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1.
Artigo em Chinês | MEDLINE | ID: mdl-31177710

RESUMO

Objective: To investigate the possible mechanism of doxycycline inhibiting paraquat-induced pulmonary fibrosis and provide a theoretical basis for its clinical application. Methods: Human lung fibroblast HFL1 cells were selected as the research object in the cell group. Divided into blank group, paraquat group, paraquat+doxycycline group. The expression of TGF-ß1, a-SMA, Smad3 and Smad2 protein was detected by ELISA using 40 ml of paraquat 40 umol/L and 3 mg/L of oleic acid 10 mg/L. In the animal group, 120 healthy and clean SD rats were randomly divided into three groups: blank group, paraquat group, paraquat+doxycycline group. The expression of TGF-ß1, a-SMA, Smad3 and Smad2 protein in lung tissue of mice at 1 day, 3 days, 7 days, 14 days and 21 days was detected by Elisa method. The expression of TGF-ß1, a-SMA, Smad3 and Smad2 protein in lung tissue of 21-day mice was detected by Western Blotting. The pathological changes of lung tissue were observed by HE staining for 1 day, 3 days, 7 days, 14 days and 21 days. Results: In the cell group experiment, the expression of TGF-ß1, a-SMA, Smad3 and Smad2 protein increased gradually with paraquat in the paraquat group, and the expression of TGF-ß1, a-SMA, Smad3 and Smad2 protein was significantly higher than that in the blank group. The difference was statistically significant (P<0.05) . The expressions of TGF-ß1, a-SMA, Smad3 and Smad2 in the paraquat+doxycycline group were significantly lower than those in the paraquat group, but still higher than the blank group, the difference was statistically significant (P<0.05) . Conclusion: Doxycycline inhibits paraquat-induced pulmonary fibrosis by inhibiting the expression of TGF-ß1, a-SMA and Smad3, Smad2 proteins.


Assuntos
Antibacterianos , Doxiciclina , Paraquat , Fibrose Pulmonar , Proteínas Smad , Fator de Crescimento Transformador beta1 , Animais , Antibacterianos/farmacologia , Doxiciclina/farmacologia , Humanos , Camundongos , Paraquat/toxicidade , Fibrose Pulmonar/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Smad/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo
2.
Eur J Med Chem ; 174: 16-32, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31022550

RESUMO

Intrinsic resistance in Pseudomonas aeruginosa, defined by chromosomally encoded low outer membrane permeability and constitutively over-expressed efflux pumps, is a major reason why the pathogen is refractory to many antibiotics. Herein, we report that heterodimeric rifampicin-tobramycin conjugates break this intrinsic resistance and sensitize multidrug and extensively drug-resistant P. aeruginosa to doxycycline and chloramphenicol in vitro and in vivo. Tetracyclines and chloramphenicol are model compounds for bacteriostatic effects, but when combined with rifampicin-tobramycin adjuvants, their effects became bactericidal at sub MIC levels. Potentiation of tetracyclines correlates with the SAR of this class of drugs and is consistent with outer membrane permeabilization and efflux pump inhibition. Overall, this strategy finds new uses for old drugs and presents an avenue to expand the therapeutic utility of legacy antibiotics to recalcitrant pathogens such as P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Cloranfenicol/farmacologia , Doxiciclina/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Rifampina/farmacologia , Tobramicina/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/toxicidade , Proteínas da Membrana Bacteriana Externa/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Células HEK293 , Células Hep G2 , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mariposas , Rifampina/análogos & derivados , Rifampina/síntese química , Rifampina/toxicidade , Suínos , Tobramicina/análogos & derivados , Tobramicina/síntese química , Tobramicina/toxicidade
3.
Exp Parasitol ; 200: 73-78, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30953626

