Improving recognition and management of acute kidney injury.

Acute kidney injury (AKI) is currently suboptimally recognised and managed in the UK, despite its association with significant patient morbidity, mortality and consequent implications for healthcare economics. Our prospective study, performed in a large urban London hospital, demonstrated that the introduction of a specially designed care bundle can significantly improve documentation of baseline creatinine, assessment and optimisation of fluid status, performance of urine dip, withholding of nephrotoxic drugs, appropriate monitoring of urine output, prescription of renal drug doses, and appropriate consideration of a renal ultrasound and urinary protein-creatinine ratio. Improved compliance of appropriate investigations and initial treatments translated to decreased requirement for intensive care admission and a trend towards shorter length of stays.

Mycobacterium avium ssp. hominissuis biofilm is composed of distinct phenotypes and influenced by the presence of antimicrobials.

Mycobacterium avium ssp. hominissuis, hereafter referred to as M. avium, forms biofilm, a property that, in mice, is associated with lung infection via aerosol. As M. avium might co-inhabit the respiratory tract with other pathogens, treatment of the co-pathogen-associated infections, such as in bronchiectasis, would expose M. avium to therapeutic compounds that may have their origin in other organisms sharing the natural environments. Incubation of M. avium with two compounds produced by environmental organisms, streptomycin and tetracycline, in vitro at subinhibitory concentrations increased biofilm formation in a number of M. avium strains, although exposure to ampicillin, moxifloxacin, rifampin and trimethoprim-sulphamethoxazole had no effect on biofilm formation. No selection of genotypically resistant clones was observed. Although incubation of bacteria in the presence of streptomycin upregulates the expression of biofilm-associated genes, the response to the antibiotics had no association with the expression of a regulator (LysR) linked to the formation of biofilm in M. avium. Biofilms are composed of planktonic and sessile bacteria. Whereas planktonic M. avium is susceptible to clarithromycin and ethambutol (clinically used antimicrobials), sessile bacteria are at least three-fold to four-fold more resistant to antibiotics. The sessile phenotype, however, is reversible, and no selection of resistant clones was observed. Mice infected through the airway with both phenotypes were infected with a similar number of bacteria, demonstrating no phenotype advantage. M. avium biofilm formation is enhanced by commonly used compounds and, in the sessile bacterial phenotype, is resistant to clarithromycin and ethambutol, in a reversible manner.

Factors influencing hospital readmission rates after acute medical treatment.

It is a concern that increasing pressure to diagnose, treat and discharge patients rapidly is leading to unacceptably high readmission rates. Readmissions were studied over a two-month period. Patients were identified through the hospital coding system, and electronic discharge summaries provided details of each admission. In total, 69 readmissions were identified, representing 4.34% of medical admissions. Readmitted patients were older than those with single admissions (median age 75 and 71 years, respectively; p < 0.05). Initial length of stay was greater in those patients who would go on to be readmitted (median six days; single admission, two days; p < 0.0001). Seventy-one per cent of readmissions were deemed avoidable, with discharge before conclusive therapy being the leading factor implicated (56%). Readmission is more likely in older patients with complex care needs. Rapid throughput of patients is not associated with readmission. The majority of readmissions can potentially be avoided with judicious medical care.

Estimating the proportion of young adults on antihypertensive treatment that have been correctly diagnosed.

