Gene expression analysis of precision-cut human liver slices indicates stable expression of ADME-Tox related genes.

In the process of drug development it is of high importance to test the safety of new drugs with predictive value for human toxicity. A promising approach of toxicity testing is based on shifts in gene expression profiling of the liver. Toxicity screening based on animal liver cells cannot be directly extrapolated to humans due to species differences. The aim of this study was to evaluate precision-cut human liver slices as in vitro method for the prediction of human specific toxicity by toxicogenomics. The liver slices contain all cell types of the liver in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process. Previously we showed that toxicogenomic analysis of rat liver slices is highly predictive for rat in vivo toxicity. In this study we investigated the levels of gene expression during incubation up to 24 h with Affymetrix microarray technology. The analysis was focused on a broad spectrum of genes related to stress and toxicity, and on genes encoding for phase-I, -II and -III metabolizing enzymes and transporters. Observed changes in gene expression were associated with cytoskeleton remodeling, extracellular matrix and cell adhesion, but for the ADME-Tox related genes only minor changes were observed. PCA analysis showed that changes in gene expression were not associated with age, sex or source of the human livers. Slices treated with acetaminophen showed patterns of gene expression related to its toxicity. These results indicate that precision-cut human liver slices are relatively stable during 24h of incubation and represent a valuable model for human in vitro hepatotoxicity testing despite the human inter-individual variability.

The human transcriptome map: clustering of highly expressed genes in chromosomal domains.

The chromosomal position of human genes is rapidly being established. We integrated these mapping data with genome-wide messenger RNA expression profiles as provided by SAGE (serial analysis of gene expression). Over 2.45 million SAGE transcript tags, including 160,000 tags of neuroblastomas, are presently known for 12 tissue types. We developed algorithms to assign these tags to UniGene clusters and their chromosomal position. The resulting Human Transcriptome Map generates gene expression profiles for any chromosomal region in 12 normal and pathologic tissue types. The map reveals a clustering of highly expressed genes to specific chromosomal regions. It provides a tool to search for genes that are overexpressed or silenced in cancer.

Palliative chemotherapy or watchful waiting? A vignettes study among oncologists.

PURPOSE: To determine the preferences of oncologists for palliative chemotherapy or watchful waiting and the factors considered important to that preference. METHODS: Sixteen vignettes (paper case descriptions), varying on eight patient and treatment characteristics, were designed to assess the oncologists' preferences. Their strength of preference was rated on a 7-point scale. An orthogonal main effects design provided a subset of all possible combinations of the characteristics, allowing estimations of the relative weights of the presented characteristics. A written questionnaire was sent to a random sample of oncologists (N = 1,235). RESULTS: The response rate was 67%, and 697 questionnaires were available for analysis. Eighty-one percent of the respondents were male. The mean age was 46 years. We found considerable variation among the oncologists. No major associations between physician characteristics and preferences were found. Of the patient and treatment characteristics affecting treatment preference, age was the strongest predictor, followed by the patient's wish to be treated and the expected survival gain. Other patient and treatment characteristics had a limited effect on preferences, except for psychologic distress, which had no independent impact. CONCLUSION: Patients will encounter different decisions depending on their oncologists' preferences and their own personal background. Therefore, to ensure adequate information for decision-making processes, decision aids are proposed.

CD44 expression predicts disease outcome in localized large B cell lymphoma.

Diffuse large B cell non-Hodgkin's lymphomas (DLCL) form a heterogeneous group of tumors with diverse morphology, clinical features, treatment response and prognosis. The biological variables underlying this heterogeneity are unknown. In the present study, we explored the value of the lymphocyte homing receptor CD44, a putative determinant of lymphoma dissemination, in predicting prognosis in DLCL. Expression of the standard form of CD44 (CD44s) and of CD44 isoforms containing exon v6 (CD44v6) on tumor cells was assessed by immunohistochemistry in a cohort of 276 DLCL patients from a population based lymphoma registry. We observed that CD44s as well as CD44v6 expression correlated with tumor dissemination in patients with primary nodal DLCL. Importantly, in patients with localized nodal disease, CD44s was a strong prognosticator predicting tumor related death independent of the other parameters of the International Prognostic Index (IPI). Incorporation of CD44s in the IPI parameter 'stage', increased the prognostic value of this parameter in nodal DLCL. Our data identify CD44 as a biological prognosticator, which can be used to 'fine-tune' the IPI for nodal DLCL.

