Development and performance of npde for the evaluation of time-to-event models.

PURPOSE: Normalised prediction distribution errors (npde) are used to graphically and statistically evaluate mixed-effect models for continuous responses. In this study, our aim was to extend npde to time-to-event (TTE) models and evaluate their performance. METHODS: Let V denote a dataset with censored TTE observations. The null hypothesis (H0) is that observations in V can be described by model M. We extended npde to TTE models using imputations to take into account censoring. We then evaluated their performance in terms of type I error and power to detect model misspecifications for TTE data by means of a simulation study with different sample sizes. RESULTS: Type I error was found to be close to the expected 5% significance level for all sample sizes tested. The npde were able to detect misspecifications in the baseline hazard as well as in the link between the longitudinal variable and the survival function. The ability to detect model misspecifications increased as the difference in the shape of the survival function became more apparent. As expected, the power also increased as the sample size increased. Imputing the censored events tended to decrease the percentage of rejections. CONCLUSIONS: We have shown that npde can be readily extended to TTE data and that they perform well with an adequate type I error.

Exogenous Lipocalin 2 Ameliorates Acute Rejection in a Mouse Model of Renal Transplantation.

Lipocalin 2 (Lcn2) is rapidly produced by damaged nephron epithelia and is one of the most promising new markers of renal injury, delayed graft function and acute allograft rejection (AR); however, the functional importance of Lcn2 in renal transplantation is largely unknown. To understand the role of Lcn2 in renal AR, kidneys from Balb/c mice were transplanted into C57Bl/6 mice and vice versa and analyzed for morphological and physiological outcomes of AR at posttransplantation days 3, 5, and 7. The allografts showed a steady increase in intensity of interstitial infiltration, tubulitis and periarterial aggregation of lymphocytes associated with a substantial elevation in serum levels of creatinine, urea and Lcn2. Perioperative administration of recombinant Lcn2:siderophore:Fe complex (rLcn2) to recipients resulted in functional and morphological amelioration of the allograft at day 7 almost as efficiently as daily immunosuppression with cyclosporine A (CsA). No significant differences were observed in various donor-recipient combinations (C57Bl/6 wild-type and Lcn2(-/-) , Balb/c donors and recipients). Histochemical analyses of the allografts showed reduced cell death in recipients treated with rLcn2 or CsA. These results demonstrate that Lcn2 plays an important role in reducing the extent of kidney AR and indicate the therapeutic potential of Lcn2 in transplantation.

Overview of model-building strategies in population PK/PD analyses: 2002-2004 literature survey.

AIMS: A descriptive survey of published population pharmacokinetic and/or pharmacodynamic (PK/PD) analyses from 2002 to 2004 was conducted and an evaluation made of how model building was performed and reported. METHODS: We selected 324 articles in Pubmed using defined keywords. A data abstraction form (DAF) was then built comprising two parts: general characteristics including article identification, context of the analysis, description of clinical studies from which the data arose, and model building, including description of the processes of modelling. The papers were examined by two readers, who extracted the relevant information and transmitted it directly to a MySQL database, from which descriptive statistical analysis was performed. RESULTS: Most published papers concerned patients with severe pathology and therapeutic classes suffering from narrow therapeutic index and/or high PK/PD variability. Most of the time, modelling was performed for descriptive purposes, with rich rather than sparse data and using NONMEM software. PK and PD models were rarely complex (one or two compartments for PK; E(max) for PD models). Covariate testing was frequently performed and essentially based on the likelihood ratio test. Based on a minimal list of items that should systematically be found in a population PK-PD analysis, it was found that only 39% and 8.5% of the PK and PD analyses, respectively, published from 2002 to 2004 provided sufficient detail to support the model-building methodology. CONCLUSIONS: This survey allowed an efficient description of recent published population analyses, but also revealed deficiencies in reporting information on model building.

Precision-cut tissue slices from transgenic mice as an in vitro toxicology system.