This paper aims to identify how many young adults on antihypertensive treatment have been misclassified as hypertensive. We identified subjects aged under 35 on antihypertensive treatment, from the Health Surveys for England, 1998-2004. Pretreatment systolic and diastolic blood pressures were calculated by adjusting on-treatment blood pressures for the effects of treatment. Treatment effects were derived from meta-analysis. Subjects were classified as hypertensive if pretreatment blood pressure was >or=160/100 mm Hg, or was >or=140/90 mm Hg in conjunction with high cardiovascular risk. We then identified the proportion of treated subjects on antihypertensive treatment who were truly eligible for treatment. From the survey data we identified 65 adults (25 men and 40 women) under 35 on diuretics, beta-blockers, angiotensin converting enzyme inhibitors, calcium blockers or other antihypertensives. Average pretreatment blood pressure was 164/100 mm Hg in those eligible for treatment, and 136/79 mm Hg in those not eligible. The analysis indicated that 29.2% of adults aged 16-34 (95% confidence interval (CI): 18.6-41.8%) were truly eligible for antihypertensive treatment: 32.0% (95% CI: 14.9-53.5%) of men and 25.0% (95% CI: 12.7-41.2%) of women. A total of 73.7% (14 of 19) of subjects eligible and 41.3% (19 of 46) of subjects not eligible for treatment either had a body mass index >30 kg m(-2) or kidney disease (chi(2)-test P=0.018). Because of biological variation in blood pressure, most young adults on treatment for hypertension have been misclassified as hypertensive. Most who have been correctly diagnosed are either clinically obese or have kidney disease.

Blood gene expression signatures predict exposure levels.

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.

Photocarcinogenesis in the Tg.AC mouse: lomefloxacin and 8-methoxypsoralen.

The Tg.AC mouse is a good predictor of carcinogenic potential when the test article is administered by dorsal painting (Tennant et al. (1995) Environ. Health Perspect. 103, 942). We have used lomefloxacin (LOME) and 8-methoxypsoralen (8-MOP) in combination with UVA to determine whether the Tg.AC transgenic mouse also responds to parenterally administered photocarcinogens. Female Tg.AC mice were given LOME (25 mg/kg intraperitoneal in normal saline) followed by UVA (25 J/cm2) 1-2 h later, five times every 2 weeks on a repetitive schedule. Other groups received LOME, UVA or vehicle alone. After 16 weeks, the mean numbers of papillomas/mouse +/- SD (% responding) were: saline, 0.3 +/- 0.5 (33%); UVA + saline, 1.3 +/- 0.6 (100%); LOME, 1.9 +/- 1.6 (86%) and LOME-UVA, 1.5 +/- 1.9 (64%). Only the 100% incidence of tumors in the UVA group and the maximum tumor yields in the LOME and UVA groups are significant (P < 0.05) when compared with the control. In a second study, Tg.AC mice were administered the classical photocarcinogen 8-MOP (8 mg/kg intragastric in corn oil) followed by 2 J/cm2 UVA 1-2 h later, five times every 2 weeks on a repetitive schedule. The second group received 8-MOP, whereas the third was exposed to UVA alone. Papillomas began to appear at 2 weeks in the 8-MOP-UVA group, and after 17 weeks the mean numbers of papillomas/mouse +/- SD (% responding) were: 8-MOP-UVA, 6.9 +/- 8.6 (93%); UVA + corn oil, 1.1 +/- 1.2 (69%) and 8-MOP, 1.1 +/- 1.6 (50%). The maximum tumor yield in the 8-MOP-UVA group was significantly higher (P < 0.01) than that in the other two groups. Our findings suggest that more studies need to be done before the Tg.AC mouse can be used with confidence to identify parenterally administered photocarcinogens.

The Tg.AC (v-Ha-ras) transgenic mouse: nature of the model.

The Tg.AC (v-Ha-ras) transgenic mouse model provides a reporter phenotype of skin papillomas in response to either genotoxic or nongenotoxic carcinogens. In common with the conventional bioassay, the Tg.AC model responds to known human carcinogens and does not respond to noncarcinogens. It also does not respond to most chemicals that are positive in conventional bioassays principally at sites of high spontaneous tumor incidence. The mechanism of response of the Tg.AC model is related to the structure and genomic position of the transgene and the induction of transgene expression through specific mediated interactions between the chemicals and target cells in the skin.

Tg.AC genetically altered mouse: assay working group overview of available data.

