RESUMO
AIMS: This study evaluated the combined effects of ozone and heat treatment to inactivate Escherichia coli O157:H7 and Salmonella Typhimurium in three types of apple juice of different soluble solids content. METHODS AND RESULTS: Three types of apple juice (18, 36, 72 °Brix) inoculated with pathogens were subjected to ozone (3·0 l min(-1) flow rate and 2·0-3·0 g m(-3) concentration) and heat treatment (25, 45, 50 and 55°C) simultaneously for 20, 40 and 60 s. Initial populations of pathogens in inoculated apple juice were approximately 10(5)-10(6) CFU ml(-1). Heat treatment alone (25, 45, 50 and 55°C) for 1 min reduced populations of E. coli O157:H7 by 0 to 4·75 log CFU ml(-1) in three types of apple juice. The combination of ozone and heat treatment for 1 min at 25 and 45°C reduced E. coli O157:H7 by 0·93-3·87 log CFU ml(-1) and below the detection limit (>1 log CFU ml(-1)) at 50 and 55°C. A similar tendency was observed for S. Typhimurium. In several instances, results showed a synergistic effect of ozone and heat treatment. Colour values were not changed during ozone and heat treatment. CONCLUSION: These results show that the combination of ozone and heat treatment can be used as a potential inactivation intervention for E. coli O157:H7 and S. Typhimurium in apple juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of ozone treatment and mild heat can be used as an alternative intervention for pasteurization of varying soluble solids content apple juice in food industries.
Assuntos
Antibacterianos/farmacologia , Bebidas/microbiologia , Escherichia coli O157 , Temperatura Alta , Malus , Ozônio/farmacologia , Salmonella typhimurium , Escherichia coli O157/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacosRESUMO
Solution-processed metal grid transparent conductors with low sheet resistance, high optical transmittance and good mechanical flexibility have great potential for use in flexible optoelectronic devices. However, there are still remaining challenges to improve optoelectrical properties and electromechanical stability of the metallic structures due to random loose packings of nanoparticles and the existence of many pores. Here we introduce a selective multi-nanosoldering method to generate robust metallic layers on the thin metal grid structures (< a thickness of 200 nm), which are generated via self-pining assisted direct inking of silver ions. The selective multi-nanosoldering leads to lowering the sheet resistance of the metal grid transparent conductors, while keeping the optical transmittance constant. Also, it reinforces the electromechanical stability of flexible metal grid transparent conductors against a small bending radius or a repeated loading. Finally, organic light-emitting diodes based on the flexible metal grid transparent conductors are demonstrated. Our approach can open a new route to enhance the functionality of metallic structures fabricated using a variety of solution-processed metal patterning methods for next-generation optoelectronic and micro/nanoelectronic applications.
RESUMO
Synthetic biomaterials can be used as instructive biological milieus to guide cellular behaviour and function. To further realize this application, we synthesized a series of structurally similar hydrogels and tested their ability to modulate angiogenesis. Hydrogels were synthesized from poly(DTE-co-x% DT carbonate) crosslinked by y% poly(ethylene glycol) (PEG). Hydrogel desaminotyrosyl tyrosine (DT) contents (x%) ranged from 10-100%, and crosslink densities (y% PEG-crosslinker) ranged from 5-80%. The hydrogels were fashioned into porous scaffolds with highly interconnected macro- and micro-pore (>100 and 10 mm in diameter, respectively) architecture using poly(DTE-co-10%DT carbonate% crosslinked with 8% PEG. Under physiological conditions (in vitro), the hydrogels degraded into three major products: desaminotyrosyl-tyrosine ethyl ester (DTE), desaminotyrosyl tyrosine (DT), and poly(ethylene glycol)-di-DT-hydrazide (PEG-di-DT hydrazide). Increasing either DT content or crosslink density brought quickened degradation. Because DT and DTE, two of the three major degradation products, have not demonstrated any noticeable cytotoxicity or angiogenic effect in previous studies, we measured the cytotoxicity of PEG-di-DT hydrazide, the third major degradation product. We found that PEG-di-DT hydrazide only displayed significant cytotoxicity at the high concentration of 100 mg/mL. Interestingly, PEG-di-DT hydrazide and its further degradation product PEG-dihydrazide stimulated in vitro endothelial cell migration and tubulogenesis, which is comparable to results found with FGF-beta treatment. Subcutaneous implantation of the PEG-crosslinked poly(DTE-co-10%DT carbonate) scaffolds into the backs of rats elicited greater tissue growth over time and superior vascularization than poly(DTE carbonate) implantation. These results show that this new class of biomaterials has a strong potential to modulate angiogenesis.