Mixed plaques: statistical evidence how plaque assays may underestimate virus concentrations.

Even at very low concentrations human pathogenic viruses may result in infection and possibly subsequent disease. Ideally, viruses are quantified by use of cell culture assays to determine their infectivity. Plaque assays are common tools for enumeration of viruses in inocula and this process is straightforward when a plaque results from the offspring of a single infectious virus particle. In the course of a study on the usefulness of sewage monitoring for surveillance of polio-virus transmission, sewage samples containing a mixture of two live polio vaccine strains (type 1 and type 3) were analyzed. The total poliovirus concentration in plaque forming units (pfu) was estimated by means of a monolayer plaque assay on L20B cells. Subsequent typing of virus directly by neutralisation of virus from excised plaques revealed the occurrence of plaques containing both type 1 and type 3 virus. This means that there must be plaques that originate from more than one initial infectious virus particle. As a consequence, the estimated virus concentration is incorrect. We present statistical methods that utilize these mixed plaque counts to estimate the concentrations of either virus type in our sewage samples. We can also calculate a correction factor for the error in virus concentration, which would result from equating a pfu to a single infectious particle. Since many quantitative methods in microbiology are based on colony counts, we conclude that such counts should be interpreted with caution, especially when data are used in quantitative microbial risk assessment to estimate the public health impact.

Reconstruction and prediction of the HIV/AIDS epidemic among adults in the European Union and in the low prevalence countries of central and eastern Europe.

OBJECTIVES: To reconstruct the HIV epidemic and to provide forecasts of AIDS incidence among adults in the European Union (EU) and in a group of low prevalence (LP) countries of central and eastern Europe (including the Asian republics of the former Soviet Union). METHODS: An empirical Bayesian back-calculation method was applied to AIDS incidence data reported by 31 March 1994. The HIV-infection curve was modelled as a yearly step function and a seven-stage Markov model of disease progression, incorporating effects of pre-AIDS treatment, was used. Estimation was by penalized maximum likelihood with empirical Bayesian smoothing. Data were analysed by transmission group and, within the EU, by country. Predictions of AIDS cases to 1998 were made assuming constant annual HIV incidence from 1993 onwards. RESULTS: Estimated HIV prevalences per 100,000 population aged 15-59 years were, at 31 December 1993, 198 (n = 447,800) in the EU and 2.7 (n = 6840) in the 22 LP countries, with increases of 41% (EU) and 71% (LP) between 1989 and 1993. Among homo/bisexual men in the EU, prevalence appears to have stabilized since 1989 and AIDS incidence appears to be reaching a peak. Among all prevalent HIV infections in the EU, 42% were estimated to be among injecting drug users, 25% among homo-/bisexual men and 18% among persons infected heterosexually, compared with 29%, 35% and 19%, respectively, in the LP countries. Without allowing for the 1993 revision of the case definition, annual AIDS incidence is predicted to increase, between 1994 and 1998, by 24% in the EU and by 48% in the LP countries, with the largest percentage increases among heterosexually-infected persons. CONCLUSION: The overall HIV prevalence rate is estimated to have been about 70-fold lower in the LP countries than in the EU in the early 1990s, but to be increasing much more rapidly in the former. Moreover, recent reports of rapidly increasing HIV infection rates suggest that back-calculation may seriously underestimate the size of the epidemic in the LP countries. Implementation of effective preventive measures is urgent if large-scale epidemics are to be avoided in the presently LP countries of the European region.