In these experiments precision-cut tissue slices from two existing transgenic mouse strains, with transgenes that couple promoting or binding elements to a reporter protein, were used for determination of reporter induction. This approach combines the power of transgenic animals with the practicality of in vitro systems to investigate the biological impact of xenobiotics. Additionally, the normal cellular architecture and heterogeneity is retained in precision-cut tissue slices. Two transgenic mouse strains, one of which couples the promoting region of CYP 1A1 to beta-galactosidase, and another which couples two forward and two backward 12-O-tetradecanoyl phorbol-13-acetate (TPA) repeat elements (TRE) to luciferase (termed AP-1/luciferase), were used to determine the feasibility of this approach. Precision-cut kidney and liver slices from both transgenic strains remain viable as determined by slice K(+) ion content and LDH enzyme release. Liver slices harvested from the CYP 1A1/beta-galactosidase transgenic mice exhibit a 14-fold increase in beta-galactosidase activity when incubated with beta-napthoflavone for 24 h. Kidney and liver slices obtained from the AP-1/luciferase transgenic mice demonstrate induction of luciferase (up to 2.5-fold) when incubated with phorbol myristate acetate (PMA or TPA) up to 4 h. These data indicate that precision-cut tissue slices from transgenic mice offer a novel in vitro method for toxicity evaluation while maintaining normal cell heterogeneity.

Human liver quality is a dominant factor in the outcome of in vitro studies.

Donated human liver in the form of precision-cut tissue slices or isolated hepatocytes, is increasingly being used to predict metabolism and toxicity of xenobiotics in man. These tissue slices or hepatocytes can also be cold-preserved and cryopreserved to prolong their use for biological experiments. The viability of human liver could substantially affect the outcome of such experimentation. The goal of this investigation was to assess the viability of donated human livers, in the form of tissue slices, as they were received and to determine how varying degrees of liver quality affect experimental outcomes. Over one hundred human livers were categorized according to initial viability, as assessed by ATP content, K+ retention, protein synthesis, and LDH leakage. Each liver was placed in a low-, a medium-, or a high-quality group. The results showed that 76% of transplant-grade tissue (procured for transplantation) fell into the high-viability classification while the majority of research-grade tissue (not procured for transplantation) fell into the lowest viability classification. It was also found that only tissue slices prepared from highly viable human liver could be cold-preserved and cryopreserved. Dichlorobenzene metabolism was also greater in slices from highly viable human livers as compared to less viable livers. This study showed that human liver tissue acquired for medical research substantially varies in its viability and that these differences will affect the experimental data obtained.

A simplified isolated perfused rat liver apparatus: characterization and measurement of extraction ratios of selected compounds.

A simplified isolated perfused rat liver (IPRL) preparation has been developed and evaluated. The liver is briefly perfused in situ prior to being placed into a 37 degrees C oven and suspended from a stand. This set-up takes about 5 min. A non-recirculatory or one-pass perfusion approach has been used. The performance of the apparatus was evaluated with use of three model compounds: antipyrine, lidocaine and ethanol. In addition, oxygen extraction was determined. The steady-state extraction ratio (ER) was determined for each compound (and oxygen) as a function of perfusate flow rate (15-35 ml/min) during sequential 45 min perfusion periods. Perfusion experiments lasted for up to 3 hr. The ERs (at 15 ml/min) of ethanol (0.65 +/- 0.15), lidocaine (0.91 +/- 0.01) and oxygen (0.65 +/- 0.10) were dependent upon perfusate flow; whereas, antipyrine ER (0.07 +/- 0.01) was independent of flow. The corresponding values for unbound intrinsic clearances (CLu,int) for antipyrine, ethanol, lidocaine and oxygen were: 1.6, 31.0, 158.0 and 27.5 ml/min, respectively. These findings are consistent with the known hepatic ER values for those compounds reported in the literature.

Sulindac derivatives inhibit growth and induce apoptosis in human prostate cancer cell lines.