Assuntos
Moduladores da Angiogênese/farmacologia , Hidrazinas/farmacologia , Hidrogéis/síntese química , Neovascularização Fisiológica/fisiologia , Polietilenoglicóis/síntese química , Polietilenoglicóis/farmacologia , Engenharia Tecidual/métodos , Tirosina/análogos & derivados , Moduladores da Angiogênese/síntese química , Animais , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Hidrazinas/síntese química , Hidrogéis/metabolismo , Microscopia Eletrônica de Varredura , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Tirosina/síntese química , Tirosina/farmacologiaRESUMO
The blood-brain barrier (BBB) plays a critical role in brain homeostasis at the cellular and global level. Mimicking the selective permeability and transport properties of the BBB to specific molecules and cells remains a significant challenge towards the development of a physiologically relevant in vitro BBB model. In this study, we developed electrospun poly (ε-caprolactone) (PCL) and polyethylene glycol (PEG) copolymer membranes that supported different cellular components of the neurovascular unit including human-derived endothelial cells, pericytes and astrocytes. Comparative analyses of thickness, morphology, biocompatibility and permeability of membranes were also conducted. We found that collagen coated 4%PEG-96%PCL membranes supported the growth of a confluent and tight endothelium confirmed by transendothelial electrical resistance measurements (TEER). Based on fabrication process and reported results, we finally discuss the adoption of these electrospun fiber membranes for in vitro and on-a-chip human BBB models.
Assuntos
Barreira Hematoencefálica , Modelos Biológicos , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/fisiologia , Permeabilidade da Membrana Celular , Células Cultivadas , Técnicas de Cocultura , Colágeno , Humanos , Poliésteres , PolietilenoglicóisRESUMO
Tanshinone II-A is a derivative of phenanthrene-quinone isolated from Salvia miltiorrhiza BUNGE, a traditional herbal medicine that is known to induce antiinflammatory, anti-oxidative and cytotoxic activity. We have examined cellular effects of Tanshione II-A on HL60 human promyelocytic leukemic cells and K562 human erythroleukemic cells. Tanshione II-A induced a dose- and time-dependent DNA fragmentation into the multiples of 180 bp and specific proteolytic cleavage of poly(ADP-ribose) polymerase in both cell lines. PI-staining and flow cytometry analysis of K562 cells following Tanshione II-A treatment showed an increase of the cells possessing hypodiploid DNA indicative of apoptotic state of cells. Caspase-3 activity was significantly increased during Tanshinone II-A treatment of both HL60 and K562 cells, whereas caspase-1 activity was not changed. These results suggest that Tanshione II-A induced HL60 and K562 cellular apoptosis that may be associated with the selective members of caspase family.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Caspases/efeitos dos fármacos , Caspases/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Leucemia/patologia , Fenantrenos/farmacologia , Abietanos , Antineoplásicos Fitogênicos/química , Caspase 3 , Ciclo Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Ativação Enzimática/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Células HL-60/patologia , Humanos , Lamiaceae/química , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Leucemia Eritroblástica Aguda/tratamento farmacológico , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Fenantrenos/química , Células Tumorais CultivadasRESUMO
The activation of Jun/Fos is a crucial factor in transmitting the tumor promoting signal from the extracellular environment to nuclear transcription machinery. One of the final steps in signal transduction is the binding of Jun/Fos to the AP-1 site in order to express gene transcription. Utilizing this concept, we screened about 100 extracts of natural plants to search for a Jun-Fos function inhibitor. The methanol extract of Ampelopsis radix reduced Jun/Foc retardation remarkably. The active principles of the extract were isolated and purified by repeated column chromatography and their structures were identified as oleanolic acid glycosides known as momordin I, Id, and Ie. These compounds reduced the Jun/Fos-DNA interaction and their activities were quantitated with liquid scintillation counting of corresponding bands. Among them, momordin I had the strongest inhibitory activity, with an IC50 value of 22.8 micrograms/ml. The methanol extract and momordin I, Id and Ie also showed cell cytotoxicity against human cancer cell lines. As expected from a gel shift assay, momordin I showed the strongest cytotoxicity and its IC50 value was from 7.280 micrograms/ml to 16.05 micrograms/ml depending on the cell line. With these data, it may be concluded that the mechanism of anticancer activity of momordin I comes from its inhibitory effect on the protein-DNA interaction. The in vivo test was done only with the methanol extract. The extract showed measurable anticancer activity against murine colon cancer. The wet tumor weight reduction rate was 17.73% at 90 mg/kg dose. We suggest that the Jun/Fos-DNA interaction results in cell cytotoxicity.
