RESUMEN
AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.
Asunto(s)
Bovinos/microbiología , Escherichia coli O157/aislamiento & purificación , Heces/microbiología , Reacción en Cadena de la Polimerasa/métodos , Animales , Escherichia coli O157/genética , Flagelos/genética , Flagelina/genética , Sensibilidad y EspecificidadRESUMEN
AIM: To determine the effect of different carbohydrate-based finishing diets on fermentation characteristics and the shedding of Escherichia coli and enterohaemorrhagic E. coli (EHEC) virulence genes in cattle faeces. METHODS AND RESULTS: The size of faecal E. coli populations and fermentation characteristics were ascertained in three experiments where cattle were maintained on a range of finishing diets including high grain, roughage, and roughage + molasses (50%) diets. Increased E. coli numbers, decreased pH and enhanced butyrate and lactate fermentation pathways were associated with grain diets, whereas roughage and roughage + molasses diets resulted in decreased concentrations of ehxA, eaeA and stx(1) genes, this trend remaining at lairage. In one experiment, faecal E. coli numbers were significantly lower in animals fed roughage and roughage + molasses, than animals fed grain (4.5, 5.2 and 6.3 mean log10 g(-1) digesta respectively). In a second experiment, faecal E. coli numbers were 2 log lower in the roughage and roughage + molasses diets compared with grain-fed animals prior to lairage (5.6, 5.5 and 7.9 mean log10 g(-1) digesta respectively) this difference increasing to 2.5 log at lairage. CONCLUSIONS: The type of dietary carbohydrate has a significant effect on E. coli numbers and concentration of EHEC virulence genes in faeces of cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a better understanding of the impact finishing diet and commercial lairage management practices may have on the shedding of E. coli and EHEC virulence factors, thus reducing the risk of carcass contamination by EHEC.
Asunto(s)
Alimentación Animal/microbiología , Escherichia coli/crecimiento & desarrollo , Heces/microbiología , Genes Bacterianos/genética , Animales , Butiratos/metabolismo , Bovinos , Recuento de Colonia Microbiana/métodos , Carbohidratos de la Dieta/administración & dosificación , Fibras de la Dieta , Grano Comestible , Escherichia coli/genética , Escherichia coli/patogenicidad , Fermentación , Concentración de Iones de Hidrógeno , Lactatos/metabolismo , Melaza , Virulencia/genéticaRESUMEN
AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.
Asunto(s)
Alimentación Animal , Microbiología Ambiental , Escherichia coli/clasificación , Escherichia coli/fisiología , Heces/microbiología , Animales , Bovinos , Análisis por Conglomerados , Farmacorresistencia Bacteriana , Proteínas de Escherichia coli/biosíntesis , Fermentación , Proteínas Hemolisinas/biosíntesis , Masculino , Distribución Aleatoria , Serotipificación , Toxinas Shiga/biosíntesisRESUMEN
Decompression sickness (DCS) is the result of bubble formation in the body due to excessive/rapid reduction in the ambient pressure. Existing models relate the decompression stress either to the inert gas load or to the size of a single bubble in a tissue compartment. This paper presents a model that uses the gas exchange equations combined with bubble dissolution physics and population balance equations to produce a new mathematical framework for DCS modeling. This framework, the population balance model for decompression sickness (PBMDS), simulates the number of bubbles with their corresponding size distributions in a compartmental tissue array. The model has a modular structure that enables one to explore different modeling results with respect to key aspects of DCS, such as gas exchange, nucleation, and surface tension. The paper's goal is to present the derivation of PBMDS in detail, however, three simple application case studies are provided. The aim of these case studies is to suggest that PBMDS supplies additional information on bubble distribution while supporting the results from current practice.
Asunto(s)
Descompresión/efectos adversos , Embolia Aérea/etiología , Embolia Aérea/fisiopatología , Modelos Biológicos , Simulación por Computador , Enfermedad de Descompresión/etiología , Enfermedad de Descompresión/fisiopatología , Buceo/fisiología , Humanos , Medicina Naval , Intercambio Gaseoso Pulmonar/fisiologíaRESUMEN
The effectiveness of antineoplastic agents has been augmented by applying pulsed electric fields directly to tumours after the administration of the drug. This treatment, known as electrochemotherapy (ECT), has been successful for cutaneous malignancies in animal models and in recent clinical trials. This study was aimed at investigating the applicability of ECT in a surgical setting for hepatocellular carcinomas induced in the livers of rats. Established tumours were injected with bleomycin, and electric pulses were then administered locally. Animals were followed based on tumour volumes and histological samples. Dose response data were obtained for both electric field intensity and bleomycin. Complete response rates for animals treated with electrochemotherapy ranged from 26.67% to 93.33 and were durable. In contrast, tumours that received no treatment, pulses only or drug only responded minimally. This supports the feasibility of using a ECT as a modality for treating hepatocellular carcinoma.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Bleomicina/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Electroporación , Neoplasias Hepáticas/tratamiento farmacológico , Animales , Carcinoma Hepatocelular/patología , Relación Dosis-Respuesta a Droga , Electroporación/métodos , Neoplasias Hepáticas/patología , Masculino , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
Gene therapy depends on safe and efficient gene delivery. The skin is an attractive target for gene delivery because of its accessibility. Recently, in vivo electroporation has been shown to enhance expression after injection of plasmid DNA. In this study, we examined the use of electroporation to deliver plasmid DNA to cells of the skin in order to demonstrate that localized delivery can result in increased serum concentrations of a specific protein. Intradermal injection of a plasmid encoding luciferase resulted in low levels of expression. However, when injection was combined with electroporation, expression was significantly increased. When performing this procedure with a plasmid encoding interleukin-12, the induced serum concentrations of gamma-interferon were as much as 10 fold higher when electroporation was used. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.