In a Government/Industry/Academic partnership to evaluate alternative approaches to carcinogenicity testing, 21 pharmaceutical agents representing a variety of chemical and pharmacological classes and possessing known human and or rodent carcinogenic potential were selected for study in several rodent models. The studies from this partnership project, coordinated by the International Life Sciences Institute, provide additional data to better understand the models' limitations and sensitivity in identifying carcinogens. The results of these alternative model studies were reviewed by members of Assay Working Groups (AWG) composed of scientists from government and industry with expertise in toxicology, genetics, statistics, and pathology. The Tg.AC genetically manipulated mouse was one of the models selected for this project based on previous studies indicating that Tg.AC mice seem to respond to topical application of either mutagenic or nonmutagenic carcinogens with papilloma formation at the site of application. This communication describes the results and AWG interpretations of studies conducted on 14 chemicals administered by the topical and oral (gavage and/or diet) routes to Tg.AC genetically manipulated mice. Cyclosporin A, an immunosuppresant human carcinogen, ethinyl estradiol and diethylstilbestrol (human hormone carcinogens) and clofibrate, an hepatocarcinogenic peroxisome proliferator in rodents, were considered clearly positive in the topical studies. In the oral studies, ethinyl estradiol and diethylstilbestrol were negative, cyclosporin was considered equivocal, and results were not available for the clofibrate study. Of the 3 genotoxic human carcinogens (phenacetin, melphalan, and cyclophosphamide), phenacetin was negative by both the topical and oral routes. Melphalan and cyclophosphamide are, respectively, direct and indirect DNA alkylating agents and topical administration of both caused equivocal responses. With the exception of clofibrate, Tg.AC mice did not exhibit tumor responses to the rodent carcinogens that were putative human noncarcinogens, (di(2-ethylhexyl) phthalate, methapyraline HCl, phenobarbital Na, reserpine, sulfamethoxazole or WY-14643, or the nongenotoxic, noncarcinogen, sulfisoxazole) regardless of route of administration. Based on the observed responses in these studies, it was concluded by the AWG that the Tg.AC model was not overly sensitive and possesses utility as an adjunct to the battery of toxicity studies used to establish human carcinogenic risk.

Age-dependent skin tumorigenesis and transgene expression in the Tg.AC (v-Ha-ras) transgenic mouse.

Transgenic Tg.AC (v-Ha-ras ) mice develop skin tumors in response to specific carcinogens and tumor promoters. The Tg.AC mouse carries the coding sequence of v-Ha ras, linked to a zeta-globin promoter and an SV40 polyadenylation signal sequence. The transgene confers on these mice the property of genetically initiated skin. This study examines the age-dependent sensitivity of the incidence of skin papillomas in Tg.AC mice exposed to topically applied 12-O:-tetradecanoylphorbol-13-acetate (TPA) treatment, full thickness skin wounding or UV radiation. Skin tumor incidence and multiplicity were strongly age-dependent, increasing with increasing age of the animal when first treated at 5, 10, 21 or 32 weeks of age. Furthermore, the temporal induction of transgene expression in keratinocytes isolated from TPA-treated mouse skin was also influenced by the age of the mice. Transgene expression was seen as early as 14 days after the start of TPA treatment in mice that were 10-32 weeks of age, but was not detected in similarly treated 5-week old mice. When isolated keratinocytes were fractionated by density gradient centrifugation the highest transgene expression was found in the denser basal keratinocytes. Transgene expression could be detected in the denser keratinocyte fraction as early as 9 days from start of TPA treatment in 32-week old mice. Using flow cytometry, a positive correlation was observed between expression of the v-Ha-ras transgene and enriched expression of the cell surface protein beta1-integrin, a putative marker of epidermal stem cells. This result suggests that, in the Tg.AC mouse, an age-dependent sensitivity to tumor promotion and the correlated induction of transgene expression are related to changes in cellular development in the follicular compartment of the skin.

Loss of heterozygosity frequency at the Trp53 locus in p53-deficient (+/-) mouse tumors is carcinogen-and tissue-dependent.