Back-calculation by birth cohort, incorporating age- specific disease progression, pre-AIDS mortality and change in European AIDS case definition. European Union Concerted Action on Multinational AIDS Scenarios.

OBJECTIVES: To adapt and improve methodology for back-calculation of AIDS in Europe and to examine the feasibility of estimating past HIV incidence by birth cohort. METHODS: Empirical Bayesian back-calculation (EBBC) used Markov disease progression models, modified to allow for three diseases added to the AIDS case definition in 1993 and for pre-AIDS mortality, and estimation by penalized maximum likelihood with a neighbour prior. EBBC by 5-year birth cohort assumed a minimum age at infection and age-dependent progression rates; three versions, with varying age effects, were investigated using AIDS cases diagnosed prior to the introduction of highly active antiretroviral therapies (HAART). RESULTS: Compared with the no age-effect version, EBBC by birth cohort tended to produce flattened HIV incidence curves in country-exposure groups with < 1000 AIDS cases, reflecting effects of the neighbour prior when data become sparse. Otherwise, birth cohort analysis, with moderate effects of age on progression, gave initially increasing incidence curves and consistent patterns across countries, with the 1960-1964 cohort most affected. In the European Union, incidence is estimated to have peaked in 1983 among homosexual men and in 1988 among injecting drug users; 460000 persons were estimated to be living with HIV/AIDS at the end of 1995. CONCLUSIONS: Our improved methodology deals effectively with the change in AIDS case definition and has allowed quantitative assessments of the HIV epidemic by birth cohort using all AIDS cases diagnosed before 1996, thus providing a sound basis for public health policy at a time when estimation of more recent prevalence is compromised by the effects of HAART.

The treatment-free incubation period of AIDS in a cohort of homosexual men.

OBJECTIVE: The treatment-free incubation period distribution was estimated from data obtained from a cohort of homo/bisexual men in Amsterdam. DESIGN: Participants in a cohort study that started in Amsterdam at the end of 1984 were seen at 3- or 6-monthly intervals. The analysis excluded time since the start of zidovudine treatment (given to 27 individuals) and no cohort member received primary Pneumocystis carinii pneumonia prophylaxis (i.e., before AIDS diagnosis) before February 1990. METHODS: Both HIV-positive (n = 269) and HIV-seroconverted (n = 79) homosexual men were included in this study. Of these, 68 developed AIDS before February 1990. The unobserved dates of seroconversion were obtained by multiple imputation. The incubation period distribution was estimated from these data by direct Kaplan-Meier analysis and by using parametric Weibull and gamma distributions (including a parameter describing the probability of never progressing to AIDS). Our results are compared with published estimates of progression to AIDS from other homo/bisexual cohort studies. RESULTS: Both the Weibull and the gamma distributions provide equally good empirical descriptions of the incubation period distribution for up to 7 years postseroconversion, but the estimated gamma distribution (median, 9.2; mean, 10.2; percentage AIDS at 7 years, 33%) should be preferred beyond that time, due to a slowing of the hazard rate. There is insufficient information to be able to estimate accurately the probability of never progressing to AIDS. CONCLUSIONS: The time-dependent pattern of HIV incidence should be considered in the analysis of prevalent cohort studies. Our results are in agreement with other homo/bisexual cohort studies, and will be valuable for future comparison with and understanding of the epidemiological consequences of clinical treatment that delays the onset of AIDS.

Comparison of three methods to assess therapeutic quality control of treatment with vitamin K antagonists.

During treatment with vitamin K antagonists, International Normalized Ratios (INR) are determined periodically to maintain a therapeutic level of anticoagulation. We evaluated two existing methods for therapeutic quality control (linear interpolation and equidivision), with regard to their validity and reproducibility. In addition, we proposed and evaluated a (hybrid) method that takes into account potential effects of dosage modifications when INRs are far out of the target range. Validity was assessed by deleting intermediary INR results and estimating this INR based on the two surrounding INRs with each of the three methods. The estimated INRs were then compared with the observed INR. Reproducibility of time spent in an INR range was evaluated for each of the three methods by deleting at random increasing proportions of INRs and comparing these estimates with the situation without deletions. We found that estimates of time spent in INR categories obtained with equidivision were most reproducible, but least valid. The hybrid method showed slightly higher validity and reproducibility in comparison with linear interpolation. Since these differences were small, linear interpolation is preferable to the hybrid method, since the calculations involved are easier.

