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1.
Knee Surg Sports Traumatol Arthrosc ; 28(12): 3912-3918, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32034427

RESUMO

PURPOSE: Periprosthetic infection is a common reason for surgical revision. Given the increasing resistance of bacteria to antibiotics (e.g., VRE, 4-MRGN) local antiseptic treatment is gaining in importance. However, no standard guideline-based treatment recommendation is yet available. The aim of this study was to investigate the effectiveness of sodium hypochlorite and chlorhexidine against bacterial biofilms. Furthermore, the toxicity of both antiseptics towards human chondrocytes was examined. METHODS: Human chondrocytes were isolated, cultivated and treated with sodium hypochlorite and chlorhexidine. The viability of cultures was assessed by determination of cell count, XTT and MTT ELISAs, and fluorescent staining with propidium iodide. Bacterial strains of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa were added to liquid media and incubated overnight. After determination of bacterial concentrations polyethylene (PE) devices were inoculated with bacteria for 48 h until biofilms formed. The devices were then washed, treated with antiseptics for 2 and 5 min and subsequently spread on agar plates. RESULTS: Sodium hypochlorite is more effective than chlorhexidine in penetrating biofilms of S. aureus, S. epidermidis and P. aeruginosa. Both antiseptics are chondrotoxic, but sodium hypochlorite damages human chondrocytes less than chlorhexidine in vitro. CONCLUSIONS: The findings confirm the effectiveness of sodium hypochlorite and chlorhexidine against bacterial biofilms. Both antiseptics can be recommended for the treatment of periprosthetic infections. The toxic effects of sodium hypochlorite and chlorhexidine towards chondrocytes may mean there is a risk of damage to cartilage tissue. LEVEL OF EVIDENCE: Controlled experimental study.


Assuntos
Anti-Infecciosos Locais/farmacologia , Biofilmes/efeitos dos fármacos , Clorexidina/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Hipoclorito de Sódio/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/microbiologia , Humanos , Infecções Relacionadas à Prótese/prevenção & controle
2.
Int J Med Microbiol ; 308(6): 640-652, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29526448

RESUMO

Infective endocarditis (IE) is a life-threatening disease, caused by septic vegetations and inflammatory foci on the surface of the endothelium and the valves. Due to its complex and often indecisive presentation the mortality rate is still about 30%. Most frequently bacterial microorganisms entering the bloodstream are the underlying origin of the intracardiac infection. While the disease was primarily restricted to younger patients suffering from rheumatic heart streptococci infections, new at risk categories for Staphylococcus (S.) aureus infections arose over the last years. Rising patient age, increasing drug resistance, intensive treatment conditions such as renal hemodialysis, immunosuppression and long term indwelling central venous catheters but also the application of modern cardiac device implants and valve prosthesis have led to emerging incidences of S. aureus IE in health care settings and community. The aetiologic change has impact on the pathophysiology of IE, the clinical presentation and the overall patient management. Despite intensive research on appropriate in vitro and in vivo models of IE and gained knowledge about the fundamental mechanisms in the formation of bacterial vegetations and extracardiac complications, improved understanding of relevant bacterial virulence factors and triggered host immune responses is required to help developing novel antipathogenic treatment strategies and pathogen specific diagnostic markers. In this review, we summarize and discuss the two main areas affected by the changing patient demographics and provide first, recent knowledge about the pathogenic strategies of S. aureus in the induction of IE, including available experimental models of IE used to study host-pathogen interactions and diagnostic and therapeutic targets. In a second focus we present diagnostic (imaging) regimens for patients with S. aureus IE according to current guidelines as well as treatment strategies and surgical recommendations.


Assuntos
Endocardite Bacteriana/fisiopatologia , Infecções Estafilocócicas/fisiopatologia , Staphylococcus aureus/patogenicidade , Animais , Modelos Animais de Doenças , Endocardite Bacteriana/cirurgia , Endocardite Bacteriana/terapia , Próteses Valvulares Cardíacas/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Incidência , Camundongos , Estudos Retrospectivos , Infecções Estafilocócicas/complicações
3.
Infection ; 46(5): 599-605, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29961209

RESUMO

BACKGROUND: Here, we report the case of an HIV positive patient under a dual antiretroviral drug regimen with tenofovir disoproxil and darunavir/ritonavir with stable clinical, virological, and immunological response over 126 weeks. Dual antiretroviral therapy has the advantage of reduced toxicity and lower health care costs, treatment failure and fostering drug resistance are perceived risks. Optimal drug combinations and indication criteria for dual treatment remain controversial. Nevertheless, first clinical trials indicate non-inferiority for combinations of nucleoside reverse transcriptase inhibitors and protease inhibitors. This case presents the combination of tenofovir disoproxil in combination with a protease inhibitor as a new potential dual treatment regimen. METHOD: We performed a systematic literature search and meta-analysis of trials comparing dual to triple ART. RESULTS: Literature review revealed nine studies in which dual therapy with a protease inhibitor and an NRTI was compared to triple therapy. We performed a meta-analysis of six trials that reported a 48-week follow-up. In treatment-naïve patients as well when ART switch was assessed, there was no difference in the treatment success in patients with dual ART versus triple. CONCLUSION: We conclude that dual therapy with a protease inhibitor and NRTI is safe and effective. The use of tenofovir in dual treatment as described in our case needs to be assessed in future clinical trials.