RESUMO

OBJECTIVE: The aim of this study was to evaluate the efficacy of oral doxycycline treatment for Brugia malayi as measured by microfilarial and filarial DNA clearance in naturally infected domestic cats. METHODS: This study included 8 domestic cats that lived with families that resided in Tak Bai District of Narathiwat Province, which is located in Southern Thailand. The study area is a known B. malayi endemic area. All study cats received doxycycline treatment doses by their respective owners according to a previously described protocol. Briefly, doxycycline (VibraVet@) was given orally once a day during weeks 1-4, 10-11, and 16-17. Blood collections were performed at baseline before treatment, and then every month for 12 months after the initial dose of doxycycline to assess microfilaraemia by Giemsa stain, and filarial DNA detection by high-resolution melt (HRM) real-time polymerase chain reaction (PCR). RESULTS: One month after the start of doxycycline treatment, five of eight cats were negative for microfilaraemia, and 4 of those were negative for filarial DNA. All cats receiving doxycycline treatment were negative for microfilaria by Giemsa stain, and for filarial DNA by HRM real-time PCR within 8 months after receiving the initial dose of doxycycline treatment. CONCLUSION: Administration of oral doxycycline to domestic cats naturally infected with B. malayi in disease endemic areas can significantly reduce microfilaraemia at 1 month and filarial DNA was undetectable by 8 months after the initial dose of doxycycline treatment. No recurrence of microfilaraemia or filarial DNA was observed in study cats at 1 year after the start of doxycycline. Included cats appeared to tolerate doxycycline (VibraVet@) well, with no adverse drug reactions reported by any study cat owner.


Assuntos
Brugia Malayi/efeitos dos fármacos , Doenças do Gato/tratamento farmacológico , Doenças do Gato/parasitologia , Doxiciclina/uso terapêutico , Filariose/veterinária , Filaricidas/uso terapêutico , Administração Oral , Animais , Gatos , Reservatórios de Doenças , Doxiciclina/administração & dosagem , Doxiciclina/farmacologia , Filariose/tratamento farmacológico , Filariose/parasitologia , Filaricidas/administração & dosagem , Filaricidas/farmacologia , Microfilárias/efeitos dos fármacos , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Parasitemia/veterinária , Tailândia
4.
Reprod Biol Endocrinol ; 17(1): 38, 2019 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-30981279

RESUMO

BACKGROUND: Matrix metalloproteinases (MMPs), especially the gelatinases MMP-2 and MMP-9, play a crucial role in the pathogenesis of endometriosis by enabling invasion. Doxycycline is a well-tolerated antibiotic and a potent MMP-inhibitor in subantimicrobial doses. METHODS: Gelatin zymography and activity assays were used to detect latent and active MMP-2 and -9 in cell culture supernatants of immortalized epithelial (12Z) and two isolates of primary endometriotic stromal cells treated with doxycycline. The invasiveness of 12Z endometriotic cells treated with doxycycline was assessed in matrigel-coated invasion chambers. The effect on latent and active MMP-2 expression of the combination of progesterone and doxycycline was tested in 12Z. RESULTS: Doxycycline significantly reduced the MMP-2 activity and pro-MMP-2 expression in 12Z and the MMP-2 and -9 activity as well as expression of pro-MMP-2 and -9 in primary endometriotic stromal cells. The percentage of 12Z cells invading through a matrigel-coated membrane was reduced to 65 and 22% of the control after treatment with doxycycline at doses of 1 µg/ml and 10 µg/ml, respectively. Furthermore, a combination of progesterone and doxycycline showed an additive effect in low doses on the reduction of MMP-2 activity and pro-MMP2 expression in 12Z endometriotic cells. CONCLUSIONS: In conclusion, the MMP-inhibiting features of subantimicrobial-dose doxycycline may be further evaluated as a well-tolerable additional therapeutic approach, e.g. in combination with progestins such as dienogest, in patients with infiltrative endometriosis with insufficient response to current medical treatment options.


Assuntos
Doxiciclina/farmacologia , Endometriose/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Células Estromais/efeitos dos fármacos
5.
Int J Mol Sci ; 20(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987262

RESUMO

Antigen-presenting cells (APCs) including dendritic cells (DCs) play a critical role in the development of autoimmune diseases by presenting self-antigen to T-cells. Different signals modulate the ability of APCs to activate or tolerize autoreactive T-cells. Since the expression of heme oxygenase-1 (HO-1) by APCs has been associated with the tolerization of autoreactive T-cells, we hypothesized that HO-1 expression might be altered in APCs from autoimmune-prone non-obese diabetic (NOD) mice. We found that, compared to control mice, NOD mice exhibited a lower percentage of HO-1-expressing cells among the splenic DCs, suggesting an impairment of their tolerogenic functions. To investigate whether restored expression of HO-1 in APCs could alter the development of diabetes in NOD mice, we generated a transgenic mouse strain in which HO-1 expression can be specifically induced in DCs using a tetracycline-controlled transcriptional activation system. Mice in which HO-1 expression was induced in DCs exhibited a lower Type 1 Diabetes (T1D) incidence and a reduced insulitis compared to non-induced mice. Upregulation of HO-1 in DCs also prevented further increase of glycemia in recently diabetic NOD mice. Altogether, our data demonstrated the potential of induction of HO-1 expression in DCs as a preventative treatment, and potential as a curative approach for T1D.