Asunto(s)
ADN/administración & dosificación , Electroporación/métodos , Interleucina-12/genética , Plásmidos , Animales , Estudios de Evaluación como Asunto , Femenino , Terapia Genética , Inyecciones Intradérmicas , Luciferasas/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.
Asunto(s)
Transfección/métodos , Electroporación , Plásmidos , TransgenesRESUMEN
BACKGROUND: Pulsed electric fields have been shown to increase the effectiveness of antineoplastic agents by temporarily increasing the permeability of cell membranes. This type of drug delivery is called electrochemotherapy, and it has been successful in the treatment of patients with cutaneous malignancies in clinical trials. This study focused on determining the applicability of electrochemotherapy to the treatment of soft tissue sarcoma, using an animal model bearing human sarcomas. The antitumor effects of single and multiple electrochemotherapy treatments were investigated using small (250 mm3) and large (4000 mm3) tumors. METHODS: Established tumors were injected with bleomycin, then electric pulses were administered to the tumor site. Animals were followed based on periodic tumor volume determinations, which were used to categorize treatment of each tumor as a complete response, a partial response, stable disease, or progressive disease. Histologic analysis was used to confirm response data. RESULTS: Animals were randomly assigned to one of four different treatment groups. These groups received no treatment, drug only, electric pulses only, or drug combined with electric pulses. A single electrochemotherapy treatment protocol for small tumors resulted in a 100% complete response rate and a 41.7% cure rate. Multiple treatments of small and large tumors resulted in complete response rates of 83.3% and 100%, respectively. These responses were identical to the cure rates. In contrast, tumors in the groups that received no treatment, electric pulses only, and drug only progressed for both single treatment and multiple treatment scenarios, regardless of tumor size. CONCLUSIONS: In this study, a single electrochemotherapy treatment had a strong cytoreductive effect on small tumors that lasted approximately 35 days, until recurrences began. Multiple treatment of small and large tumors resulted in high complete response rates that lasted at least 100 days after treatment. This indicates the feasibility of electrochemotherapy as a modality of limb-preserving treatment for patients with sarcoma of the extremities.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Bleomicina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Sarcoma/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/uso terapéutico , Bleomicina/uso terapéutico , Electroporación , Humanos , Masculino , Trasplante de Neoplasias , Ratas , Ratas Desnudas , Sarcoma/patología , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
BACKGROUND: Electrochemotherapy (ECT) is performed by locally administering a chemotherapeutic agent in combination with electric pulses. Previous clinical studies have demonstrated the effectiveness of ECT. In these initial trials, the drug was administered intravenously, followed by administration of electric pulses directly to the tumor. This study was initiated to determine whether an intralesional injection of the drug in combination with electric pulses could provide an improved result. A group of 34 patients was studied. METHODS: The dose of intralesional bleomycin was based on tumor volume. This was followed 10 minutes later by 6 or 8 99-microsec pulses of electricity at an amplitude of 1.3 kV/cm. Both the bleomycin and the electric pulses were administered after 1% lidocaine with epinephrine solution was injected around the treatment site. RESULTS: All patients responded to the treatment. Responses were observed in 142 (99%) of 143 metastatic nodules or primary tumors within 12 weeks, with complete responses observed in 130 (91%) of the nodules. No complete responses were observed in nodules treated with bleomycin only or electric pulses only. Random biopsies confirmed the clinical findings. All patients tolerated the procedure well, and no significant side effects were noted. Muscle contraction was evident during administration of each electric pulse but promptly subsided after the pulse. CONCLUSIONS: ECT was shown to be an effective local treatment for cutaneous malignancies. The results suggest that ECT may have a tissue-sparing effect and result in minimal scarring. ECT may be a suitable alternative therapy for the treatment of basal cell carcinoma, local or regional recurrent melanoma, and other skin cancers.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Bleomicina/uso terapéutico , Terapia por Estimulación Eléctrica , Neoplasias Cutáneas/terapia , Adulto , Anciano , Carcinoma Basocelular/patología , Carcinoma Basocelular/terapia , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Femenino , Humanos , Masculino , Melanoma/patología , Melanoma/terapia , Persona de Mediana Edad , Neoplasias Cutáneas/patologíaRESUMEN
Direct current pulses for electrochemotherapy treatment are typically administered using two parallel plate electrodes that are placed on either side of the tumor. This simple design has produced high response rates (70 to 85%) in animal studies and in clinical trials. However, parallel plate electrodes are not suitable for all situations. This study describes five novel electrode designs and compares their effectiveness to a parallel plate design for treating melanoma tumors in mice. Results for the 2 x 2 needle array design showed 50% increases in doubling time and in complete response rate compared to the standard parallel plate electrode.