Assuntos
Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores da Protease de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Darunavir/administração & dosagem , Infecções por HIV/imunologia , Inibidores da Protease de HIV/administração & dosagem , HIV-1/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Transcriptase Reversa/administração & dosagem , Ritonavir/administração & dosagem , Tenofovir/administração & dosagem , Fatores de Tempo , Resultado do Tratamento , Carga Viral
4.
Eur J Clin Microbiol Infect Dis ; 36(6): 1033-1040, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28063000

RESUMO

The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla CTX-M family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.


Assuntos
Bacteriemia/diagnóstico , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Hemocultura , Testes de Sensibilidade Microbiana/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Bactérias/genética , Farmacorresistência Bacteriana , Técnicas de Genotipagem/métodos , Humanos , Sensibilidade e Especificidade , Fatores de Tempo
5.
J Cardiovasc Magn Reson ; 19(1): 39, 2017 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28359292

RESUMO

BACKGROUND: Time resolved 4D phase contrast (PC) cardiovascular magnetic resonance (CMR) in mice is challenging due to long scan times, small animal ECG-gating and the rapid blood flow and cardiac motion of small rodents. To overcome several of these technical challenges we implemented a retrospectively self-gated 4D PC radial ultra-short echo-time (UTE) acquisition scheme and assessed its performance in healthy mice by comparing the results with those obtained with an ECG-triggered 4D PC fast low angle shot (FLASH) sequence. METHODS: Cardiac 4D PC CMR images were acquired at 9.4 T in healthy mice using the proposed self-gated radial center-out UTE acquisition scheme (TE/TR of 0.5 ms/3.1 ms) and a standard Cartesian 4D PC imaging sequence (TE/TR of 2.1 ms/5.0 ms) with a four-point Hadamard flow encoding scheme. To validate the proposed UTE flow imaging technique, experiments on a flow phantom with variable pump rates were performed. RESULTS: The anatomical images and flow velocity maps of the proposed 4D PC UTE technique showed reduced artifacts and an improved SNR (left ventricular cavity (LV): 8.9 ± 2.5, myocardium (MC): 15.7 ± 1.9) compared to those obtained using a typical Cartesian FLASH sequence (LV: 5.6 ± 1.2, MC: 10.1 ± 1.4) that was used as a reference. With both sequences comparable flow velocities were obtained in the flow phantom as well as in the ascending aorta (UTE: 132.8 ± 18.3 cm/s, FLASH: 134.7 ± 13.4 cm/s) and pulmonary artery (UTE: 78.5 ± 15.4 cm/s, FLASH: 86.6 ± 6.2 cm/s) of the animals. Self-gated navigator signals derived from information of the oversampled k-space center were successfully extracted for all animals with a higher gating efficiency of time spent on acquiring gated data versus total measurement time (UTE: 61.8 ± 11.5%, FLASH: 48.5 ± 4.9%). CONCLUSIONS: The proposed self-gated 4D PC UTE sequence enables robust and accurate flow velocity mapping of the mouse heart in vivo at high magnetic fields. At the same time SNR, gating efficiency, flow artifacts and image quality all improved compared to the images obtained using the well-established, ECG-triggered, 4D PC FLASH sequence.


Assuntos
Técnicas de Imagem de Sincronização Cardíaca , Coração/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Imagem Cinética por Ressonância Magnética/métodos , Animais , Artefatos , Velocidade do Fluxo Sanguíneo , Técnicas de Imagem de Sincronização Cardíaca/instrumentação , Circulação Coronária , Eletrocardiografia , Frequência Cardíaca , Imagem Cinética por Ressonância Magnética/instrumentação , Masculino , Camundongos Endogâmicos C57BL , Modelos Animais , Imagens de Fantasmas , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de Tempo
6.
J Antimicrob Chemother ; 71(2): 438-48, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26589581