Evidence for VIP(1)/PACAP receptors in the afferent pathway mediating surgery-induced fundic relaxation in the rat.

We previously reported activation of an inhibitory adrenergic and a non-adrenergic non-cholinergic (NANC) pathway during abdominal surgery relaxing the rat gastric fundus. In the present study, we investigated the possible role of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) in the NANC part of the surgery-induced fundic relaxation. The effect of the NO biosynthesis inhibitor N(G)-nitro-L-arginine (L-NOARG), the non-selective VIP receptor antagonist [D-p-Cl-Phe(6),Leu(17)]-VIP and the selective VIP(1) receptor antagonist [Acetyl-His(1),D-Phe(2),Lys(15),Arg(16), Leu(17)]-VIP was investigated on the non-adrenergic fundic relaxation induced by manipulation of the small intestine followed by resection of the caecum. Guanethidine partly reduced the manipulation-induced fundic relaxation. Addition of L-NOARG reduced this non-adrenergic component, whereas the non-selective VIP receptor antagonist had no significant effect. Combination of L-NOARG and the non-selective VIP antagonist however further reduced the relaxation to manipulation. The selective VIP(1) receptor antagonist reduced the mean and maximal relaxation induced by abdominal surgery in the presence of guanethidine. When combined with L-NOARG, the relaxation of the gastric fundus was almost completely abolished. The VIP(1) receptor antagonist alone had no significant effect on the mean and maximal relaxation, but enhanced recovery of fundic tone. In conclusion, as VIP(1) receptors are not present in the rat gastric fundus, these results suggest that the NANC inhibitory pathway activated during abdominal surgery involves VIP(1) receptors, most likely in the afferent limb. The inhibitory neurotransmitters released at the level of the gastric fundus smooth muscle are NO and a substance different from VIP.

Neuroendocrine reactivity and recovery from work with different physical and mental demands.

OBJECTIVES: The purpose of this study was to examine the extent to which the type or nature (physical, mental or mixed mental and physical) of work and work characteristics is related to the course of neuroendocrine reactivity and recovery from work. METHODS: Neuroendocrine reactivity and recovery were studied by measuring the urinary excretion of adrenaline, noradrenaline, and cortisol during and after 3 workdays, 1 consecutive day off, and a baseline day. The assessment was made in 3 groups of Dutch male workers (N=60) who differed in the nature (mental, physical, and combined mental and physical demands) of their work. Multilevel analyses were performed to fit linear mixed-effects models for each hormone. RESULTS: Main or interaction effects with time of day were found between the workers in combined mental and physical work and the 2 other groups of workers for cortisol, adrenaline, and noradrenaline excretion. In addition, the baseline levels of the 3 hormones were higher in the workers with combined mental and physical work when compared with the other 2 groups. The excretion rates during the workdays were higher than those on the day off, but a trend towards mobilizing less activity was found from the 1st to the 3rd workday. Job demands were negatively related to cortisol excretion. Job control and social demands at work did not affect the excretion rates of the hormones. CONCLUSIONS: Unfavorable effects on cortisol and adrenaline reactivity or recovery was found for workers with combined mental and physical demands when compared with workers doing mainly mental or mainly physical work. The results of the present study are in accordance with the cognitive activation theory and the allostatic load model.

[Analysis of the AIDS epidemic in The Netherlands, 1982-1993]. / Analyse van de AIDS-epidemie in Nederland, 1982-1993.