We examined the activity of two metabolites of sulindac (a nonsteroidal anti-inflammatory drug), sulindac sulfide and sulindac sulfone (exisulind, Prevatec), and a novel highly potent analog of exisulind (CP248) on a series of human prostate epithelial cell lines. Marked growth inhibition was seen with the BPH-1, LNCaP, and PC3 cell lines with IC50 values of about 66 microM, 137 microM, and 64 nM for sulindac sulfide, exisulind, and CP248, respectively. DNA flow cytometry and 4',6'-diamido-2-phenylindole (DAPI) staining indicated that these three compounds also induced apoptosis in all of these cell lines. Similar growth inhibition also was seen with the PrEC normal human prostate epithelial cell line, but these cells were resistant to induction of apoptosis at concentrations up to 300 microM, 1 mM, and 750 nM of sulindac sulfide, exisulind, and CP248, respectively. Derivatives of LNCaP cells that stably overexpress bcl-2 remained sensitive to growth inhibition and induction of apoptosis by these compounds. In vitro enzyme assays indicated that despite its high potency in inhibiting growth and inducing apoptosis, CP248, like exisulind, lacked cyclooxygenase (COX-1 and COX-2) inhibitory activity even at concentrations up to 10 mM. Moreover, despite variations of COX-1 and COX-2 expression, the three benign and malignant prostate cell lines showed similar sensitivity to growth inhibition and induction of apoptosis by these three compounds. Therefore, sulindac derivatives can cause growth inhibition and induce apoptosis in human prostate cancer cells by a COX-1 and -2 independent mechanism, and this occurs irrespective of androgen sensitivity or increased expression of bcl-2. These compounds may be useful in the prevention and treatment of human prostate cancer.

Benzo(a)pyrene-induced alterations in growth-related gene expression and signaling in precision-cut adult rat liver and kidney slices.

Benzo(a)pyrene (BaP) and related aromatic hydrocarbons are suspected carcinogens; however, the molecular basis underlying tumorigenesis remains unclear. To identify acute molecular targets of BaP within the liver and kidney, precision-cut slices harvested from naive, adult female Sprague-Dawley rats were challenged with BaP (0.3-30 microM) for 0.5 to 24 h. BaP did not elicit cytotoxicity, as assessed by intracellular K+ and ATP content and histological evaluation over the 24-h period. To determine if molecular signaling pathways were maintained in precision-cut slices, induction of the aryl hydrocarbon receptor (AhR) pathway was assessed following BaP challenge. Induction of cytochrome P450IA1 (P450IA1) mRNA and protein expression was observed in both liver and kidney slices. c-fos and c-Ha-ras gene expression was enhanced in liver, but not kidney, slices by BaP. c-jun mRNA levels were decreased in liver and kidney slices, although the effect was earlier (0.5 h) in liver slices compared to kidney slices. BaP increased the DNA binding of nuclear proteins to the AP-1 consensus recognition element in liver, but decreased DNA binding in kidney slices. In contrast, DNA binding of NF-kappa B was not affected by BaP in either liver or kidney slices. These results suggest that acute BaP challenge is associated with altered expression of several growth-related genes and AP-1 signaling and establish precision-cut slices as a useful in vitro system to investigate the molecular basis of BaP-induced tumorigenesis, including organ-specific differences.

Sampling designs for xerophthalmia prevalence surveys.