RESUMO

OBJECTIVES: Staphylococcus aureus osteomyelitis often develops to chronicity despite antimicrobial treatments that have been found to be susceptible in in vitro tests. The complex infection strategies of S. aureus, including host cell invasion and intracellular persistence via the formation of dynamic small colony variant (SCV) phenotypes, could be responsible for therapy-refractory infection courses. METHODS: To analyse the efficacy of antibiotics in the acute and chronic stage of bone infections, we established long-term in vitro and in vivo osteomyelitis models. Antibiotics that were tested include ß-lactams, fluoroquinolones, vancomycin, linezolid, daptomycin, fosfomycin, gentamicin, rifampicin and clindamycin. RESULTS: Cell culture infection experiments revealed that all tested antibiotics reduced bacterial numbers within infected osteoblasts when treatment was started immediately, whereas some antibiotics lost their activity against intracellular persisting bacteria. Only rifampicin almost cleared infected osteoblasts in the acute and chronic stages. Furthermore, we detected that low concentrations of gentamicin, moxifloxacin and clindamycin enhanced the formation of SCVs, and these could promote chronic infections. Next, we treated a murine osteomyelitis model in the acute and chronic stages. Only rifampicin significantly reduced the bacterial load of bones in the acute phase, whereas cefuroxime and gentamicin were less effective and gentamicin strongly induced SCV formation. During chronicity none of the antimicrobial compounds tested showed a beneficial effect on bone deformation or reduced the numbers of persisting bacteria. CONCLUSIONS: In all infection models rifampicin was most effective at reducing bacterial loads. In the chronic stage, particularly in the in vivo model, many tested compounds lost activity against persisting bacteria and some antibiotics even induced SCV formation.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Osteomielite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Feminino , Humanos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Osteoblastos/microbiologia , Staphylococcus aureus/isolamento & purificação
7.
Infection ; 44(4): 499-504, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26797915

RESUMO

PURPOSE: There is increasing clinical evidence from observational studies, that combination therapy of daptomycin with rifampin is a valuable treatment option for biofilm-associated difficult to treat Staphylococcus aureus infections such as osteomyelitis, prosthetic joint infection and endocarditis. However, two studies analyzing a limited number of S. aureus isolates reported an antagonism of those two drugs questioning the benefit of this combination. METHODS: To estimate the frequency of this possible antagonism, we performed in vitro checkerboard assays on 58 consecutive clinical isolates of S. aureus (MSSA n = 9, MRSA n = 49). We determined the fractional inhibitory concentration index (FICI) and the susceptible breakpoint index (SBPI). All isolates were characterized by a microprobe array detecting 336 different genes/alleles to ensure their non-clonal origin. RESULTS: For all isolates, the FICI was between 1.00 and 1.25 indicating additive effects for the daptomycin/rifampin combination. Neither antagonism nor synergism as defined by the FICI was found for any of the isolates. CONCLUSION: Based on these data, there is no evidence to advise against the daptomycin/rifampin combination therapy.


Assuntos
Antibacterianos/farmacologia , Daptomicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Rifampina/farmacologia , Infecções Estafilocócicas/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Pré-Escolar , Antagonismo de Drogas , Feminino , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Staphylococcus aureus/efeitos dos fármacos , Adulto Jovem
8.
Hautarzt ; 65(1): 15-25, 2014 Jan.
Artigo em Alemão | MEDLINE | ID: mdl-24445941

RESUMO

Staphylococcus aureus is one of the major pathogens causing chronic skin and soft tissue infections. Particularly isolates producing Panton-Valentine leukocidin (PVL) comprising methicillin-susceptible and community-associated methicillin-resistant S. aureus (CA-MRSA) have been associated with more aggressive and persistent or relapsing courses. Beyond classical resistance mechanisms, functional resistance as shown by the small colony-variant (SCV) phenotype could be also responsible for treatment failures, despite the administration of antibiotics tested in vitro as susceptible. Also this phenotype has been associated with chronic courses of infections often with multiple exacerbations. Due to their ability to persist intracellularly, SCVs are protected from host defense and antibiotic treatment if only extracellularly active agents are administered. Reduced growth, abnormal colony morphology and changes in the metabolism of the SCVs aggravate drastically their identification, differentiation and susceptibility testing. The diagnostic and therapeutic challenges of PVL-positive and SCV isolates necessitate close collaboration with microbiological and infectious disease specialists.


Assuntos
Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Leucocidinas/metabolismo , Infecções dos Tecidos Moles/diagnóstico , Infecções dos Tecidos Moles/microbiologia , Infecções Cutâneas Estafilocócicas/diagnóstico , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Antibacterianos/uso terapêutico , Doença Crônica , Humanos , Recidiva , Infecções dos Tecidos Moles/tratamento farmacológico , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/classificação
9.
J Hosp Infect ; 100(3): 309-315, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29253623