OBJECTIVE: Description of the epidemiology and transmission categories of AIDS in the Netherlands. DESIGN: Descriptive. SETTING: The Netherlands. METHOD: Analysis of all registered AIDS patients until 31 December 1993. Trends in the composition of this population were studied with respect to age and sex, risk groups, geographic distribution across the country, heterosexual transmission, AIDS-defining diseases and reporting pattern. RESULTS: From the first patient in 1982 until December 31, 1993, a cumulative total of 2912 patients was diagnosed and reported in the Netherlands (2995 when corrected for reporting delay). The numbers of reported AIDS cases in the Netherlands are smaller than previously predicted by mathematical models. The proportion of homosexual men in the incidence of AIDS dropped from 89 to 73 per cent, the proportions of intravenous drug users and heterosexual transmission rose to 11 per cent each. Patients in the category of heterosexual transmission are mainly individuals from countries where heterosexual contact is the dominant mode of transmission and their sex partners, and to a lesser extent the sex partners of intravenous drug users (whether or not in relation to prostitution). The proportion of women is rising (229 patients or 8 per cent by December 1993), with most cases transmitted initially by intravenous drug use but later by heterosexual contact. CONCLUSION: The number of AIDS cases in all risk groups combined is levelling off. However, more detailed analysis shows that the numbers of cases of heterosexual transmission and those in young homosexual men are still rising. For a better quantitation of the quality of the AIDS data, specific research into underreporting and non-diagnosis of AIDS cases in the Netherlands is warranted.

[Practice of systematic reviews. VII. Pooling of results from observational studies]. / De praktijk van systematische reviews. VII. Het combineren van de resultaten van observationele onderzoeken.

Knowledge concerning aetiologic hypotheses can be obtained through systematic reviews of observational studies. In observational studies, heterogeneity between studies is expected, because of differences in study design and analysis. Data from observational studies have to be made comparable before pooling of results from component studies is possible. In this, definition of exposure and disease and adjustment for confounding have to be taken into account. Three methods to deal with heterogeneity are presented: ignoring (fixed effects model), modelling (random effects model) and exploring (meta regression). Any combination of data in meta-analysis of observational studies has to be based on statistical, methodological and clinical considerations. The same considerations are applied for the choice which method is used.

Estimation and prediction of the HIV-AIDS-epidemic under conditions of HAART using mixtures of incubation time distributions.

The estimation of the HIV-AIDS epidemic by means of back-calculation (BC) has been difficult since the introduction of highly active anti-retroviral therapy (HAART) because the incubation time distributions needed for BC were poorly known. Moreover, it has been assumed that if the general public is aware that effective treatments are available then the majority of infected people would be known, and therefore a hidden epidemic was assumed not to exist. Nevertheless, it was suspected that not every infected person would come to the attention of health-care providers, and therefore estimates independent of the patients' registration were necessary. In this paper, the incubation time distributions for HIV treated with the HAART regimen are derived from a cohort study. By using estimates of the proportion treated according to the HAART regimen and the incubation time distributions estimated in the era before the implementation of HAART (pre-HAART), new marginal population incubation time distributions for each of the three risk groups (homosexuals, drug users and others) were constructed. The BC was performed using an empirical Bayesian approach based on the latter incubation time distribution.

Dose dependency of adjuvant activity of particulate matter from five European sites in three seasons in an ovalbumin-mouse model.

Various particulate matter (PM) samples were tested for their adjuvant potency in an animal model of allergy (ovalbumin) in the European Union study entitled Respiratory Allergy and Inflammation Due to Ambient Particles. Coarse and fine ambient particles were collected during spring, summer, and winter in Rome, Oslo, Lodz, Amsterdam, and De Zilk. De Zilk, at the Dutch seaside, has mainly westerly winds and served as a negative pollution control. EHC-93 (Ottawa dust) was used as a positive control. We studied the adjuvant potency of the particle antibody responses to ovalbumin and histopathological changes in the lung. After a sensitization phase by coexposure to EHC-93 and ovalbumin, the antibody response to ovalbumin and inflammatory responses in the lung were huge. There was more adjuvant activity in reaction to 9-mg/ml samples than to 3-mg/ml samples. A best-fit analysis of these samples shows that the ambient coarse and fine particles at these sites, in combination with allergens, have severe to mild adjuvant activity in the order Lodz, Rome, Oslo, and Amsterdam. A high dose of the fine fraction was more potent than a high dose of the coarse fraction, except at De Zilk, where the reverse was true. Spring and winter PM was more potent than summer PM. Depending on the site, either a water-soluble or a water-insoluble fraction was responsible for the adjuvant activity. A concentration of 3 mg/ml is effective for screening high-activity samples, as is a concentration of 9 mg/ml for screening low-activity samples in the ovalbumin-mouse model.