BACKGROUND: The purpose of this study was to estimate the bias and design effects associated with the Expanded Program on Immunization's (EPI) sampling design when estimating xerophthalmia prevalence, and to estimate the savings associated with EPI in terms of distance travelled within selected clusters. METHODS: Computer simulation of the EPI sampling strategy was done using maps from a xerophthalmia survey of 40 wards in Sarlahi district, Nepal. Samples of fixed cluster sizes of 7, 10, 15, 20 and 25 were compared. The estimated prevalence using the EPI design was compared with the true prevalence in the 40 wards to estimate the bias. The design effect was estimated by taking the ratio of the variance under EPI sampling to that of stratified random sampling (SRS) with fixed cluster sizes. The EPI was also modified by increasing the distance between selected houses from nearest neighbour to skipping 1-4 houses between selected ones. The difference between the distance travelled within clusters using SRS compared with EPI was weighed against the bias and increased variance. RESULTS: The prevalence of xerophthalmia was 2.8%. The EPI design overestimated xerophthalmia prevalence by between 0.27% and 1.16%. The design effects of EPI cluster sampling within wards varied between 0.73 and 1.35. Neither the bias nor the design effect was related to distance between households or cluster size. Distance travelled within wards was always less for EPI designs with cluster sizes of 7 or 10. There was no saving in terms of distance travelled for designs with cluster sizes from 15 to 25 if there were two or more houses between selected ones. For fixed cluster sizes of 15 or fewer, the EPI sampling design using nearest or next nearest neighbours is a better choice than SRS in terms of minimizing the distance travelled and the mean square error. CONCLUSIONS: The choice of an optimum method would need to account for the density of clusters and difficulty of travel between clusters, as well as the costs of travel within clusters. Based on certain assumptions, EPI with 15 children per cluster would be favoured over examining all children in selected wards unless the travel time between wards was more than 2 days.

Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac, have cancer chemopreventive properties by a mechanism that has been suggested to involve cyclooxygenase inhibition and reduction of prostaglandin (PGE2) levels in the target tissue. To test this hypothesis, we studied the effect of dietary sulindac sulfone (500-2000 ppm), a metabolite of sulindac reported to lack cyclooxygenase inhibitory activity, on tumor formation and PGE2 levels in the azoxymethane model of colon carcinogenesis. Rats treated with sulindac at 400 ppm and piroxicam at 150 ppm were used as positive controls. Rats received two s.c. injections of azoxymethane (15 mg/kg) for 2 weeks and were fed either experimental or control diets until necropsy. After 31 weeks of sulfone treatment, a dose-related increase in sulfone levels in both serum and cecal contents was measured; there was no evidence of metabolic conversion to sulindac or other metabolites. Rats treated with sulfone at 1000 and 2000 ppm, sulindac, and piroxicam had significantly fewer colonic adenomas and carcinomas compared with rats fed control diet as measured by tumor incidence, multiplicity, and tumor burden. Sulfone-treated rats also showed a dose-response relationship for inhibiting all tumor parameters. Colons from rats treated with sulindac or piroxicam contained PGE2 levels that ranged from approximately 16-49% of control levels. PGE2 levels in rats treated with sulfone up to 2000 ppm ranged from 78-118% of control levels. Moreover, the effects of sulindac sulfone on various enzymes responsible for regulating prostaglandin levels were evaluated. No significant inhibitory effects were observed for cyclooxygenase, lipoxygenase, or phospholipase A2. These results suggest that reduction of prostaglandin levels in the target tissue may not be necessary for the chemopreventive properties of sulindac.

Human and rat lung biotransformation of cyclosporin A and its derivatives using slices and bronchial epithelial cells.

Lung biotransformation of the immunosuppressants, cyclosporin A (CSA), the hydroxyethyl derivative SDZ IMM 125 (IMM), and the methylcarbonate derivative SDZ SCP 764 (SCP), was demonstrated in slices from human and rat. The major biotransformation pathway for CSA and IMM (0.1-10 microM) was hydroxylation at amino acid 1 to form AM1 or IMM1, while for SCP it was an esterase cleavage of the methylcarbonate group to form AM1 in both species. The initial rate (0-1 hr) of human total metabolite formation increased proportionally with substrate concentration. AM1 formation was five times greater from SCP, an esterase pathway, than CSA, an oxidative pathway which was inhibited (50%) by ketoconazole. At 24 hr human lung CSA metabolite formation was greater than IMM (3-fold) or SCP (2-fold), whereas rat lung and liver and human bronchial epithelial cell SCP metabolite formation generally exceeded CSA or IMM metabolism. CSA biotransformation is expected to occur throughout the human lung as demonstrated by the similar metabolite profile and extent of metabolism by slices derived from five different regions. The scaling of slice total metabolism to organ metabolism revealed that initially lung CSA metabolite formation would be equal to liver but with time liver metabolism would exceed lung for human and rat. This study has demonstrated that human and rat lung are metabolically active, exhibiting oxidative and esterase pathways toward cyclosporin derivatives. The lung will play an important role in this metabolism, particularly when administered via inhalation; however, the liver will also be a major organ involved in the total clearance of these compounds.