RESUMO

BACKGROUND: The prevalence of nasopharyngeal colonization with Staphylococcus aureus can reach 20-30% among the population, which can lead to invasive infection. AIM: To investigate the prevalence of colonization among different age groups, and analyse S. aureus strain-specific virulence patterns. METHOD: For analysis of the prevalence of colonization, groups consisting of newborns, healthy volunteers aged 5-60 years, and nursing home residents aged >80 years were examined with nasopharyngeal swabs. After S. aureus was cultured, genetic analysis and phenotypic virulence testing were performed by cell-based assays. FINDINGS: Among 924 volunteers, the overall colonization rate was approximately 30%, with a peak in subjects aged 5-10 years (49%). Neonates and subjects aged >80 years showed different distributions of clonal clusters. Overall, the strains of all age groups exhibited virulence characteristics that can contribute to the development of infection. In particular, the neonatal strains exhibited a high incidence of toxin genes that resulted in increased cytotoxic effects compared with the other strains tested. CONCLUSIONS: Colonizing strains showed a virulence profile in all age groups, which may lead to the establishment of invasive infection. Consequently, decolonization measures could be considered for selected patients depending on the risk of infection.


Assuntos
Portador Sadio/epidemiologia , Nasofaringe/microbiologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/isolamento & purificação , Fatores de Virulência/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Portador Sadio/microbiologia , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Fenótipo , Prevalência , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Virulência , Fatores de Virulência/genética , Adulto Jovem
10.
BMJ Open ; 7(4): e013976, 2017 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-28391236

RESUMO

INTRODUCTION: Staphylococcus aureus bacteraemia (SAB) is a frequent infection with high mortality rates. It requires specific diagnostic and therapeutic management such as prolonged intravenous administration of antibiotics and aggressive search for and control of infectious sources. Underestimation of disease severity frequently results in delayed or inappropriate management of patients with SAB leading to increased mortality rates. According to observational studies, patient counselling by infectious disease consultants (IDC) improves survival and reduces the length of hospital stay as well as complication rates. In many countries, IDC are available only in some tertiary hospitals. In this trial, we aim to demonstrate that the outcome of patients with SAB in small and medium size hospitals that do not employ IDC can be improved by unsolicited ID phone counselling. The SUPPORT trial will be the first cluster-randomised controlled multicentre trial addressing this question. METHODS AND ANALYSIS: SUPPORT is a single-blinded, multicentre interventional, cluster-randomised, controlled crossover trial with a minimum of 15 centres that will include 250 patients with SAB who will receive unsolicited IDC counselling and 250 who will receive standard of care. Reporting of SAB will be conducted by an electronic real-time blood culture registry established for the German Federal state of Thuringia (ALERTSNet) or directly by participating centres in order to minimise time delay before counselling. Mortality, disease course and complications will be monitored for 90 days with 30-day all-cause mortality rates as the primary outcome. Generalised linear mixed modelling will be used to detect the difference between the intervention sequences. We expect improved outcome of patients with SAB after IDC. ETHICS AND DISSEMINATION: We obtained ethics approval from the Ethics committee of the Jena University Hospital and from the Ethics committee of the State Chamber of Physicians of Thuringia. Results will be published in a peer-reviewed journal and additionally disseminated through public media. TRIAL REGISTRATION NUMBER: DRKS00010135.


Assuntos
Aconselhamento , Infecções Estafilocócicas , Staphylococcus aureus/efeitos dos fármacos , Administração Intravenosa , Antibacterianos/uso terapêutico , Protocolos Clínicos , Análise por Conglomerados , Aconselhamento/métodos , Estudos Cross-Over , Alemanha/epidemiologia , Humanos , Educação de Pacientes como Assunto , Avaliação de Programas e Projetos de Saúde , Garantia da Qualidade dos Cuidados de Saúde , Encaminhamento e Consulta , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/prevenção & controle , Telefone
11.
Clin Microbiol Infect ; 22(9): 799-809, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27393124

RESUMO

Host cell invasion is a major feature of Staphylococcus aureus and contributes to infection development. The intracellular metabolically active bacteria can induce host cell activation and death but they can also persist for long time periods. In this study a comparative analysis was performed of different well-characterized S. aureus strains in their interaction with a variety of host cell types. Staphylococcus aureus (strains 6850, USA300, LS1, SH1000, Cowan1) invasion was compared in different human cell types (epithelial and endothelial cells, keratinocytes, fibroblasts, osteoblasts). The number of intracellular bacteria was determined, cell inflammation was investigated, as well as cell death and phagosomal escape of bacteria. To explain strain-dependent differences in the secretome, a proteomic approach was used. Barrier cells took up high amounts of bacteria and were killed by aggressive strains. These strains expressed high levels of toxins, and possessed the ability to escape from phagolysosomes. Osteoblasts and keratinocytes ingested less bacteria, and were not killed, even though the primary osteoblasts were strongly activated by S. aureus. In all cell types S. aureus was able to persist. Strong differences in uptake, cytotoxicity, and inflammatory response were observed between primary cells and their corresponding cell lines, demonstrating that cell lines reflect only partially the functions and physiology of primary cells. This study provides a contribution for a better understanding of the pathomechanisms of S. aureus infections. The proteomic data provide important basic knowledge on strains commonly used in the analysis of S. aureus-host cell interaction.


