RESUMO
Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to promote wound healing; it also exhibits antimicrobial properties that may aid in the healing of infected wounds. The purpose of this study was to quantify the efficacy of human platelet gel against the opportunistic bacterial wound pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus on skin. These opportunistic pathogens may exhibit extensive antibiotic resistance, necessitating the development of alternative treatment options. The antimicrobial efficacy of platelet gel supernatants was quantified using an in vitro broth dilution assay, an ex vivo inoculated skin assay, and in an in vivo skin decontamination assay. Human platelet gel supernatants were highly bactericidal against A. baumannii and moderately but significantly bactericidal against S. aureus in vitro and in the ex vivo skin model. P. aeruginosa was not inactivated in vitro; a low but significant inactivation level was observed ex vivo. These supernatants were quite effective at inactivating a model organism on skin in vivo. These results suggest application of platelet gel has potential clinical applicability, not only in the acceleration of wound healing, but also against relevant bacteria causing wound infections.
Assuntos
Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Plaquetas/metabolismo , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/microbiologia , Plasma Rico em Plaquetas/metabolismo , Ferimentos e Lesões/tratamento farmacológico , Ferimentos e Lesões/microbiologia , Acinetobacter baumannii , Animais , Modelos Animais de Doenças , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Ratos , Pele/lesões , Staphylococcus aureus , CicatrizaçãoRESUMO
3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug with cardiovascular effects that have not been fully described. In the current study, we observed the effects of acute MDMA on rabbit left ventricular function. We also observed the effects of MDMA on nuclear factor-kappa B (NF-κB) activity in cultured rat ventricular myocytes (H9c2). In the rabbit, MDMA (2 mg/kg) alone caused a significant increase in heart rate and a significant decrease in the duration of the cardiac cycle. Inhibition of nitric oxide synthase (NOS) by pretreatment with L-NAME (10 mg/kg) alone caused significant dysfunction in heart rate, systolic pressure, diastolic pressure, duration of relaxation, duration of cardiac cycle, and mean left ventricular pressure. Pretreatment with L-NAME followed by treatment with MDMA caused significant dysfunction in additional parameters that were not abnormal upon exposure to either compound in isolation: duration of contraction, inotropy, and pulse pressure. Exposure to 1.0 mM MDMA for 6 h or 2.0 µM MDMA for 12 h caused increased nuclear localization of NF-κB in cultured H9c2 cells. The current results suggest that MDMA is acutely detrimental to heart function and that an intact cardiovascular NOS system is important to help mitigate early sequelae in some functional parameters. The delayed timing of NF-κB activation suggests that this factor may be relevant to MDMA induced cardiomyopathy of later onset.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Metanfetamina/análogos & derivados , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Metanfetamina/farmacologia , Miócitos Cardíacos/patologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Coelhos , Ratos , Fatores de TempoRESUMO
Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological responses (e.g., apoptosis, stimulation of calcium (Ca2+) fluxes, changes in membrane potential). In this study, we investigated the effect of nanosecond pulses, which generate intense electric fields (nsPEFs), on human platelet aggregation, intracellular free Ca2+ ion concentration ([Ca2+]i) and platelet-derived growth factor release. When platelet rich plasma was pulsed with one 300ns pulse with an electric field of 30kV/cm, platelets aggregated and a platelet gel was produced. Platelet aggregation was observed with pulses as low as 7kV/cm with maximum effects seen with approximately 30kV/cm. The increases in intracellular Ca2+ release and Ca2+ influx were dose dependent on the electrical energy density and were maximally stimulated with approximately 30kV/cm. The increases in [Ca2+]i induced by nsPEF were similar to those seen with thapsigargin but not thrombin. We postulate that nsPEF caused Ca2+ to leak out of intracellular Ca2+ stores by a process involving the formation of nanopores in organelle membranes and also caused Ca2+ influx through plasma membrane nanopores. We conclude that nsPEFs dose-dependently cause platelets to rapidly aggregate, like other platelet agonists, and this is most likely initiated by the nsPEFs increasing [Ca2+]i, however by a different mechanism.
Assuntos
Cálcio/metabolismo , Membrana Celular/efeitos da radiação , Campos Eletromagnéticos , Eletroporação/métodos , Nanotecnologia/métodos , Ativação Plaquetária/efeitos da radiação , Fator de Crescimento Derivado de Plaquetas/efeitos da radiação , Membrana Celular/metabolismo , Relação Dose-Resposta à Radiação , Estimulação Elétrica , Humanos , Ligantes , Ativação Plaquetária/fisiologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de TempoRESUMO
To determine the cardiovascular molecular events associated with acute exposure to cocaine, the present study utilized in vivo analysis of left-ventricular heart function in adult rabbits, fluorescence confocal microscopy of fluo-2, rhod-2, (5-(and-6) carboxy 2',7' dichlorodihydrofluores-cein diacetate (carboxy-H2DCFDA), and JC-1 in H9C2 cells and gene expression microarray technology for analysis of gene activation in both rabbit ventricular tissue and H9C2 cells. In the rabbit, acute cocaine exposure (2 mg/kg) caused left-ventricular dysfunction and 0.1-10 mM cocaine increased cytosolic and mitochondrial calcium activity and mitochondrial membrane depolarization in H9C2 cells. A 3-min pretreatment of H9C2 cells by 10 microM verapamil, nifedipine, or nadolol inhibited calcium increases, but only 1 mM N-acetylcysteine (NAC) or 1 mM glutathione blocked mitochondrial membrane depolarization. Cocaine induced activation of genes in the rabbit heart and H9C2 cells including angiotensinogen, ADRB1, and c-reactive protein (CRP). In H9C2 cells, NAC pretreatment blocked cocaine-mediated increases in CRP, FAS, FAS ligand, and cytokine receptor-like factor1 (CRLF1) expression. Collectively, these data suggest that acute cocaine administration initiates cellular and genetic changes that, if chronically manifested, could cause cardiac deficits similar to those seen in heart failure and ischemia, such as ventricular dysfunction, cardiac arrhythmias, and cardiac remodeling.