Assuntos
Inibidores do Crescimento/farmacologia , Ácido Oleanólico/farmacologia , Extratos Vegetais/farmacologia , Fator de Transcrição AP-1/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Humanos , Ressonância Magnética Nuclear Biomolecular , Ácido Oleanólico/análogos & derivados , Relação Estrutura-Atividade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Some, but not all, organophosphorus pesticides are more acutely toxic to the young as compared to adults. We have developed an in vitro assay that measures the detoxification potential (via carboxylesterase and A-esterases) of tissues. Previous results using this in vitro screen correlated with the marked in vivo sensitivity of the young to chlorpyrifos and also correlated with the equal sensitivity of the young and adult to methamidophos (Padilla et al., 2000). We have now extended these observations to two other pesticides that have already been shown in the literature to be more toxic to the young: parathion (paraoxon) and malathion (malaoxon). In our in vitro assay, liver or plasma from 7-d-old rats were much less efficacious than adult tissues at detoxification of the active metabolites of these two pesticides. Using our in vitro assay we also tested the active metabolite of diazinon, diazoxon, and again found that young liver or plasma possessed much less detoxification capability than adult tissues. From these results, we predicted that young animals would be more sensitive to diazinon, which, in fact, was the case: When postnatal day (PND) 17 or adult rats were given a dosage of 75 mg/kg diazinon, adult brain cholinesterase (ChE) was only inhibited 38%, while the brain ChE in the PND 17 animals showed much more inhibition (75%). We conclude that our in vitro screen may prove to be a useful, quick, convenient test for identifying which organophosphorus pesticides may be more acutely toxic to the young as compared to adults.
Assuntos
Bioensaio/métodos , Inativação Metabólica , Fígado , Malation/análogos & derivados , Programas de Rastreamento/métodos , Modelos Animais , Plasma , Testes de Toxicidade/métodos , Fatores Etários , Animais , Animais Recém-Nascidos , Bioensaio/normas , Feminino , Técnicas In Vitro , Inseticidas/metabolismo , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Malation/toxicidade , Masculino , Programas de Rastreamento/normas , Compostos Organofosforados/toxicidade , Paraoxon/toxicidade , Plasma/efeitos dos fármacos , Plasma/metabolismo , Gravidez , Radiometria/métodos , Radiometria/normas , Ratos , Ratos Long-Evans , Espectrofotometria/métodos , Espectrofotometria/normas , Testes de Toxicidade/normasRESUMO
Apoptosis is a new therapeutic target of cancer research. Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE, a traditional oriental medical herb, was observed to induce apoptosis in HL60 human premyelocytic leukemia cell line. Tanshinone IIA induced DNA fragmentation into the multiples of 180 bp and increased the percentage of hypodiploid cells in flow cytometry after propidium iodide (PI) staining. Tanshinone IIA-induced apoptosis is accompanied by the specific proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of caspase-3, a major component in apoptotic cell death mechanism.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia , Fenantrenos/uso terapêutico , Extratos Vegetais/uso terapêutico , Abietanos , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Corantes/química , Fragmentação do DNA/efeitos dos fármacos , Citometria de Fluxo , Humanos , Técnicas In Vitro , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Propídio/química , Fatores de Tempo , Células Tumorais CultivadasRESUMO
A number of fish species have been used for studies on endocrine disrupting chemicals (EDCs). However, despite the widespread use of oviparous fish, relatively little attention has been given to viviparous species. This study investigated the effects of EDCs in a viviparous fish and examined the possible usefulness of the fish as an alternative model for the studies on EDCs. Swordtails (Xiphophorus helleri) were exposed to nonylphenol (NP), bisphenol A (BPA), and their mixture. Both short-term (3-d) and relatively long-term (60-d) exposures were carried out using adult male and 30-d-old juvenile fish, respectively. Following the short-term exposure, both NP and BPA caused vitellogenin mRNA expression. Flow cytometric analysis and terminal deoxynucleotidyl transferase assay on the testes of treated fish indicated reproductive damage. Histopathological analysis found degenerative and necrotic cells in seminiferous tubules following the exposure to 100 ppb NP. The testes with lesions were also associated with highly suppressed spermatogenesis. Following the long-term exposure, both NP and BPA exposures significantly affected the growth of swordtails. In all cases, the results showed that the mixture was always more potent than a single chemical and that swordtail fish can be a useful model for the study of endocrine disruptors.