Assuntos
Staphylococcus aureus/fisiologia , Morte Celular , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Progressão da Doença , Interações Hospedeiro-Patógeno , Humanos , Lisossomos/metabolismo , Especificidade de Órgãos , Fagossomos/metabolismo , Proteômica/métodos , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia
12.
Biochim Biophys Acta ; 1003(3): 225-31, 1989 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-2742858

RESUMO

A basically new approach is presented for purifying lysosomal phospholipase A1 (EC 3.1.1.32) from rat liver. This procedure not only simplifies and speeds up the purification process, but also improves the yield in comparison to the most efficient methods reported so far. A high recovery of about 88% was achieved by (1) homogenisation of whole rat liver in a hypotonic medium, (2) acid precipitation, (3) combined dye binding chromatography on triacinyl dyes (Yellow H-A and Red HE-3B) immobilised to agaroses, and (4) combined concanavalin A-Sepharose and phenyl-Sepharose chromatography. Ethylene glycol was required for enzyme stabilisation as well as for enzyme elution in dye-binding and hydrophobic chromatography. In SDS-polyacrylamide slab gel electrophoresis, the purified material showed two major protein bands of 56 and 33 kDa, which amounted to about 85 and 12%, respectively, of the total protein visualised. Under reductive conditions, the 56 kDa protein decomposed completely into three subunits of 30, 21 and 20 kDa. The 33 kDa protein in the non-reduced material seems to be identical with the 30 kDa protein in the reduced material. Native polyacrylamide gel electrophoresis provided strong evidence that the 56 kDa protein is the active form of PLA1. The purified material displayed a specific activity of approximately 7.7 mumol fatty acid released per min per mg of protein using 200 microM phosphatidylethanolamine as a substrate.


Assuntos
Fígado/enzimologia , Fosfolipases A/isolamento & purificação , Fosfolipases/isolamento & purificação , Animais , Cromatografia de Afinidade , Concanavalina A , Eletroforese em Gel de Poliacrilamida , Lisossomos/enzimologia , Peso Molecular , Fosfolipases A1 , Ratos , Solubilidade
13.
Biochim Biophys Acta ; 835(3): 448-55, 1985 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-4016141

RESUMO

Activities of membrane-associated phospholipases A1 and A2, and membrane-associated as well as soluble lysophospholipases were measured in different subcellular fractions of rat liver, using suspensions of stereospecifically labelled radioactive phospholipids as substrates. Plasma membranes and endoplasmic reticulum were shown to contain phospholipase A1 and lysophospholipase activities, both of which could be stimulated by Ca2+, mitochondria Ca2+-dependent phospholipase A2 and cytosol Ca2+-independent lysophospholipase activities. Each of these lipolytic enzymes could be inhibited by antimalarial drugs (chloroquine, mepacrine, primaquine) at concentrations above 1 x 10(-4) M. Inhibition of the alkaline cytosolic lysophospholipase by these drugs was noncompetitive with respect to the substrate, and the inhibitory potency increased, when the pH was raised.


Assuntos
Antimaláricos/farmacologia , Fígado/ultraestrutura , Lisofosfolipase/antagonistas & inibidores , Fosfolipases A/antagonistas & inibidores , Fosfolipases/antagonistas & inibidores , Animais , Membrana Celular/enzimologia , Cloroquina/farmacologia , Citosol/enzimologia , Retículo Endoplasmático/enzimologia , Masculino , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Fosfolipases A1 , Fosfolipases A2 , Primaquina/farmacologia , Quinacrina/farmacologia , Ratos , Ratos Endogâmicos
14.
Circulation ; 99(20): 2665-8, 1999 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-10338460

RESUMO

BACKGROUND: Exaggerated pulmonary hypertension is thought to play an important part in the pathogenesis of high-altitude pulmonary edema (HAPE). Endothelin-1 is a potent pulmonary vasoconstrictor peptide that also augments microvascular permeability. METHODS AND RESULTS: We measured endothelin-1 plasma levels and pulmonary artery pressure in 16 mountaineers prone to HAPE and in 16 mountaineers resistant to this condition at low (580 m) and high (4559 m) altitudes. At high altitude, in mountaineers prone to HAPE, mean (+/-SE) endothelin-1 plasma levels were approximately 33% higher than in HAPE-resistant mountaineers (22.2+/-1.1 versus 16.8+/-1.1 pg/mL, P<0.01). There was a direct relationship between the changes from low to high altitude in endothelin-1 plasma levels and systolic pulmonary artery pressure (r=0.82, P<0.01) and between endothelin-1 plasma levels and pulmonary artery pressure measured at high altitude (r=0.35, P=0.05). CONCLUSIONS: These findings suggest that in HAPE-susceptible mountaineers, an augmented release of the potent pulmonary vasoconstrictor peptide endothelin-1 and/or its reduced pulmonary clearance could represent one of the mechanisms contributing to exaggerated pulmonary hypertension at high altitude.


