farmacologia , Desoxirribonuclease I/uso terapêuticoRESUMO
The pathogenesis of Streptococcus uberis is attributed to a combination of extracellular factors and properties such as adherence and biofilm formation. The aim of this work was to evaluate the influence of different factors, additives and bovine milk compounds on S. uberis biofilm formation, as the presence of the sua gene by PCR. Additionally, extracellular DNA and the effect of DNaseI were evaluated in the biofilms yielded. Optimal biofilm development was observed when the pH was adjusted to 7.0 and 37 °C. Additives as glucose and lactose reduced biofilm formation as bovine milk compounds tested. PCR assay showed that not all the isolates yielded sua gene. Extrachromosomal ADN was found in cell-free supernatants, suggesting that DNA released spontaneously to the medium. The results contribute to a better understanding of the factors involved in biofilm production of this important pathogen associated with mastitis in order to promote the design of new therapeutic approaches.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Streptococcus/genética , Animais , Argentina , Caseínas/farmacologia , Bovinos , Meios de Cultura/química , DNA Bacteriano/genética , Desoxirribonuclease I/farmacologia , Feminino , Genes Bacterianos/genética , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Lactose/farmacologia , Mastite/microbiologia , Mastite Bovina/microbiologia , Leite/química , Leite/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Soroalbumina Bovina/farmacologia , Infecções Estreptocócicas/microbiologia , Streptococcus/efeitos dos fármacos , Streptococcus/isolamento & purificação , Streptococcus/patogenicidade , TemperaturaRESUMO
Background Cell-free DNA circulates in cancer patients and induces in vivo cell transformation and cancer progression in susceptible cells. Based on this, we hypothesized that depletion of circulating DNA with DNAse I and a protease mix could have antitumor effects. Study design The study aimed to demonstrate that DNAse I and a protease mix can degrade in vitro DNA and proteins from the serum of healthy individuals and cancer patients, and in vivo in serum of Wistar rats,. Moreover, the antitumor effect of the systemically administered enzyme mix treatmentwas evaluated in nude mice subcutaneously grafted with the human colon cancer cell line SW480. Results The serum DNA of cancer patients or healthy individuals was almost completely degraded in vitro by the enzymatic treatment, but no degradation was found with the enzymes given separately. The intravenous administration of the enzymes led to significant decreases in DNA and proteins from rat serum. No antitumor effect was observed in immunodeficient mice treated with the enzymes given separately. In contrast, the animals that received both enzymes exhibited a marked growth inhibition of tumors, 40% of them having pathological complete response. Conclusion This study demonstrated that systemic treatment with DNAse I and a protease mix in rats decreases DNA and proteins from serum and that this treatment has antitumor effects. Our results support the hypothesis that circulating DNA could have a role in tumor progression, which can be offset by depleting it. Further studies are needed to prove this concept.
Assuntos
Desoxirribonuclease I/farmacologia , Peptídeo Hidrolases/farmacologia , Adulto , Animais , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/sangue , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , DNA/sangue , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas/metabolismo , Ratos , Ratos WistarRESUMO
The radiosensitive mutant cell line IRS-20, its wild type counterpart CHO and a derivative of IRS-20 with a transfected YAC clone (YAC-IRS) that restores radioresistance were tested for DNAse I sensitivity. The three cell lines were cultured under the same conditions and had a mitotic index of 2-5%. One drop of fixed cells from the three lines was always spread on the same microscopic slide. After one day of ageing, slides were exposed to DNAse I and stained with DAPI. Images from every field were captured and the intensity of blue fluorescence was measured with appropriate software. For untreated cells, the fluorescence intensity was similar for all of the cell lines. After DNAse I treatment, CHO and YAC-IRS had an intensity of 85% but IRS-20 had an intensity of 60%, when compared with the controls. DNAse I sensitivity differences between the cell lines indicate that overall conformation of chromatin might contribute to radiation sensitivity of the IRS-20 cells.
Assuntos
Células CHO/efeitos da radiação , Proteínas de Ligação a DNA , DNA/efeitos da radiação , Desoxirribonuclease I/farmacologia , Conformação de Ácido Nucleico , Tolerância a Radiação/genética , Animais , Células CHO/metabolismo , Cromossomos Artificiais de Levedura/genética , Cromossomos Humanos Par 8/genética , Cricetinae , Cricetulus , DNA/efeitos dos fármacos , Dano ao DNA , Reparo do DNA , Proteína Quinase Ativada por DNA , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Teste de Complementação Genética , Humanos , Índice Mitótico , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , TransfecçãoRESUMO
A part of sperm glycosidase activities was detected as detergent-insoluble after sequential extractions with Triton X-100. Sixty per cent of total beta-glucuronidase activity was found in the detergent-insoluble fraction. This portion of beta-glucuronidase was resistant to extractions in the presence of 1 M KCl, chaotropic agents, colchicine or cytochalasine B, being only partially solubilized by 3 M KCl or DNAse I treatment. Results demonstrate that beta-glucuronidase is tightly associated to the Triton X-100 resistant fraction.
Assuntos
Glucuronidase/química , Glucuronidase/isolamento & purificação , Octoxinol/química , Espermatozoides/enzimologia , Actinas/química , Actinas/efeitos dos fármacos , Fracionamento Celular , Proteínas do Citoesqueleto/efeitos dos fármacos , Proteínas do Citoesqueleto/isolamento & purificação , Desoxirribonuclease I/farmacologia , Humanos , Masculino , Octoxinol/farmacologia , Polímeros , SolubilidadeRESUMO
Chinese hamster ovary (CHO) cells were treated with bovine pancreatic DNase I using the method of electroporation. The enzyme induced chromosomal aberrations in a S-phase independent manner. The frequencies of polycentric chromosomes induced in the G1 phase of the cell cycle are positively correlated with the dose of DNase I. The distributions of DNase I-induced polycentric chromosomes were overdispersed.
Assuntos
Aberrações Cromossômicas , Desoxirribonuclease I/farmacologia , Animais , Linhagem Celular , Desoxirribonuclease I/administração & dosagem , Relação Dose-Resposta a Droga , Fase G1/fisiologia , Pâncreas/enzimologia , Fase S/fisiologiaRESUMO
Cryoprecipitates from systemic lupus erythematosus (SLE) patients with high levels of anti DNA antibodies show a sharply migrating large circulating DNA species of about 17-20 kb (M. Rieber et al., Clin. exp. Immunol. (1986) 66, 61). We have now used Southern blot analysis of circulating DNA from different individuals to analyse the relative cross-hybridization of circulating DNA from different individuals, as well as their homology with genomic DNA from different species. Molecular hybridization showed significant homology of the various circulating DNA examined, only with human genomic DNA, but limited cross-reactivity among circulating DNA from different individuals. This suggests that the circulating DNA is composed of sequences repeated in human genomic DNA and by specific sequences unique to circulating DNA from some individuals. Our data suggests the possibility of using probes derived from the specific sequences now reported in the circulating DNA, in gene typing and in the analysis of susceptibility to disease.