Assuntos
Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/prevenção & controle , Heme Oxigenase-1/genética , Animais , Antígeno CD11c/metabolismo , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/complicações , Doxiciclina/farmacologia , Hiperglicemia/complicações , Hiperglicemia/prevenção & controle , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Regulação para Cima/efeitos dos fármacos
6.
Mol Biotechnol ; 61(6): 427-431, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30941576

RESUMO

Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in the regulation of lipid and glucose homeostasis and inflammation. PPARγ expression level has been widely studied in multiple tissues; however, there are few reports of preceding attempts to produce full-length human PPARγ (hPPARγ) in cellular models, and generally, expression level is not known or measurable. We propose an alternative strategy to express recombinant hPPARγ1, using a transient transfection with an inducible Tet-On 3G system where target and reporter gene were cloned in the same open reading frame. We transiently co-transfected human embryonic kidney 293T (HEK293T) cells with pTRE-ZsGreen1-IRES2-hPPARγ1 and pCMV-TET3G for inducible expression of hPPARγ1. Relative expression of the transcript was evaluated by RT-qPCR 48 h after transfection, obtaining a high expression level of hPPARγ (530-fold change, p < 0.002) in co-transfected HEK293T cells in the presence of doxycycline (1 µg/mL); also a significantly increased production of the reporter protein ZsGreen1 (3.6-fold change, p < 0.05) was determined by fluorescence analysis. These data indicated that HEK293T cells were successfully co-transfected and it could be an alternative model for hPPARγ expression in vitro. Additionally, this model will help to validate the quantification of inducible hPPARγ expression in vivo models for future research.


Assuntos
Clonagem Molecular/métodos , Vetores Genéticos/metabolismo , PPAR gama/genética , Proteínas Recombinantes de Fusão/genética , Doxiciclina/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Vetores Genéticos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Fases de Leitura Aberta , PPAR gama/biossíntese , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transfecção
7.
Emerg Microbes Infect ; 8(1): 339-352, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866787

RESUMO

Acute undifferentiated fever (AUF) is frequently observed in tropical settings, but diagnosing the cause of AUF is often a challenge for local physicians and the physicians treating returning travellers. We conducted a case-control study in central Vietnam in 2016. A total of 378 febrile adult patients (AUFs) with a fever for ≤21 days, no evidence of localized infection and negative screening tests for dengue and malaria, and 384 afebrile adult patients (Controls) were prospectively enrolled. Whole blood, plasma, eschar swab, throat swab and urine specimens were collected and analysed. Quantitative PCR and RT-PCR were used to test for 55 bacteria, viruses and their subtypes. Serological tests were also used to test for rickettsial agents. The most common aetiology was influenza virus (20.9% in AUFs vs. 0% in Controls), followed by rickettsial agents (mainly Orientia tsutsugamushi and Rickettsia typhi) (10.8% vs. 0.3%), dengue virus (7.7% vs. 0.5%), Leptospira (4.8% vs. 0.8%), adenovirus (4.8% vs. 1.0%), and enterovirus (2.1% vs. 0%) (p < .05). The real proportion of dengue in AUF cases was underestimated because patients with dengue-positive rapid diagnosis tests were excluded from the study. The emerging agent Rickettsia felis, which had not been previously observed in Vietnam, was detected in this study. In total, 216 patients (57.1%) were given causative diagnoses, comprising 143 (66.2%) monoinfections and 73 (33.8%) coinfections. The infections caused by these agents should be considered in clinical practice and further studies. Additionally, agents susceptible to doxycycline were detected in 15.6% of AUFs; thus, this drug should be included in the panel used to treat AUF patients.


Assuntos
Bactérias/classificação , Coinfecção/epidemiologia , Febre/etiologia , Vírus/classificação , Adulto , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Estudos de Casos e Controles , Coinfecção/microbiologia , Doxiciclina/farmacologia , Feminino , Febre/sangue , Febre/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/isolamento & purificação , Estudos Prospectivos , Rickettsia/efeitos dos fármacos , Rickettsia/isolamento & purificação , Vietnã/epidemiologia , Vírus/efeitos dos fármacos , Vírus/genética , Vírus/isolamento & purificação
8.
Molecules ; 24(6)2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30909480