Assuntos
Altitude , Endotelina-1/metabolismo , Edema Pulmonar/etiologia , Edema Pulmonar/metabolismo , Adulto , Pressão Sanguínea/fisiologia , Suscetibilidade a Doenças , Endotelina-1/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Montanhismo/fisiologia , Artéria Pulmonar/fisiopatologia , Edema Pulmonar/fisiopatologia , Sístole
15.
Circulation ; 99(14): 1802-9, 1999 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-10199875

RESUMO

BACKGROUND: An activated endothelin (ET) system may be of pathophysiological relevance in human heart failure. We characterized the functional effects of ET-1, ET receptors, and ET-1 peptide concentration in left ventricular myocardium from 10 nonfailing hearts (NF) and 27 hearts in end-stage failure due to idiopathic dilative cardiomyopathy (DCM). METHODS AND RESULTS: Inotropic effects were characterized in isolated muscle strips (1 Hz; 37 degrees C). ET-1 0.0001 to 0.3 micromol/L significantly (P<0.05) increased twitch force by maximally 59+/-10% in NF and by 36+/-11% in DCM (P<0.05 versus NF). Preincubation with propranolol 1 micromol/L and prazosin 0.1 micromol/L did not affect the response to ET-1, but the mixed ET receptor antagonist bosentan and the ETA receptor antagonist BQ-123 shifted the concentration-response curves for ET-1 rightward. The ETB receptor agonist sarafotoxin S6c 0.001 to 0.3 micromol/L had no functional effects. The inotropic response to ET-1 was not associated with increased intracellular Ca2+ transients, as assessed in aequorin-loaded muscle strips. ET receptor density (Bmax; radioligand binding) was 62.5+/-12.5 fmol/mg protein in NF and 122. 4+/-24.3 fmol/mg protein in DCM (P<0.05 versus NF). The increase in Bmax in DCM resulted from an increase in ETA receptors without change in ETB receptors. ET-1 peptide concentration (radioimmunoassay) was higher in DCM than in NF (14 447+/-2232 versus 4541+/-1340 pg/mg protein, P<0.05). CONCLUSIONS: ET-1 exerts inotropic effects in human myocardium through ETA receptor-mediated increases in myofibrillar Ca2+ responsiveness. In DCM, functional effects of ET-1 are attenuated, but ETA receptor density and ET-1 peptide concentration are increased, indicating an activated local cardiac ET system and possibly a reduced postreceptor signaling efficiency.


Assuntos
Baixo Débito Cardíaco/metabolismo , Endotelina-1/farmacologia , Miocárdio/metabolismo , Receptores de Endotelina/metabolismo , Equorina/farmacologia , Cálcio/metabolismo , Cálcio/fisiologia , Baixo Débito Cardíaco/etiologia , Baixo Débito Cardíaco/fisiopatologia , Cardiomiopatia Dilatada/complicações , Antagonistas dos Receptores de Endotelina , Endotelina-1/metabolismo , Humanos , Técnicas In Vitro , Medições Luminescentes , Contração Miocárdica/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Valores de Referência
16.
Cardiovasc Res ; 31(2): 287-95, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8730406

RESUMO

OBJECTIVES: Recent studies have shown that beside elevated arterial blood pressure, humoral factors such as angiotensin II, aldosterone, endothelin or bradykinin might play a role in the cardiac hypertrophy and fibrosis secondary to hypertension. In addition, it seems that perivascular fibrosis and interstitial fibrosis are controlled by independent mechanisms. Therefore, the goal of our study was to evaluate the respective role of the increased arterial pressure and of humoral factors on cardiac remodeling in an experimental hypertension model. METHODS: Uninephrectomized rats received DOCA, a high salt diet, and when hypertension was installed, they were treated for 6 weeks with either a long-acting calcium antagonist, mibefradil (30 mg/kg day-1), an ACE inhibitor, enalapril (3 mg/kg day-1), or a mixed ETA-ETB endothelin receptor antagonist, bosentan (100 mg/kg day-1). A group of hypertensive rats was left untreated and a sham-operated group of normotensive rats was used for control. At the end of treatment, maximal coronary blood flow was measured in isolated perfused hearts. Cardiac hypertrophy and interstitial as well as perivascular fibrosis were evaluated by quantitative morphometry. RESULTS: DOCA-salt hypertensive rats exhibited a marked cardiac hypertrophy associated with a decrease of maximal coronary blood flow and interstitial and perivascular fibrosis. The calcium antagonist nearly normalized arterial pressure and suppressed all these changes. Enalapril had no effect on arterial pressure and perivascular fibrosis but decreased subendocardial fibrosis. Bosentan had a very small effect on arterial pressure but decreased cardiac hypertrophy and both perivascular and subendocardial fibrosis. CONCLUSIONS: We conclude that in DOCA salt hypertension, humoral factors such as endothelin may play a role beside high blood pressure in cardiac remodeling. In addition, the different components of this remodeling (decrease of vascular reserve, cardiac hypertrophy and cardiac fibrosis) are controlled independently.