Assuntos
Cálcio/metabolismo , Cocaína/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Entorpecentes/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Relação Dose-Resposta a Droga , Feminino , Glutationa/farmacologia , Insuficiência Cardíaca/genética , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microscopia Confocal , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Coelhos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Ativação Transcricional , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Esquerda/genética , Remodelação Ventricular/genéticaRESUMO
3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug that has gained immense popularity among teenagers and young adults. The cardiovascular toxicological consequences of abusing this compound have not been fully characterized. The present study utilized a transient transfection/dual luciferase genetic reporter assay, fluorescence confocal microscopy, and gene expression macroarray technology to determine nuclear factor-kappaB (NF-kappaB) activity, intracellular calcium balance, mitochondrial depolarization, and gene transcription profiles, respectively, in cultured rat striated cardiac myocytes (H9c2) exposed to MDMA. At concentrations of 1 x 10(-3) M and 1 x 10(-2) M, MDMA significantly enhanced NF-kappaB reporter activity compared with 0 M (medium only) control. This response was mitigated by cotransfection with IkappaB for 1 x 10(-3) M but not 1 x 10(-2) M MDMA. MDMA significantly increased intracellular calcium at concentrations of 1 x 10(-3) M and 1 x 10(-2) M and caused mitochondrial depolarization at 1 x 10(-2) M. MDMA increased the transcription of genes that are considered to be biomarkers in cardiovascular disease and genes that respond to toxic insults. Selected gene activation was verified via temperature-gradient RT-PCR conducted with annealing temperatures ranging from 50 degrees C to 65 degrees C. Collectively, these results suggest that MDMA may be toxic to the heart through its ability to activate the myocardial NF-kappaB response, disrupt cytosolic calcium and mitochondrial homeostasis, and alter gene transcription.
Assuntos
Cálcio/metabolismo , Expressão Gênica/efeitos dos fármacos , Alucinógenos/farmacologia , Miócitos Cardíacos/metabolismo , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , NF-kappa B/biossíntese , Animais , Células Cultivadas , Genes Reporter/efeitos dos fármacos , Genes Reporter/genética , Luciferases/metabolismo , Microscopia Confocal , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Acinetobacter baumannii is an environmentally resilient healthcare-associated opportunistic pathogen responsible for infections at many body sites. In the last 10 years, clinical strains resistant to many or all commonly used antibiotics have emerged globally. With few antimicrobial agents in the pharmaceutical pipeline, new and alternative agents are essential. Platelets secrete a large number of proteins, including proteins with antimicrobial activity. In a previous study, we demonstrated that donor platelet supernatants and plasma significantly inhibited the growth of a reference strain of A. baumannii in broth and on skin. This inhibition appeared to be unrelated to the platelet activation state. In this study, we demonstrate that this growth inhibition extends to clinical multidrug resistant isolates. We also demonstrate that there is no relationship between this activity and selected platelet-derived antimicrobial proteins. Instead, the donor plasma components complement and alpha-2 macroglobulin are implicated.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/metabolismo , Atividade Bactericida do Sangue , Proteínas do Sistema Complemento/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Plasma/química , alfa-Macroglobulinas/metabolismo , Acinetobacter baumannii/fisiologia , HumanosRESUMO
This study was designed to determine levels of NF-kappaB reporter gene activity and free radical generation in cultured striated myocytes (H9C2 cells) exposed to cocaine or morphine in the presence of free radical scavengers. Cells were transiently transfected with a NF-kappaB reporter gene and changes in luciferase activity were detected by bioluminescence. Using confocal microscopy and 2',7'-dichlorofluorescin diacetate, cocaine-induced or morphine-induced free radicals were quantified in H9C2 cells. Cocaine and morphine (0-1 x 10(-2) M) were tested separately. Cocaine but not morphine significantly activated NF-kappaB reporter gene activity in H9C2 cells. Overexpression of IkappaB inhibited NF-kappaB reporter activity at low (1 x 10(-4) M) but not high (1 x 10(-2) M) cocaine concentrations. Free radicals were generated in H9C2 cells stimulated with cocaine but not with morphine. The production of free radicals and NF-kappaB reporter gene activity could be blocked with N-acetylcysteine, glutathione, and, to a lesser extent, lipoic acid. The results suggest that cocaine induces free radical production, which leads to the activation of NF-kappaB signal transduction and possible inflammatory responses.