Mutagenic carcinogens rapidly induced tumors in the p53 haploinsufficient mouse. Heterozygous p53-deficient (+/-) mice were exposed to different mutagenic carcinogens to determine whether p53 loss of heterozygosity (LOH) was carcinogen-and tissue-dependent. For 26 weeks, C57BL/6 (N4) [corrected] p53-deficient (+/-) male or female mice were exposed to p-cresidine, benzene or phenolphthalein. Tumors were examined first for loss of the wild-type p53 allele. p-cresidine induced p53 LOH in three of 13 bladder tumors, whereas hepatocellular tumors showed p53 LOH in carcinomas (2/2), but not in adenomas (0/3). Benzene induced p53 LOH in 13 of 16 tumors examined. Finally, phenolphthalein induced p53 LOH in all tumors analyzed (21/21). Analysis of the p-cresidine-induced bladder tumors by cold single-strand conformation polymorphism (SSCP) analysis of exon 4-9 amplicons failed to demonstrate polymorphisms associated with mutations in tumors that retained the p53 wild-type allele. p-cresidine induced a dose-related increase in lacI mutations in bladder DNA. In summary, these data demonstrate that loss of the wild-type allele occurred frequently in thymic lymphomas and sarcomas, but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas other mechanisms may be operational. These might include (i) other mechanisms of p53 inactivation, (ii) inactivating mutations occurring outside exons 4-9 or (iii) p53 haploinsufficiency creating a condition that favors other critical genetic events which drive bladder carcinogenesis, as evidenced by the significant decrease in tumor latency. Understanding the mechanisms of p53 LOH and chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of potential human carcinogens and the role of the p53 tumor suppressor gene in different pathways of chemical carcinogenesis.

Statistical analysis of skin tumor data from Tg.AC mouse bioassays.

New strategies for identifying chemical carcinogens and assessing risk have been proposed based on the Tg.AC (zetaglobin promoted v-Ha-ras) transgenic mouse. Preliminary studies suggest that the Tg. AC mouse bioassay may be an effective means of quickly evaluating the carcinogenic potential of a test agent. The skin of the Tg.AC mouse is genetically initiated, and the induction of epidermal papillomas in response to dermal or oral exposure to a chemical agent acts as a reporter phenotype of the activity of the test chemical. In Tg.AC mouse bioassays, the test agent is typically applied topically for up to 26 weeks, and the number of papillomas in the treated area is counted weekly. Statistical analyses are complicated by within-animal and serial dependency in the papilloma counts, survival differences between animals, and missing data. In this paper, we describe a statistical model for the analysis of skin tumor data from a Tg.AC mouse bioassay. The model separates effects on papilloma latency and multiplicity and accommodates important features of the data, including variability in expression of the transgene and dependency in the tumor counts. Methods are described for carcinogenicity testing and risk assessment. We illustrate our approach using data from a study of the effect of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on tumorigenesis.

Use of transgenic animals for carcinogenicity testing: considerations and implications for risk assessment.

Advances in genetic engineering have created opportunities for improved understanding of the molecular basis of carcinogenesis. Through selective introduction, activation, and inactivation of specific genes, investigators can produce mice of unique genotypes and phenotypes that afford insights into the events and mechanisms responsible for tumor formation. It has been suggested that such animals might be used for routine testing of chemicals to determine their carcinogenic potential because the animals may be mechanistically relevant for understanding and predicting the human response to exposure to the chemical being tested. Before transgenic and knockout mice can be used as an adjunct or alternative to the conventional 2-year rodent bioassay, information related to the animal line to be used, study design, and data analysis and interpretation must be carefully considered. Here, we identify and review such information relative to Tg.AC and rasH2 transgenic mice and p53+/- and XPA-/- knockout mice, all of which have been proposed for use in chemical carcinogenicity testing. In addition, the implications of findings of tumors in transgenic and knockout animals when exposed to chemicals is discussed in the context of human health risk assessment.