Assuntos
Cardiomegalia/etiologia , Hipertensão/complicações , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Benzimidazóis/farmacologia , Bosentana , Bloqueadores dos Canais de Cálcio/farmacologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Colágeno/análise , Circulação Coronária/efeitos dos fármacos , Desoxicorticosterona , Enalapril/farmacologia , Antagonistas dos Receptores de Endotelina , Fibrose , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Mibefradil , Miocárdio/química , Miocárdio/patologia , Perfusão , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Tetra-Hidronaftalenos/farmacologia
17.
Hypertension ; 30(5): 1204-9, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9369277

RESUMO

Endothelin-1 (0.25 nmol/kg, injected into the left cardiac ventricle) induces a protracted increase of mean arterial pressure that is significantly reduced by the selective ET(A) receptor antagonist BQ-123 (1 and 10 mg/kg) in the anesthetized rabbit. The sole administration of the selective ET(B) antagonist BQ-788 (0.25 mg/kg) induces a pressor response abolished by BQ-123 (1 mg/kg). Concomitant to the increase in mean arterial pressure, BQ-788 induces a significant increase in plasma levels of endothelin-1 and its precursor big endothelin-1. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) also increases arterial blood pressure, and the response is reduced dose-dependently by BQ-123 (1 and 10 mg/kg). In addition, the administration of BQ-788 in the presence of L-NAME induced a further increase in arterial blood pressure. The duration of the pressor response to L-NAME is also significantly reduced by an endothelin-converting enzyme inhibitor, phosphoramidon (10 mg/kg). Finally, L-NAME induces an increase in plasma levels of big endothelin-1 but not endothelin-1. Our results illustrate that blockade of either nitric oxide synthase or ET(B) receptors triggers a raise in plasma levels of endothelin-1 or its precursor. These later moieties are suggested to be significantly involved, through the activation of ET(A) receptors, in the pressor effects of L-NAME and BQ-788 in the anesthetized rabbit.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Óxido Nítrico Sintase/fisiologia , Peptídeos Cíclicos/farmacologia , Receptores de Endotelina/fisiologia , Animais , Antagonistas dos Receptores de Endotelina , Endotelina-1 , Endotelinas/sangue , Inibidores Enzimáticos/farmacologia , Feminino , Hemodinâmica/efeitos dos fármacos , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Oligopeptídeos/farmacologia , Piperidinas/farmacologia , Precursores de Proteínas/sangue , Coelhos , Receptor de Endotelina B
18.
Hypertension ; 28(3): 379-85, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8794820

RESUMO

High blood pressure results in cardiac hypertrophy and fibrosis, increased thickness and stiffness of large artery walls, and decreased renal function. The objective of our study was to assess the role of endothelin, angiotensin II, and high blood pressure in the end-organ damage observed in spontaneously hypertensive rats (SHR). For this purpose, SHR were treated for 10 weeks with either a mixed endothelin-A and endothelin-B receptor antagonist, bosentan (100 mg/kg per day), an angiotensin-converting enzyme inhibitor, enalapril (10 mg/kg per day), or a long-acting calcium antagonist, mibefradil (20 mg/kg per day). A group of SHR was left untreated, and a group of normotensive Wistar rats was used as control. At the end of treatment, maximal coronary blood flow was measured in isolated perfused hearts. Cardiac hypertrophy and fibrosis, aortic medial thickness, and extracellular matrix content were evaluated by quantitative morphometry. Proteinuria and urea and creatinine clearances were measured, and renal histopathology was assessed. SHR exhibited cardiac hypertrophy, perivascular fibrosis, and decreased maximal coronary blood flow. Aortic medial thickness was increased, whereas elastin density was decreased. Finally, SHR showed decreased urinary excretion and decreased urea and creatinine clearances. No renal histological lesions were observed. Although bosentan did not affect blood pressure, it normalized renal function and slightly decreased left ventricular hypertrophy and fibrosis. Enalapril and mibefradil were both effective in significantly decreasing blood pressure, left ventricular hypertrophy, and aortic medial thickness and improving coronary blood flow, but in contrast to bosentan, they did not improve creatinine clearance. We conclude that in SHR, high blood pressure plays a major role in end-organ damage and that endothelin may partly mediate renal dysfunction and cardiac remodeling independently of a direct hemodynamic effect.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Endotelinas/antagonistas & inibidores , Hipertensão/patologia , Sulfonamidas/farmacologia , Animais , Aorta/metabolismo , Aorta/patologia , Benzimidazóis/farmacologia , Peso Corporal/efeitos dos fármacos , Bosentana , Cardiomegalia/patologia , Circulação Coronária/efeitos dos fármacos , Enalapril/farmacologia , Fibrose , Ventrículos do Coração , Hemodinâmica/efeitos dos fármacos , Hormônios/sangue , Hipertensão/sangue , Hipertensão/fisiopatologia , Rim/patologia , Rim/fisiopatologia , Mibefradil , Miocárdio/patologia , Ratos , Ratos Endogâmicos SHR , Tetra-Hidronaftalenos/farmacologia
19.
Hypertension ; 36(2): 282-90, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10948091