Efficiency of EPI cluster sampling for assessing diarrhoea and dysentery prevalence.

This study examines the efficiency of EPI cluster sampling in assessing the prevalence of diarrhoea and dysentery. A computer was used to simulate fieldwork carried out by a survey taker. The bias and variance of prevalence estimates obtained using EPI cluster sampling were compared with those obtained using simple random sampling and cluster (stratified random) sampling. Efficiency ratios, calculated as the mean square error divided by total distance travelled, were used to compare EPI cluster sampling to simple random sampling and standard cluster sampling. EPI cluster sampling may be an appropriate low-cost tool for monitoring trends in the prevalence of diarrhoea and dysentery over time. However, it should be used with caution when estimating the prevalence of diarrhoea at a single point in time because of the bias associated with this cluster sampling method.

PIP: Diarrhea is one of the major causes of morbidity and mortality among children in developing countries, and more than 12,000 people are estimated to die per day from diarrheal disease in such countries. Diarrhea has also been shown to affect child growth and is an important cause of malnutrition which can adversely affect child mortality. The infectious nature of diarrhea suggests that fecal contamination of water and food contributes to its transmission. That is, a contaminated source of water, if serving several families, can cause a cluster of illness. Simple random sampling and cluster sampling are discussed as examples of strategies which can be used to measure the prevalence of diarrhea and dysentery in a community. The authors investigated the cost of conducting World Health Organization (WHO) Expanded Program on Immunization (EPI) cluster sample surveys, expressed as the distance travelled to visit the sample subjects, and the bias and variance of the estimates derived with that design. The efficiency of EPI cluster sampling was specifically evaluated with regard to the assessment of the prevalence of diarrhea and dysentery. A computer was used to simulate fieldwork conducted by a survey taker. The bias and variance of prevalence estimates obtained using EPI cluster sampling were compared with those obtained using simple random sampling and cluster sampling. EPI cluster sampling was determined to be an appropriate low-cost tool with which to monitor trends in the prevalence of diarrhea and dysentery over time. It should, however, be used with caution when estimating the prevalence of diarrhea at a single point in time because of bias.

Vitamin A potentiation of vinylidene chloride hepatotoxicity in rats and precision-cut rat liver slices.

Pretreatment of large doses of vitamin A (VA) is known to potentiate the hepatotoxicity of carbon tetrachloride. Therefore the effects of 1-day VA pretreatment on VDC hepatotoxicity was examined both in vivo and in an in vitro system of precision-cut rat liver slices. Male Sprague-Dawley rats were pretreated with 250,000 IU/kg VA by oral gavage. After 24 hr rats were administered 50, 100, or 200 mg/kg VDC ip. Precision-cut liver slices were prepared from VA pretreated rats 24 hr later and the liver slices were exposed for 2-8 hr to 0.025-1.0 microliter VDC evaporated into the gas phase of the incubation vials. VA pretreatment resulted in an enhancement of VDC toxicity, both in vivo and in vitro. There was a dose-dependent increase in plasma ALT 24 hr after VDC treatment of rats and an increase in K+ leakage from liver slices after VDC exposure. Histological analysis of the liver or the liver slices revealed that VA + VDC treatment resulted in centrilobular necrosis of the liver. When GdCl3 (10 mg/kg iv) was administered just before VA pretreatment of rats, VDC toxicity was partially reversed as observed by a decrease in ALT in vivo and a decrease in the loss of K+ in vitro. These results indicated that Kupffer cells, the resident macrophages of the liver, were partially responsible for the VA-potentiated VDC hepatotoxicity. One-day pretreatment of VA induced cytochrome P450IIE1 protein content as well as its enzymatic activity as measured by pnitrophenol hydroxylation. Because VDC is bioactivated by cytochrome P450IIE1, the increase in VDC hepatotoxicity after VA may be due to an increased bioactivation of VDC in the liver and in precision-cut liver slices. Thus, more than one mechanism may be involved in the VA enhancement of VDC hepatotoxicity.