Responses of transgenic mouse lines p53(+/-) and Tg.AC to agents tested in conventional carcinogenicity bioassays.

The haplo-insufficient p53 knockout (p53+/-) and zetaglobin v-Ha-ras (Tg.AC) transgenic mouse models were compared to the conventional two rodent species carcinogen bioassay by prospectively testing nine chemicals. Seven of the chemicals classified as carcinogens in the conventional bioassay induced tumors in the liver or kidneys of B6C3F1 mice, and one (pentachlorophenol) also induced tumors in other tissues. Only three chemicals, furfuryl alcohol, pyridine, and pentachlorophenol, induced tumors in rats. The tumorigenic effect of pyridine was seen in F344 rats but not in Wistar strain rats. None of the chemicals induced tumors in the p53+/- transgenic mice, which is consistent with the absence of genotoxicity of these chemicals. Only two of the seven nongenotoxic carcinogens were positive in the Tg.AC model (lauric acid diethanolamine and pentachlorophenol). These results show that these transgenic models do not respond to many chemicals that show strain- or species-specific responses in conventional bioassays.

A farnesyl transferase inhibitor suppresses TPA-mediated skin tumor development without altering hyperplasia in the ras transgenic Tg.AC mouse.

The Tg.AC mouse carries an activated v-Ha-ras oncogene fused to an embryonic zeta-globin promoter and develops cutaneous papillomas in response to specific chemicals, full thickness wounding, and ultraviolet radiation. Papilloma development in these mice has been suggested to be dependent upon activation of ras transgene expression, thus providing a potential model for studying ras-inhibitory compounds. Farnesyl transferase inhibitors (FTIs) prevent a critical posttranslational modification step necessary for activation of ras proteins. Our studies demonstrated that a tricyclic FTI (SCH 56582) applied directly to the skin of homozygous Tg.AC mice 1 h prior to administration of the tumor promoter TPA decreased tumor multiplicity compared to TPA-only controls. In addition, a reduction of TPA-induced tumor development was seen in similarly treated hemizygous Tg.AC mice either on an FVB/N strain background or 50% C57BL/6. Histological examination of skin from Tg. AC(+/-):FVB/N mice revealed no differences with respect to 12-O-tetradecamoylpharbol-13-acetate (TPA)-mediated hyperplasia. Keratinocytes isolated from treated and control skin were assayed for ras transgene expression by reverse transcription-polymerase chain reaction, and expression was detected in both TPA- and FTI+TPA-treated tissue, although the appearance of transgene positive pre-papillomas was observed only in histological sections taken 21 d after the first treatment. In summary, we have used a regimen of topical application of an FTI (SCH 56582) to suppress TPA-mediated papillomagenesis in v-Ha-ras transgenic Tg.AC mice. These studies demonstrate that TPA-induced epidermal hyperplasia is a ras-independent process, while papilloma development in response to TPA treatment requires the function of activated ras.

Radial transformation-associated recombination cloning from the mouse genome: isolation of Tg.AC transgene with flanking DNAs.

Transformation-associated recombination (TAR) cloning allows entire genes and large chromosomal regions to be specifically, accurately, and quickly isolated from total genomic DNA. We report the first example of radial TAR cloning from the mouse genome. Tg.AC mice carry a zeta-globin promoter/v-Ha-ras transgene. Fluorescence in situ hybridization localized the transgene integrant as a single site proximal to the centromere of chromosome 11. Radial TAR cloning in yeast was utilized to create orientation-specific yeast artificial chromosomes (YACs) to explore the possibility that cis-flanking regions were involved in transgene expression. YACs containing variable lengths of 5' or 3' flanking chromosome 11 DNA and the Tg.AC transgene were specifically chosen, converted to bacterial artificial chromosomes (BACs), and assayed for their ability to promote transcription of the transgene following transfection into an FVB/N carcinoma cell line. A transgene-specific reverse transcription-polymerase chain reaction assay was utilized to examine RNA transcripts from stably transfected clones. All Tg.AC BACs expressed the transgene in this in vitro system. This report describes the cloning of the v-Ha-ras transgene and suggests that transcriptional activity may not require cis elements flanking the transgene's integration site.