RESUMO

Doxycycline (DOXY) is an antibiotic routinely prescribed in human and veterinary medicine for antibacterial treatment, but it has also numerous side effects that include oxidative stress, inflammation, cancer or hypoxia-induced injury. Endogenously produced hydrogen sulfide (H2S) and polysulfides affect similar biological processes, in which reactive oxygen species (ROS) play a role. Herein, we have studied the interaction of DOXY with H2S (Na2S) or polysulfides (Na2S2, Na2S3 and Na2S4) to gain insights into the biological effects of intermediates/products that they generate. To achieve this, UV-VIS, EPR spectroscopy and plasmid DNA (pDNA) cleavage assay were employed. Na2S or Na2S2 in a mixture with DOXY, depending on ratio, concentration and time, displayed bell-shape kinetics in terms of producing/scavenging superoxide and hydroxyl radicals and decomposing hydrogen peroxide. In contrast, the effects of individual compounds (except for Na2S2) were hardly observable. In addition, DOXY, as well as oxytetracycline and tetracycline, interacting with Na2S or other studied polysulfides reduced the •cPTIO radical. Tetracyclines induced pDNA cleavage in the presence of Na2S. Interestingly, they inhibited pDNA cleavage induced by other polysulfides. In conclusion, sulfide and polysulfides interacting with tetracyclines produce/scavenge free radicals, indicating a consequence for free radical biology under conditions of ROS production and tetracyclines administration.


Assuntos
Clivagem do DNA/efeitos dos fármacos , Doxiciclina/química , Doxiciclina/farmacologia , Radical Hidroxila/química , Sulfetos/química , Sulfetos/farmacologia , Superóxidos/química , Interações de Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Análise Espectral
9.
In Vitro Cell Dev Biol Anim ; 55(3): 211-219, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30756235

RESUMO

Attenuation of fibroblast growth factor receptor (FGFR) 2b signaling suppresses the differentiation of oral epithelial stem cells to ameloblasts, their survival and viability remaining unaffected; however, its effect on dentin formation is unknown. This study aimed to clarify the effect of attenuation of FGFR2b signaling on odontoblast differentiation and dentin formation. Initially, we used a murine rtTA transactivator/tetracycline promoter system for inducible and reversible attenuation of FGFR2b signaling in adult mice. Experimental animals overexpressed soluble FGFR2b (sFGFR2b), and wild-type controls were selected from the same litter (WT group). Histological analysis of CMV mice confirmed the obliteration of the enamel and ameloblast layer, and micro CT analysis revealed a significant increase in dentin thickness in CMV mice rather than in WT mice (P < 0.05). On analyzing the expression of dentin-related differentiation factors, DSPP, nestin, and OCN were upregulated in CMV mice compared to WT mice after 2 weeks of attenuation of FGFR2b signaling. Thereafter, on overexpressing sFGFR2b in dental pulp stem cells, RUNX2 and ALP were upregulated; however, DSPP, nestin, and OCN were downregulated in CMV mice compared to WT mice. The present results show that attenuation of FGFR2b signaling in the oral epithelium specifically induced odontoblast differentiation and promotes early-stage dentin calcification in dental pulp tissue.


Assuntos
Dentina/crescimento & desenvolvimento , Odontoblastos/citologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Esmalte Dentário/diagnóstico por imagem , Polpa Dentária/citologia , Dentina/metabolismo , Doxiciclina/farmacologia , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos Mutantes , Odontoblastos/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Microtomografia por Raio-X
10.
Methods Mol Biol ; 1940: 63-76, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788818

RESUMO

Terminally differentiated somatic cells can be reprogrammed into an embryonic stem cell-like state by the forced expression of four transcription factors: Oct4, Klf4, Sox2, and c-Myc (OKSM). These so-called induced pluripotent stem (iPS) cells can give rise to any cell type of the body and thus have tremendous potential for many applications in research and regenerative medicine. Herein, we describe (1) a protocol for the generation of iPS cells from mouse embryonic fibroblasts (MEFs) using a doxycycline (Dox)-inducible lentiviral transduction system; (2) the derivation of clonal iPS cell lines; and (3) the characterization of the pluripotent potential of iPS cell lines using alkaline phosphatase staining, flow cytometry, and the teratoma formation assays.


Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular/genética , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/virologia , Lentivirus/genética , Animais , Diferenciação Celular , Células Cultivadas , Doxiciclina/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução Genética/métodos
11.
J Antibiot (Tokyo) ; 72(4): 225-236, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30737453

RESUMO

In bacteria, RNase III cleaves the initial long primary ribosomal RNA transcripts/precursors (pre-rRNAs), thereby releasing the pre-16S and pre-23S rRNAs for maturation. This cleavage is specified by the double-stranded secondary structures flanking the mature rRNAs, and not necessarily by the nucleotide sequences. Inhibition of this cleavage would lead to a build-up of pre-rRNA molecules. Doxycycline has earlier been shown to bind synthetic double-stranded RNAs and inhibit their cleavage by RNase III. Since bacterial rRNA processing is primarily dependent on RNase III cleavage (which is inhibited by doxycycline), doxycycline could therefore inhibit the normal processing of bacterial rRNA. In this study, the effect of doxycycline on bacterial rRNA processing was investigated by analyzing the amounts of various rRNAs in growing Escherichia coli cells treated with doxycycline. The results showed a doxycycline dose-dependent decrease in mature 16S and 23S rRNAs, concurrent with an accumulation of the initial rRNA transcripts and long precursors. Morphologically, treated cells were elongated at low drug concentrations, while nucleoid degeneration indicative of cell death occurred at higher drug concentrations. These observations suggest that doxycycline inhibits the cleavage and processing of bacterial rRNA transcripts/precursors, leading to impaired formation of mature rRNAs, and the consequent inhibition of protein synthesis for which the tetracycline group of antibiotics are renowned. Since rRNA structure and processing pathway is conserved among bacterial species, this mechanism may account for the broad spectrum of antibiotic activity and selective microbial protein synthesis inhibition of doxycycline and the tetracyclines.


Assuntos
Antibacterianos/farmacologia , Doxiciclina/farmacologia , Escherichia coli/efeitos dos fármacos , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo
12.
J Antibiot (Tokyo) ; 72(5): 291-297, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30755738

RESUMO

Legionella pneumophila is a waterborne intracellular pathogenic bacterium, the most frequent cause of human legionellosis and a relatively common cause of community-acquired and nosocomial pneumonia. Some legionellosis outbreaks are related to the presence of biofilms, which provide a reservoir for L. pneumophila strains. We investigated the in vitro activities of antibiotics; erythromycin and doxycycline, antimicrobial peptides AMPs; melittin, LL-37 and CAMA (cecropin A (1-7)-Melittin A (2-9) and ceragenins; CSA-8, CSA-13, CSA-44, CSA-131 and CSA-138 against L. pneumophila. Isolation of Legionella strains was conducted according to ISO 1998. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum biofilm eradication concentrations (MBECs) were determined using microbroth dilution techniques. MIC ranges for melittin, LL-37, and CAMA were 0.25-1, 1-4, and 2-8 µg ml-1, respectively. MIC ranges for CSA-8, 13, 44, 131, and 138 were 0.5-2, 0.5-1, 1-4, 0.5-2, and 1-2 µg ml-1, respectively, and MBEC values for the ceragenins were 10-160 µg ml-1. These results demonstrate that AMPs and ceragenins display broad-spectrum, in vitro activity against L. pneumophila. In particular, CSA-8, CSA-13 and melittin gave the lowest MICs and MBCs. We also observed that ceragenins are active against established L. pneumophila biofilms.


Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Legionella pneumophila/efeitos dos fármacos , Esteroides/farmacologia , Biofilmes/efeitos dos fármacos , Doxiciclina/farmacologia , Eritromicina/farmacologia , Legionella pneumophila/isolamento & purificação , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos
13.
J Antibiot (Tokyo) ; 72(5): 253-259, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30760838

RESUMO

The rise in antibiotic resistance among pathogenic microorganisms has created an imbalance in the drugs available for treatment, in part due to the slow development of new antibiotics. Cystic fibrosis (CF) patients are highly susceptible to antibiotic-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Phloroglucinols and related polyketide natural products have demonstrated antimicrobial activity against a number of Gram-positive bacteria including S. aureus. In this study, we investigated a series of acylated phloroglucinol derivatives to determine their potential as lead compounds for the design of novel therapeutics. To assess the activity of these compounds, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively), the minimum biofilm inhibitory and biofilm eradication concentration (MBIC and MBEC, respectively), and evaluated hemolytic activity, as well as their interaction with clinically relevant antibiotics. Of the 12 compounds tested against MRSA and methicillin-susceptible strains, four showed MIC values ranging from 0.125 to 8 µg ml-1 and all of them were bactericidal. However, none of the compounds were able to eradicate biofilms at the concentrations tested. Three of the four did not display hemolytic activity under the conditions tested. Further studies on the interactions of these compounds with clinically relevant antibiotics showed that phlorodipropanophenone displayed synergistic activity when paired with doxycycline. Our results suggest that these acylated phloroglucinols have potential for being further investigated as antibacterial leads.


Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Floroglucinol/análogos & derivados , Floroglucinol/farmacologia , Antibacterianos/síntese química , Antibacterianos/toxicidade , Biofilmes/efeitos dos fármacos , Doxiciclina/farmacologia , Interações de Medicamentos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Floroglucinol/síntese química , Floroglucinol/toxicidade
14.
Arch. Soc. Esp. Oftalmol ; 94(2): 100-104, feb. 2019. ilus
Artigo em Espanhol | IBECS | ID: ibc-180373

RESUMO

Caso clínico: Una paciente con antecedente de resección quirúrgica de un neurinoma del acústico presentó compromiso tanto del nervio facial como del nervio trigémino izquierdos. Inicialmente consultó por queratitis de exposición, pero 2 semanas después presentó una queratitis infecciosa. Tras la toma de la muestra corneal cursó con un defecto epitelial persistente, que no respondió al manejo médico. Se indicó insulina tópica con lo que se evidenció disminución del área de la lesión en los siguientes 5 días. Se colocó además, en ese momento, una lente de contacto terapéutica y, finalmente, 2 semanas después de haberse iniciado la insulina, el defecto epitelial cerró por completo. Discusión: Se trata de un caso complejo por la confluencia de parálisis facial, queratitis neurotrófica y queratitis infecciosa, que finalmente tuvo un resultado exitoso. La insulina tópica puede ser una terapia coadyuvante efectiva en casos de úlceras neurotróficas que no respondan a la terapia convencional


Case report: A patient with a history of surgical resection of an acoustic neuroma presented with involvement of both the left facial nerve and the left trigeminal nerve. She initially consulted for exposure keratitis, but two weeks later presented with an infectious keratitis. After taking the corneal sample, she presented with persistent epithelial defect, which did not respond to medical management. Topical insulin was indicated, and a decrease in the area of the lesion was seen in the following 5 days. A therapeutic contact lens was also placed at that time and finally, two weeks after the initiation of insulin, the epithelial defect completely closed. Discussion: This was a complex case due to the confluence of facial paralysis, neurotrophic keratitis, and infectious keratitis, which finally had a successful outcome. Topical insulin can be an effective adjuvant therapy in cases of neurotrophic ulcers that do not respond to standard therapy


Assuntos
Feminino , Pessoa de Meia-Idade , Insulina/farmacologia , Insulina/uso terapêutico , Ceratite/classificação , Neuroma Acústico/diagnóstico , Paralisia Facial/classificação , Paralisia Facial/diagnóstico , Células de Schwann/patologia , Staphylococcus aureus/classificação , Vancomicina/análise , Doxiciclina/farmacologia , Ácido Ascórbico/farmacologia , Edema da Córnea/diagnóstico
15.
Mater Sci Eng C Mater Biol Appl ; 97: 84-95, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30678975

RESUMO

The purpose of the present work was to evaluate in vivo different antimicrobial therapies to eradicate osteomyelitis created in the femoral head of New Zealand rabbits. Five phosphate-based cements were evaluated: calcium phosphate cements (CPC) and calcium phosphate foams (CPF), both in their pristine form and loaded with doxycycline hyclate, and an intrinsic antimicrobial magnesium phosphate cement (MPC; not loaded with an antibiotic). The cements were implanted in a bone previously infected with Staphylococcus aureus to discern the effects of the type of antibiotic administration (systemic vs. local), porosity (microporosity, i.e. <5 µm vs. macroporosity, i.e. >5 µm) and type of antimicrobial mechanism (release of antibiotic vs. intrinsic antimicrobial activity) on the improvement of the health state of the infected animals. A new method was developed, with a more comprehensive composite score that integrates 5 parameters of bone infection, 4 parameters of bone structural integrity and 4 parameters of bone regeneration. This method was used to evaluate the health state of the infected animals, both before and after osteomyelitis treatment. The results showed that the composite score allows to discern statistically significant differences between treatments that individual evaluations were not able to identify. Despite none of the therapies completely eradicated the infection, it was observed that macroporous materials (CPF and CPFd, the latter loaded with doxycycline hyclate) and intrinsic antimicrobial MPC allowed a better containment of the osteomyelitis. This study provides novel insights to understand the effect of different antimicrobial therapies in vivo, and a promising comprehensive methodology to evaluate the health state of the animals was developed. We expect that the implementation of such methodology could improve the criteria to select a proper antimicrobial therapy.