Protein arylation precedes acetaminophen toxicity in a dynamic organ slice culture of mouse kidney.

Acetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals. In vivo studies suggest that in situ activation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution of in situ activation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0-25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhydryls (NPSH), ATP content, and K+ retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier, in vivo results and demonstrates the importance of in situ activation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity.

Increased resistance to acetaminophen-induced hepatotoxicity in retrovirus-infected mice.

In this research we examined the influence of chronic retrovirus infection on the hepatic metabolism of a model substrate, acetaminophen (APAP), and its induced liver injury in mice inoculated with LP-BM5 murine leukemia viruses. Female C57BL/6 mice at 15-17 wks after LP-BM5 retrovirus inoculation and age-matched control animals were used in the studies. APAP treatment (300 mg kg-1, p.o.) resulted in moderate to severe centrilobular necrosis in control animals, with the necrotic area accounting for 40-60% of the total area. In contrast, the APAP-treated (300 mg kg-1, p.o.) infected animals exhibited mild zonal necrosis with the necrotic area accounting for 1-6% of the total area. In the same study, a statistically significant higher percentage of APAP glucuronide and a lower percentage of unchanged APAP were recovered from the urine of the LP-BM5-inoculated animals than from that of controls. No statistically significant differences between infected and uninfected animals in the urinary recovery of APAP sulfate, APAP cysteine, or APAP mercapturate were observed. The formation of APAP metabolites and APAP-associated biochemical changes were also determined from liver slice preparation to avoid in vivo complicating factors. Consistently more significant depletion of the intracellular glutathione levels and K+ content were observed in slices from the uninfected animals at high concentrations of APAP (1 and 2 mM) than in slices from the retrovirus-infected animals. The differences in APAP-associated biochemical changes were accompanied by a 1.4-1.5-fold increase in the formation of APAP glucuronide, sulfate, and glutathione metabolites in slices prepared from animals inoculated with LP-BM5. We concluded that, based on histological examination and hepatic biochemical measurements, the retrovirus-infected animals were more resistant to APAP-induced liver injury. This could be due, in part, to alterations in the detoxification and activation metabolic pathways of APAP.

Cold- and cryopreservation of dog liver and kidney slices.

The use of tissue slices in culture could decrease the number of animals used in health-related research and decrease experimental variation. This reduction may come about particularly if the methods of cold- and cryopreserving tissue slices are perfected, and one can conduct sequential in vitro experiments into xenobiotic metabolism, organ-specific toxicity, or organ-specific biochemical processes with tissue slices. With this goal in mind, dog liver and kidney slices were placed in cold storage at 0 degrees C using Viaspan (UW), Euro-Collins (EC), Sacks + prostacyclin (SP), and V-7 (V7) cold-preservation solutions for 10 days. Viability was assessed each day by measuring K+ content and protein synthesis after 4 h of incubation in Waymouth + 10% fetal calf serum (FCS). Dog liver slices can be cold-preserved in V7 for up to 7 days using K+ retention as the viability criterion but only up to 4 days using protein synthesis. Dog kidney slices can be cold-preserved in UW, EC, and V7 for up to 10 days using K+ retention, but only V7 could maintain protein synthesis for 10 days. Cryopreserved dog liver and kidney slices retained 63-68% of control viability after 4 h of incubation in FCS. The cryopreservation regimen included using 10% dimethyl sulfoxide in FCS as the cryoprotectant, a freezing rate of 0.5 degrees C/min for liver slices and 12 degrees C/min for kidney slices, and thawing in 37 degrees C FCS. Continued development of cold- and cryopreserving tissue slices could reduce the numbers of animals used and provide accurate and reproducible data.