Asunto(s)
Electrodos , Melanoma/terapia , Neoplasias Cutáneas/terapia , Animales , Terapia Combinada , Terapia por Estimulación Eléctrica/instrumentación , Electroporación , Diseño de Equipo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Neoplasias Cutáneas/patologíaRESUMEN
The use of in vivo electroporation in combination with a chemotherapeutic agent (electrochemotherapy) for the treatment of liver tumors was examined. Induced rat hepatomas were treated with a 0.5 unit intratumor bleomycin dose followed by rectangular direct current pulses. Six pulses were administered during treatment using a needle array electrode that rotated the applied electric field around the tumors. A 84.6% objective response rate resulted for tumors that received both bleomycin and electric pulses. Control groups that received partial or no treatment showed less than 10% objective response rates. These results indicate that electrochemotherapy can be translated from the treatment of cutaneous cancers to the treatment of liver tumors and other internal tumors.
Asunto(s)
Neoplasias Hepáticas Experimentales/terapia , Animales , Antibióticos Antineoplásicos/administración & dosificación , Bleomicina/administración & dosificación , Terapia Combinada , Terapia por Estimulación Eléctrica/instrumentación , Electrodos , Electroporación , Femenino , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/patología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Electroporation is a process that causes a transient increase in the permeability of cell membranes. It can be used to increase the intracellular concentration of chemotherapeutic agents in tumor cells (electrochemotherapy; ECT). A clinical study was initiated to determine if this mode of treatment would be effective against certain primary and metastatic cutaneous malignancies. A group of six patients, three with malignant melanoma, two with basal cell carcinoma, and one with metastatic adenocarcinoma, were enrolled in the study. the treatment was administered in a two-step process. METHODS: Each patient received a 10 unit/m2 dose of bleomycin administered intravenously at 1 to 1.5 units/minute. This was followed by eight 99 microsecond pulses at an amplitude of 1.3 kV/cm administered directly to the tumors 5 to 15 minutes after the bleomycin was completely infused. Pulses were administered after the injection of 1% lidocaine solution around the treatment site. RESULTS: Two of three melanoma patients had objective responses. In these two patients, five of six treated tumors decreased in size, and three completely responded. Untreated tumors displayed continued growth. Objective responses were observed in both basal cell carcinoma (BCC) patients. One patient had partial responses in both treated tumors. The other patient had one of four primary BCCs respond completely, and the remaining three respond partially. Patients with metastatic breast adenocarcinoma showed complete responses in both treated nodules after ECT. All patients tolerated the treatment well with no residual effects from the electric pulses. CONCLUSIONS: ECT was an effective local treatment in the majority of nodules treated. The results thus far are very encouraging and the study is being continued.
Asunto(s)
Adenocarcinoma/terapia , Antibióticos Antineoplásicos/uso terapéutico , Bleomicina/uso terapéutico , Carcinoma Basocelular/terapia , Melanoma/terapia , Neoplasias Cutáneas/terapia , Estimulación Eléctrica Transcutánea del Nervio , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adulto , Anciano , Antibióticos Antineoplásicos/efectos adversos , Antibióticos Antineoplásicos/farmacocinética , Bleomicina/efectos adversos , Bleomicina/farmacocinética , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Electrofisiología , Femenino , Humanos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Persona de Mediana Edad , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Estimulación Eléctrica Transcutánea del Nervio/efectos adversosRESUMEN
To investigate the use of teleradiology in the quality assurance programme of a multicentre radiotherapy practice, we installed image acquisition and display workstations at each of two affiliated radiation oncology clinics. A commercial diagnostic teleradiology system was successfully modified to suit the requirements of the radiotherapy subspecialty. The system allowed intersite transmission of images, access to high-resolution images from each site and, by use of laser film scanners, made accessible all types of radiation therapy image. Transmission speed and storage capacity were better than expected. Using the system, radiation oncology residents and staff reviewed 83 complex cases over eight months. Case presentation and discussion were enhanced. In the same period, 276 cases were reviewed by conference in person. Case conferences for quality assurance conducted with the teleradiology system influenced changes in treatment planning as effectively as those conducted in person. Equivalent treatment outcomes were produced. The teleradiology system facilitated quality assurance through review of patients' radiation treatments by allowing natural interactive consultation.