Assuntos
Antibacterianos/farmacologia , Cimentos para Ossos/farmacologia , Osteomielite/terapia , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Cimentos para Ossos/química , Doenças Ósseas Infecciosas/tratamento farmacológico , Doenças Ósseas Infecciosas/terapia , Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/química , Doxiciclina/administração & dosagem , Doxiciclina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Implantes de Medicamento/química , Implantes de Medicamento/farmacologia , Liberação Controlada de Fármacos , Feminino , Fêmur/diagnóstico por imagem , Fêmur/patologia , Osteomielite/tratamento farmacológico , Porosidade , Coelhos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/terapia , Resultado do Tratamento , Substâncias Viscoelásticas/química
16.
Wounds ; 31(2): 49-54, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30664497

RESUMO

INTRODUCTION: Tetracycline molecules comprise a group of broad-spectrum antibiotics whose primary mechanism of action is the inhibition of protein synthesis through the binding of the bacterial ribosome. In addition, tetracyclines inhibit matrix metalloproteases (MMPs), a family of zinc-dependent proteases that contribute to tissue remodeling, inflammation, and angiogenesis and are overexpressed in certain pathophysiologies such as diabetic foot ulcers (DFUs). OBJECTIVE: This study aims to develop a liquid chromatography and mass spectrometry (LC-MS/MS) doxycycline quantification methodology to facilitate the development of a stable topical doxycycline hyclate (DOXY) formulation as well as evaluate the topical DOXY formulation for the efficacy in MMP-9 inhibition in vitro and in a clinical application of diabetic lower extremity wounds. MATERIALS AND METHODS: A simple quantification method utilizing LC-MS/MS was used to develop a topical DOXY formulation, a sample of which was analyzed in stability testing. The formulation was evaluated in vitro for MMP-9 activity using a commercial assay and compared with internal kit controls as well as in a clinical setting for wound healing. RESULTS: Two formulations of 2% (w/w) DOXY demonstrated acceptable stability (±10% target concentration) for 70 days when stored at 4°C. Using an in vitro assay of MMP-9 enzyme activity, the 2% DOXY formulation imparted a ~30% decrease in MMP-9 inhibitory potential as compared with the control drug alone (IC50 values 62.92 µM and 48.27 µM, respectively). This topical product was evaluated for clinical utility in a patient with a DFU, and preliminary data suggest this intervention may promote wound healing. CONCLUSIONS: In summary, novel DOXY formulations may be stable and biologically active tools amenable to complex wound care.


Assuntos
Antibacterianos/farmacologia , Compostos Cromogênicos/farmacologia , Pé Diabético/tratamento farmacológico , Doxiciclina/administração & dosagem , Doxiciclina/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Cicatrização/efeitos dos fármacos , Administração Tópica , Antibacterianos/administração & dosagem , Cromatografia Líquida , Compostos Cromogênicos/administração & dosagem , Pé Diabético/patologia , Feminino , Humanos , Espectrometria de Massas , Inibidores de Metaloproteinases de Matriz/administração & dosagem , Pessoa de Meia-Idade , Resultado do Tratamento , Cicatrização/fisiologia
17.
Carbohydr Polym ; 207: 1-10, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30599988

RESUMO

Bacterial cellulose (BC) has shown high potential as innovative wound dressing and drug delivery system. Bringing both together, drug-loaded BC was investigated for applications in dental therapies such as dental extraction or mucosal transplantation. Both applications would benefit from a material which degrades under physiological conditions, and from an antibiotic environment. Consequently, periodate-oxidation of BC was investigated to facilitate modified degradation behaviour. A periodate concentration of 0.14 mol/L at ϑ = 25 °C and t = 8 h resulted in a material loss of <10%, but at the same time a sufficient degree of degradation. Additionally, native and oxidised BC loaded with doxycycline was tested for prophylaxis against infection. An in vitro-toxicity test (MTT assay) provided a first confirmation of biocompatibility, whereas agar diffusion tests proved antibiotic efficiency against pathogenic oral bacteria. Release studies of the drug from native and oxidised BC confirmed a comparative biphasic release behaviour.