RESUMO

Reports on the effectiveness of endothelin receptor blockers in angiotensin (Ang) II-induced end-organ damage are conflicting, and the mechanisms involved are uncertain. We tested the hypothesis that endothelin (ET)(A/B) receptor blockade with bosentan (100 mg/kg by gavage after age 4 weeks) ameliorates cardiac and renal damage by decreasing inflammation in rats harboring both human renin and angiotensinogen genes (dTGR). Furthermore, we elucidated the effect of bosentan on tissue factor (TF), which is a key regulator of the extrinsic coagulation cascade. We compared bosentan with hydralazine (80 mg/L in the drinking water for 3 weeks) as a blood pressure control. Untreated dTGR featured hypertension, focal necrosis in heart and kidney, and a 45% mortality rate (9 of 20) at age 7 weeks. Compared with Sprague-Dawley controls, both systolic blood pressure and 24-hour albuminuria were increased in untreated dTGR (203+/-8 versus 111+/-2 mm Hg and 67.1+/-8.6 versus 0.3+/-0.06 mg/d at week 7, respectively). Bosentan and hydralazine both reduced blood pressure and cardiac hypertrophy. Mortality rate was markedly reduced by bosentan (1/15) and partially by hydralazine (4/15). However, only bosentan decreased albuminuria and renal injury. Untreated and hydralazine-treated dTGR showed increased nuclear factor (NF)-kappaB and AP-1 expression in the kidney and heart; the p65 NF-kappaB subunit was increased in the endothelium, vascular smooth muscles cells, infiltrating cells, glomeruli, and tubules. In the heart and kidney, ET(A/B) receptor blockade inhibited NF-kappaB and AP-1 activation compared with hydralazine treatment. Macrophage infiltration, ICAM-1 expression, and the integrin expression on infiltrating cells were markedly reduced. Renal vasculopathy was accompanied by increased tissue factor expression on macrophages and vessels of untreated and hydralazine-treated dTGR, which was markedly reduced by bosentan. Thus, ET(A/B) receptor blockade inhibits NF-kappaB and AP-1 activation and the NF-kappaB- and/or AP-1-regulated genes ICAM-1, VCAM-1, and TF, independent of blood pressure-related effects. We conclude that Ang II-induced NF-kappaB and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET(A/B) receptors.


Assuntos
Coração/efeitos dos fármacos , Inflamação/prevenção & controle , Rim/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Sulfonamidas/farmacologia , Tromboplastina/efeitos dos fármacos , Albuminúria/prevenção & controle , Angiotensina II/efeitos adversos , Angiotensinogênio/genética , Animais , Animais Geneticamente Modificados , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Bosentana , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , Cardiomegalia/prevenção & controle , Fibronectinas/análise , Coração/fisiopatologia , Humanos , Hidralazina/farmacologia , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/análise , Rim/metabolismo , Rim/patologia , Macrófagos/patologia , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Renina/genética , Tromboplastina/metabolismo , Fator de Transcrição AP-1/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Molécula 1 de Adesão de Célula Vascular/análise
20.
FEBS Lett ; 216(1): 51-6, 1987 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-3582666

RESUMO

Human liver was homogenised and fractionated by differential centrifugation, and the subcellular fractions were characterised biochemically. Absolute values and distribution patterns of protein and marker enzyme activities obtained from human liver have also been compared with those from rat liver. In addition, acid phospholipase activities have been studied in human liver. On the basis of product formation from stereo-specifically radiolabeled phosphatidylethanolamine substrates, lysosomal phospholipases A1 and A2 with optimal activities at pH 4.7 have been identified in human liver. Acid phospholipase C and lysophospholipase activities, however, were not found in human liver. Cationic amphiphilic drugs inhibited the activities of the acid phospholipases A in human and rat liver lysosomes to about the same extent.


Assuntos
Fígado/enzimologia , Lisossomos/enzimologia , Fosfolipases/isolamento & purificação , Animais , Humanos , Fosfatidiletanolaminas/metabolismo , Fosfolipases/metabolismo , Ratos , Especificidade da Espécie , Frações Subcelulares/enzimologia
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