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Apoptose/efeitos dos fármacos , Ciprinodontiformes/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fenóis/toxicidade , Reprodução/efeitos dos fármacos , Vitelogeninas/genética , Poluentes Químicos da Água/toxicidade , Animais , Compostos Benzidrílicos , Ciprinodontiformes/crescimento & desenvolvimento , Interações Medicamentosas , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Masculino , Modelos Biológicos , Necrose , RNA Mensageiro/genética , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/patologia , Testículo/efeitos dos fármacos , Testículo/patologia , Transcrição Gênica/efeitos dos fármacosRESUMO
To determine applicability of the cryopreservation procedure for vessel grafts, the viability of endothelial cells (ECs) among the whole cells in three kinds of organs artery, vein, trachea in mongrel dogs was evaluated on the basis of histological analysis. The Griffonia simplicifolia agglutins-fluorescein isothiocyanate (GSA-FITC) and propidium iodide (PI) double staining methods were combined with flow cytometry (FCM), which was able to simultaneously determine the viability of whole cells and ECs from the same tissue, were performed after harvesting, after antibiotic solution treatment, and after cryopreservation and thawing. In most cases, the viability of ECs is lower than that of whole cells from veins and arteries. The viability of whole cells in veins was maintained until the antibiotic solution treatment and then decreased significantly after cryopreservation and thawing, while the ECs began to decrease significantly after the antibiotic solution treatment and more markedly decreased after thawing. The viability of ECs and whole cells from arteries was similar to that of the veins' conditions. The viability of whole cells from the trachea decreased with a similar pattern to that of the ECs from vessels. In consideration of maintaining cell viability among the three kinds of organs, the viability of arteries was better than that of the others. The cells in the trachea demonstrated a lower viability than the vessels. The effect of antibiotic solution treatment on the reduction of cell viability depends on the treatment time and temperature.
Assuntos
Vasos Coronários , Criopreservação , Endotélio Vascular/fisiologia , Traqueia , Animais , Artérias/transplante , Sobrevivência Celular , Vasos Coronários/transplante , Cães , Endotélio Vascular/citologia , Feminino , Masculino , Traqueia/transplante , Veias/transplanteRESUMO
Pyribenzoxim, benzophenone O-[2,6-bis(4,6-dimethoxypyrimidin-2-yloxy)benzoyl]oxime, is a new post-emergence herbicide providing broad-spectrum weed control in rice fields. [14C]Pyribenzoxim was used to study the pharmacokinetics of the compound after oral administration of a dose of 1000 mg kg-1 to male Sprague-Dawley rats. The material balance ranged from 97.3 to 99.7% of the administered dose and urinary and fecal recovery accounted for 97.1%, with the majority of radioactivity recovered in feces (88.6%) by 168 h after treatment. Elimination as volatile products or as carbon dioxide was negligible. The following values were obtained for the compound in the blood: AUC0-168 h, 28,400 micrograms equiv hg-1; Tmax, 12 h; Cmax, 372 micrograms equiv g-1; half-life, 53 h. Radioactivity in tissue decreased from 96.1% of applied radiocarbon at 6 h to 0.4% at 168 h and the highest concentration of radioactivity among the tissues was observed in liver while the lowest residues were found in brain. The elimination half-lives of radioactivity from tissues was in the range of 7 to 77 h and Tmax values of 12, 24 and 12 h were observed for blood, liver and kidney, respectively. Except for that in the digestive tract, the tissue-to-blood ratio (TBR) was highest in the liver.