Oral administration of dimethylvinyl chloride increases frequency of forestomach papillomas in Tg.AC mice.

This work was initiated to determine the potential for the Tg.AC mouse model to identify chemical carcinogens by an oral route of administration. Tg.AC v-Ha-ras transgenic mice were exposed to dimethyvinyl chloride (DMVC; 1-chloro-2-methylpropene), a structural analog of the human carcinogen vinyl chloride. In the National Toxicology Program 2-yr bioassay, DMVC induced tumors in the oral, nasal, and gastric epithelia of rats and mice. Initial studies were performed in female Tg.AC mice to determine an appropriate oral dose of DMVC to evaluate the potential for stratified gastric or oral epithelia of Tg.AC mice to serve as a target tissue for a transgene-dependent induced tumorigenic response. DMVC was administered to 13- to14-wk-old Tg.AC mice by gavage at doses of 0, 50, 100, and 200 mg/kg five times a week for 20 wk. The forestomachs of DMVC-treated Tg.AC mice had an increasing number of papillomas, which were associated with an increase in the dose of DMVC. The average numbers of papillomas per mouse per dose were 2.4, 7.6, 14.1, and 12.6 for the 0, 50, 100, and 200-mg/kg dose groups, respectively. The optimum papillomagenic dose of 100 mg/kg DMVC was established and administered for 5, 10, and 15/wk to investigate the kinetics of papilloma induction in Tg.AC mice. The average numbers of papillomas per animal were 1.8, 8.8, and 19.0 at 5, 10, and 15 wk, respectively. Reverse transcription-polymerase chain reaction assays determined that the v-Ha-ras transgene was transcriptionally active in all tumor tissues but not in nontumor tissues. In situ hybridization assays performed in conjunction with bromodeoxyuridine in vivo labeling localized the transgene-expressing cells of the forestomach papillomas to the proliferating cellular component of the tumors, as previously seen in skin papillomas of Tg.AC mice. The present results confirm that DMVC is tumorigenic and that oral routes of administration can be used to rapidly elicit a transgene-associated tumor response in the forestomach of Tg.AC mice.

Transponder-induced sarcoma in the heterozygous p53+/- mouse.

Heterozygous p53+/- transgenic mice are being studied for utility as a short-term alternative model to the 2-yr rodent carcinogenicity bioassay. During a 26-wk study to assess the potential carcinogenicity of oxymetholone using p-cresidine as a positive control, glass/polypropylene microchips (radio transponder identification devices) were subcutaneously implanted into male and female p53+/- mice. During week 15, the first palpable mass was clinically observed at an implant site. This rapidly growing mass virtually quadrupled in size by week 25. Microscopic examination of all implant sites revealed that 18 of 177 animals had a subcutaneous histologically malignant sarcoma. The neoplasms were characterized as undifferentiated sarcomas unrelated to drug treatment, as indicated by the relatively even distribution among dose groups, including controls. An unusual preneoplastic mesenchymal change characterized by the term "mesenchymal dysplasia" was present in most groups and was considered to be a prodromal change to sarcoma development. The tumors were observed to arise from dysplastic mesenchymal tissue that developed within the tissue capsule surrounding the transponder. The preneoplastic changes, including mesenchymal dysplasia, appeared to arise at the transponder's plastic anchoring barb and then progressed as a neoplasm to eventually surround the entire microchip. Capsule membrane endothelialization, inflammation, mesenchymal basophilia and dysplasia, and sarcoma were considered unequivocal preneoplastic/neoplastic responses to the transponder and were not related to treatment with either oxymetholone or p-cresidine.