Disease mapping using empirical Bayes and Bayes methods on mortality statistics in The Netherlands.

Data from the 64 public health service districts in the Netherlands, describing the health status of the Dutch population, were used to construct maps for several causes of deaths. The choice and estimation of the relative risk measure is described. The prior expected number of deaths was modelled using a Poisson regression approach based on a model with main effects of district and age. Desirable properties of risk parameters for disease mapping are that they both reflect the level of risk and cope with the instability in the observed measure caused by the numbers at risk in each district. Different techniques for estimation of parameters were applied: empirical Bayes estimation (EB) using a nonparametric prior and a gamma prior, and a Bayesian approach (B) with a uniform prior. For the parametric EB also a constrained estimator was used. The EB techniques studied in this paper shift or smooth the values of the risk parameter towards a global mean. In the Bayesian method applied here, spatial dependence among districts can be modelled, that is the estimates are smoothed towards a local mean. The three EB estimates gave by and large similar results, although the constrained EB estimate smoothed less, as was expected. The Bayesian estimates smoothed the estimates more or less similarly to the constrained EB.

Assessing health impact of environmental pollution sources using space-time models.

We used disease mapping for health impact assessment of the national airport of the Netherlands. Spatio-temporal models were used to relate hospital discharge data for acute myocardial infarction and bronchitis in 1991, 1992 and 1993 to noise and distance from the airport. To compare models a discrepancy measure (expected predictive deviance) proposed by Carlin and Louis was used. The best fitting model was the most general one with inclusion of spatial and temporal components. Results on the effects of the covariates noise and distance from the airport were somewhat inconsistent between men and women and between the two diseases: for women no association between bronchitis and distance from the airport was found, whereas for men no association between acute myocardial infarction and noise was found.

EC-collaborative study on the determination of aflatoxin B1 in animal feeding stuffs.

A collaborative study was conducted to test a method, proposed in the European Community (EC) as a candidate-official method for the determination of aflatoxin B1 in compounded feeding stuffs. The study was undertaken on behalf of the European Commission's Community Bureau of Reference (BCR). It involved 25 laboratories from 11 EC countries. The method, based on chloroform extraction and Sep-Pak Florisil and C18 cartridge clean-up, offered either reverse-phase high performance liquid chromatography (HPLC) with iodine post-column derivatization, or two-dimensional thin-layer chromatography (TLC) as determinative steps. In the study, 22 laboratories applied HPLC, three laboratories applied TLC. The study involved six unknown samples. These consisted of blind duplicate samples of compounded feeding stuff, with target concentrations of aflatoxin B1 at less than 2, 8 and and 14 micrograms/kg. Statistical analysis of the HPLC data was carried out according to ISO 5725. For the less than 2 micrograms/kg sample, all reported aflatoxin concentrations were less than 2 micrograms/kg. At the 8 and 14 micrograms/kg level (pooled), repeatability (r) and reproducibility (R), expressed as ratios, were 1.4 and 1.7 respectively, and within-laboratory and between-laboratory coefficients of variation were 11% and 18% respectively. The study revealed that admittance of daylight in the laboratory caused losses of aflatoxin B1 and must be avoided. New glassware coming into contact with aqueous solutions containing aflatoxin B1 was found to be a potential cause of loss of aflatoxin B1. The method has been recommended to the European Commission to be considered for adoption in EC regulations.