Assuntos
Antibacterianos/farmacologia , Bandagens , Celulose/química , Instrumentos Odontológicos , Doxiciclina/farmacologia , Portadores de Fármacos/química , Acetobacteraceae/química , Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Animais , Antibacterianos/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Plásticos Biodegradáveis , Linhagem Celular , Celulose/toxicidade , Doxiciclina/química , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Camundongos , Oxirredução , Ácido Periódico/química , Staphylococcus aureus/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacos
18.
Nat Commun ; 10(1): 137, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30635584

RESUMO

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Antígenos de Histocompatibilidade Menor/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Processamento de RNA/efeitos dos fármacos , Proteína bcl-X/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Sinergismo Farmacológico , Compostos de Epóxi/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Macrolídeos/farmacologia , Melanoma/genética , Camundongos , Camundongos Nus , Interferência de RNA , Processamento de RNA/genética , RNA Interferente Pequeno/genética , Spliceossomos/efeitos dos fármacos , Spliceossomos/genética , Sequenciamento Completo do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
19.
G3 (Bethesda) ; 9(2): 591-599, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30591434

RESUMO

The modification of the mouse genome by site-specific gene insertion of transgenes and other genetic elements allows the study of gene function in different developmental stages and in the pathogenesis of diseases. Here, we generated a "genomic safe harbor" Hipp11 (H11) locus-specific knock-in transgenic mouse line in which the albumin promoter is used to drive the expression of the reverse tetracycline transactivator (rtTA) in the liver. The newly generated H11-albumin-rtTA transgenic mice were bred with tetracycline-operator-Histone-2B-green fluorescent protein (TetO-H2BGFP) mice to assess inducibility and tissue-specificity. Expression of the H2BGFP fusion protein was observed exclusively upon doxycycline (Dox) induction in the liver of H11-albumin-rtTA/TetO-H2BGFP double transgenic mice. To further analyze the ability of the Dox-inducible H11-albumin-rtTA mice to implement conditional DNA recombination, H11-albumin-rtTA transgenic mice were crossed with TetO-Cre and Ai14 mice to generate H11-albumin-rtTA/TetO-Cre/Ai14 triple transgenic mice. We successfully confirmed that the Cre-mediated recombination efficiency was as strong in Dox-induced H11-albumin-rtTA /TetO-Cre/Ai14 mice as in the control albumin-Cre/A14 mice. Finally, to characterize the expression-inducing effects of Dox in H11-albumin-rtTA/TetO-H2BGFP mice in detail, we examined GFP expression in embryos at different developmental stages and found that newly conceived H11-albumin-rtTA/TetO-H2BGFP embryos of Dox-treated pregnant female mice were expressing reporter GFP by E16.5. Our study demonstrates that these new H11-albumin-rtTA transgenic mice are a powerful and efficient tool for the temporally and spatially conditional manipulation of gene expression in the liver, and illustrates how genetic crosses with these new mice enable the generation of complex multi-locus transgenic animals for mechanistic studies.


Assuntos
Técnicas de Introdução de Genes/métodos , Fígado/metabolismo , Camundongos Transgênicos/genética , Albuminas/genética , Albuminas/metabolismo , Animais , Doxiciclina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Regiões Promotoras Genéticas , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos
20.
Mol Med Rep ; 19(2): 967-973, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569122

RESUMO

Uncontrolled proliferation and defective apoptosis are two major factors responsible for maintaining the malignant properties of melanoma cells. Our previous study demonstrated that induced expression of four reprogramming factors remodeled the phenotype of B16­F10 mouse melanoma cells into melanoma stem cells. The present study was conducted to investigate the effect of the four Yamanaka reprogramming factors, namely Oct4, Sox2, Klf4 and c­Myc (OSKM), on the proliferation and apoptosis of melanoma cells, and to identify the responsible molecular signals. The results identified that expression of the four reprogramming factors was highly induced by doxycycline treatment in the stable melanoma cell clone that was transfected with a plasmid expressing these factors, driven by the Tet­On element. It was further confirmed that induced expression of these factors enhanced the proliferation and suppressed the apoptosis of the melanoma cells. In addition, induced OSKM expression increased cell proliferation, accelerated the progression of the cell cycle, and upregulated the mRNA expression levels of Janus kinase 2 (JAK2) and Cyclin­B1. Induced expression of these factors also decreased the apoptosis, as well as upregulated B­cell lymphoma 2 (BCL­2) and downregulated BCL­2­associated X (BAX) mRNA expression levels. Taken together, the results suggested that upregulated JAK2 and Cyclin­B1 may be responsible for the enhanced proliferation of melanoma cells, and that BCL­2 upregulation and BAX downregulation may account for the suppressed apoptosis of these cells.


Assuntos
Reprogramação Celular , Doxiciclina/farmacologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina B1/genética , Ciclina B1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Fatores de Transcrição Kruppel-Like/agonistas , Fatores de Transcrição Kruppel-Like/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/agonistas , Fator 3 de Transcrição de Octâmero/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/agonistas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/agonistas , Fatores de Transcrição SOXB1/metabolismo , Transfecção , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
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