Assuntos
Benzofenonas/farmacologia , Herbicidas/farmacocinética , Administração Oral , Algoritmos , Animais , Área Sob a Curva , Benzofenonas/sangue , Benzofenonas/química , Dióxido de Carbono/metabolismo , Radioisótopos de Carbono , Cromatografia Líquida de Alta Pressão , Fezes/química , Herbicidas/sangue , Herbicidas/química , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Análise Espectral , Distribuição TecidualRESUMO
Capecitabine, a prodrug of 5-FU, has been reported to generate maximal tumour activity at tumour sites and/or to improve drug tolerability as compared with 5-FU infusion, and it has also been demonstrated to act synergistically with irinotecan against some solid cancers. A previous study concluded that dose-intensified biweekly capecitabine seems to be more effective at increasing both response rate and progression-free survival time than conventional dose and schedule of capecitabine in colon cancer. We conducted this study to ascertain the efficacy and toxicity of dose-intensified biweekly capecitabine and irinotecan combination chemotherapy in chemotherapy-naïve advanced or metastatic gastric cancer patients. Patients were treated with irinotecan 130 mg m(-2) intravenously for 90 min on days 1 and 15. Capecitabine at 3500 mg m(-2) day(-1), divided into two sessions per day, was administered for seven consecutive days from days 1 and 15, and followed by a 7-day drug-free period, respectively. Fifty-five eligible patients were enrolled in this study from November 2003 to April 2006. There were 22 women and 33 men: median patient age was 54 years (range: 27-81). A total of 200 treatment cycles were administered at a median number of four per patient (range: 1-9). Intent-to-treatment analysis showed that one patient achieved complete response (1.8%), 23 partial response (41.8%), 15 stable disease (27.3%), 10 progressive disease (18.2%) and 6 were non-evaluable (10.9%). The overall response rate was 43.6% (95% confidence interval: 30.2-56.9). The common grade 3-4 toxicities were neutropenia in 12 (21.8%), nausea/vomiting in 3 (5.4%) and diarrhea in 4 (7.2%) patients. Median time to progression was 5 months (range: 0.5-11 months), median survival duration was 11 months (range: 0.5-45 months) and median response duration was 6 months (range: 0.5-9 months). Biweekly dose-intensified capecitabine and irinotecan combination chemotherapy was active for the treatment of advanced or metastatic gastric cancers with a tolerable safety profile.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/análogos & derivados , Desoxicitidina/análogos & derivados , Fluoruracila/análogos & derivados , Neoplasias Gástricas/tratamento farmacológico , Adenocarcinoma/patologia , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Capecitabina , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Irinotecano , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Gástricas/patologia , Resultado do TratamentoAssuntos
Doenças do Cão/virologia , Vírus da Influenza A Subtipo H3N2 , Infecções por Orthomyxoviridae/veterinária , Animais , Doenças do Cão/epidemiologia , Cães , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , República da Coreia/epidemiologia , Estudos RetrospectivosRESUMO
Although recent studies have shown that several pro-inflammatory proteins can be used as biomarkers for atherosclerosis, the mechanism of atherogenesis is unclear and little information is available regarding proteins involved in development of the disease. Atherosclerotic tissue samples were collected from patients in order to identify the proteins involved in atherogenesis. The protein expression profile of atherosclerosis patients was analysed using two-dimensional electrophoresis-based proteomics. Thirty-nine proteins were detected that were differentially expressed in the atherosclerotic aorta compared with the normal aorta. Twenty-seven of these proteins were identified in the MS-FIT database. They are involved in a number of biological processes, including calcium-mediated processes, migration of vascular smooth muscle cells, matrix metalloproteinase activation and regulation of pro-inflammatory cytokines. Confirmation of differential protein expression was performed by Western blot analysis. Potential applications of the results include the identification and characterization of signalling pathways involved in atherogenesis, and further exploration of the role of selected identified proteins in atherosclerosis.
