Real-time monitoring of Escherichia coli O157:H7 adherence to beef carcass surface tissues with a bioluminescent reporter.

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.

Stationary headband for clinical time-of-flight optical imaging at the bedside.

Conventional brain-imaging modalities may be limited by high cost, difficulty of bedside use, noncontinuous operation, invasiveness or an inability to obtain measurements of tissue function, such as oxygenation during stroke. Our goal was to develop a bedside clinical device able to generate continuous, noninvasive, tomographic images of the brain using low-power nonionizing optical radiation. We modified an existing stage-based time-of-flight optical tomography system to allow imaging of patients under clinical conditions. First, a stationary head-band consisting of thin, flexible optical fibers was constructed. The headband was then calibrated and tested, including an assessment of fiber lengths, the existing system software was modified to collect headband data and to perform simultaneous collection of data and image reconstruction, and the existing hardware was modified to scan optically using this headband. The headband was tested on resin models and allowed for the generation of tomographic images in vitro; the headband was tested on critically ill infants and allowed for optical tomographic images of the neonatal brain to be obtained in vivo.

Visualizing gene expression in living mammals using a bioluminescent reporter.

Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA-based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV-40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real-time, noninvasive study of gene expression in living animal models for human development and disease is possible.

Photonic detection of bacterial pathogens in living hosts.

The study of pathogenic processes is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurlum that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bioluminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo.

Effects of ketoconazole on cholesterol synthesis.

Studies have demonstrated that ketoconazole and related imidazoles block gonadal and adrenal steroidogenesis in humans by inhibiting several cytochrome P-450-dependent enzymes. Moreover, recent evidence suggests that cholesterol production in humans is also affected by ketoconazole. In the present experiments cultured normal human fibroblasts have been used to explore the effects of ketoconazole on cholesterol synthesis. Ketoconazole inhibited cholesterol synthesis (greater than 90% suppression in 1 hr) rapidly by blocking the conversion of methyl sterols to cholesterol. Dihydrolanosterol was the major methyl sterol which accumulated with ketoconazole. At high concentrations of ketoconazole, the conversion of squalene to methyl sterols was also inhibited. The inhibition of cholesterol synthesis was dose-dependent with an IC50 approximately 2.8 X 10(-8) M. In parallel to the inhibition of cholesterol synthesis, there was a reciprocal increase in methyl sterol production. The related imidazole antimycotic, clotrimazole, had similar effects, whereas the imidazole anesthetic, etomidate, had little effect on cholesterol synthesis. Confluent cells exposed to ketoconazole had a 90% fall in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that declined with a T1/2 approximately 3.7 hr. In conclusion, ketoconazole has multiple effects on cholesterol synthesis, directly inhibiting late steps by blocking the conversion of methyl sterols to cholesterol and indirectly suppressing total sterol synthesis via feedback inhibition by sterol intermediates of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

Hydroxylation of [3H]5 alpha-androstane-3 beta,17 beta-diol by whole tissue, epithelial cells and fibroblasts from the same hyperplastic human prostate.

Hydroxylations of 3 beta-hydroxy 5 alpha-dihydro C19-steroids are terminal reactions by which male accessory sex organs dispose of intracellular androgens. Cellular androgen egress is of particular interest in benign prostatic hyperplasia (BPH) where the elevated nuclear 5 alpha-dihydrotestosterone-receptor content may be implicated in the etiology of the disease. We here report substitution of hydroxyl groups at C-6 alpha, C-7 beta and predominantly at C-7 alpha of [3H]5 alpha-androstane-3 beta,17 beta-diol on incubation of 3 and 8.5 nM substrate concentrations with minced and explanted human BPH tissue. Fibroblasts isolated from the same prostatectomy specimen hydroxylated 3 nM radiosubstrate mainly at C-6 alpha, with extensive metabolism to 17-oxosteroids. Epithelial cells from the same tissue source substituted to the same extent at the three positions. Competing 3 beta-hydroxysteroid dehydrogenase exceeded hydroxylase activity only in epithelial-cell cultures. Our findings support previous evidence that prostatic epithelial and stromal cells make different contributions to androgen disposition by the 3 beta-hydroxysteroid pathway.

Metabolism of 2-acetylaminofluorene in primary rat hepatocyte cultures.

Primary cultures of adult rat parenchymal hepatocytes were developed as an in vitro model to investigate the biochemical fate of 2-acetylaminofluorene (AAF), a potent hepatocarcinogen. More than 5 x 10(8) viable cells were routinely isolated by collagenase perfusion in rat liver; the cells were cultured 2-5 d on collagen-coated dishes in serum-free culture medium containing hormones and other factors to retard the decline of cytochrome P-450. All of 137 ng or 13.7 microgram AAF was metabolized in 21-24 h by 2 x 10(6) cultured hepatocytes in 4.0 ml defined medium. At the higher dose, water-soluble metabolites appeared at 70% of the rate of metabolism at the lower dose, which was 17 ng/h for the initial 4 h. As the parent compound was consumed, bound AAF residues were recovered with exhaustively extracted, trichloro-acetic acid-precipitated hepatocellular macromolecules, accounting for a maximum of 5% of the 137-ng dose. Addition of hormones to the culture medium stimulated the rate of appearance of water-soluble metabolites, AAF, correlating with the enhanced cytochrome P-450 levels of hormone-treated cells. Metabolism of AAF was diminished 50% during 3 h of incubation with 10(-4) M SKF 525A and 100% with 10(-3) M SKF 525A. At a dose of 40 microgram AAF per 2 X 10(6) cells, only 31% of the carcinogen was recovered from the culture medium as water-soluble products after 24 h; the cells were sown to be capable of metabolizing a subsequent 40-microgram dose at an undiminished rate, suggesting that saturation of metabolizing enzymes rather than toxicity occurred. These results support the validity of primary hepatocyte cultures as a model system for quantitative investigations of the biochemical fate of AAF in mammalian cells, and provide preliminary characterization of the cells' processes of detoxification and metabolic activation of a chemical carcinogen.