Assuntos
Aterosclerose/genética , Regulação da Expressão Gênica , Proteômica , Doenças da Aorta , Eletroforese em Gel Bidimensional , Humanos , Proteínas/análise , Proteômica/métodos , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Apoptosis is a new therapeutic target of cancer research. Shikonin isolated from Lithospermum erythrorhizon, a traditional oriental medicinal herb, was observed to induce apoptosis in HL60 human premyelocytic leukemia cell line. Shikonin induced DNA fragmentation into the multiples of 180 bp and increased the percentage of hypodiploid cells in flow cytometry after propidium iodide staining. The increase of apoptotic cells was preceded by the activation of caspase-3, which was reported to play a central role in apoptotic process. The DNA fragmentation induced by shikonin was completely inhibited by the pretreatment of z-VAD-fmk, a specific inhibitor of caspase, clearly showing that the mode of cell death is apoptotic.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Naftoquinonas/farmacologia , Plantas Medicinais , Caspase 3 , Caspases/metabolismo , Células HL-60 , Humanos , Coreia (Geográfico) , Medicina Tradicional do Leste Asiático , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Pretreatment of tissue by using antibiotics is a critical step to prevent microbial contamination before venous transplantation. In this study, the optimal time and temperature of antibiotic solution treatment for maintaining cellular viability with antibacterial effect were investigated. The antibiotic-nutrient solutions were composed of cefoxitin, lincomycin, vancomycin, and polymyxin B in RPMI-1640 medium. After various antibiotic solution treatment times (4, 8, and 12 h) and temperatures (4, 25, and 37 degrees C), the viabilities of cells dissociated from veins (jugular vein, femoral vein, superior vena cava, and inferior vena cava) were determined. Double staining by Griffonia simplicifolia agglutins-fluorescein isothiocyanate (GS1-FITC) and propidium iodide was used. To measure the antibacterial effect of the antibiotic solution, canine veins were artificially infected by 3 kinds of bacteria (Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae) and were treated by antibiotic solutions as viability test conditions. After the treatment with the antibiotic solution, the tissue was minced, and the homogenized tissue fraction was cultured on standard method agar. The colony that seemed to be resistant to the antibiotic solution was counted. At 37 and 25 degrees C, the viability of whole cells decreased significantly Asymptotic Significance 2-tailed (Asymp.Sig 2-tailed) < 0.05 after 4 h of antibiotic solution treatment, whereas at 4 degrees C it began to reduce significantly after 8 h of treatment. By antibiotic solution treatment at all 3 temperatures for 4 h, no significant difference in viability of the endothelial cells and whole cells was observed. To maintain the donor vein's cellular viability until transplantation, antibiotic solution treatment for 4 h at 4 degrees C is assumed to be appropriate.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/prevenção & controle , Veias/efeitos dos fármacos , Animais , Cefoxitina/farmacologia , Cães , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Lincomicina/farmacologia , Polimixina B/farmacologia , Soluções , Estatísticas não Paramétricas , Vancomicina/farmacologia , Veias/transplanteRESUMO
Abstract: An efficient method for specifically determining the viability of endothelial cells (EC) from cells dissociated from the human saphenous vein was investigated. Three different methods, trypan blue staining assay, [3H]-proline incorporation assay, and flow cytometry (FCM), combined with the fluorescein isothiocyanate conjugated with Griffonia simplicifolia agglutins (GS1-FITC)/propidium iodide (PI) double staining, were used. Both trypan blue staining and [3H] proline incorporation assays demonstrated less sensitivity to determine viability of EC differentially from the other cells. FITC-GS1 showed prominent binding to the vascular EC and could be counted by FCM including PI on dead cells. Following the cryopreservation process, the GS1-FITC/PI FCM analytical method was adopted to test simultaneously the viability of whole cells and EC from the same tissue, human saphenous veins, and mongrel dogs' femoral veins after harvesting, antibiotic solution treatment, and thawing. The viability of the whole cells from veins decreased with a significant difference (p < 0.05) from that of EC after thawing.