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Objectives Paper dust has previously been linked to adverse health effects. However, a comprehensive dataset of paper dust exposures does not appear to have been published previously. Our study was intended to address this need by describing a large dataset of measurements made in Swedish soft tissue paper mills. Methods We compiled personal and area total dust exposure measurements collected from a range of operations by our research staff at four soft tissue paper mills in Sweden. We also compiled measurements made by the occupational health staff at each mill and the Swedish Work Environment Authority. We analyzed these measurements to describe patterns and trends in exposures and used mixed-effects regression models to identify measurement characteristics that predicted exposure levels. Results We compiled 1578 measurements from 1971 to 2009, of which 1026 (65%) were personal samples. Statistically significant differences were found between measurements made by research, mill, and Swedish Work Environment Authority staff, as well as between personal and area measurements. The measurement data suggest that, beginning in the 1980s, exposures declined at three of the four mills, but that overexposures were still common at the end of the period. Papermaking and converting operations had the highest observed dust exposures. One mill had significantly lower exposures than the others. Type of measurement (personal versus area) and source of measurement (research staff, company, or regulatory agency) were not significant predictors of measured total dust exposure after controlling for mill, operation, and time. Conclusions Our analysis of measured paper dust exposures may be useful for historical and contemporary exposure assessment in our own and other epidemiological studies. We have identified specific characteristics (i.e. papermaking operations and mill) and time trends that are important data features to consider, and documented continuing overexposure situations. Our results highlight the ongoing need for application of exposure controls to reduce paper dust exposures in the soft tissue paper industry.

Objectives The aim of this study was to characterize personal occupational exposure to endotoxin in size-separated airborne particles of MWF aerosol, using a Sioutas cascade impactor (SCI). Methods Exposure to inhalable fractions of MWF aerosol and endotoxin was measured by personal sampling of 52 individuals over an 8-h work shift using a PAS-6 sampler in parallel with a SCI ( Results A total of 117 measurements of inhalable MWF aerosols were made among 52 workers. The geometric mean of inhalable MWF aerosol was 0.16 mg m−3 air. The geometric mean of endotoxin concentration on the inhalable sampler was 0.15 EU m−3. Airborne endotoxin was found on all size fractions from the impactor, with the major part seen in the fraction (2.5–10 µm). There was a correlation between the inhalable fraction of endotoxin measured by the PAS-6 sampler and on the SCI sampler (2.5–10 µm), estimated to be 0.51 for all samples (P < 0.0001). The concentration of endotoxin varied between the MWFs, as did the proportion of Gram-negative bacteria among the culturable bacteria (>80% in one MWF and Conclusions The personal exposure to inhalable fractions of endotoxin contained in the MWF aerosol were low, where most of the endotoxin were found in fraction (2.5–10 µm), measured by SCI. There are differences between factories and MWF systems regarding the distribution of endotoxin and so results from one context should not be generalized to other plants and systems. Compressed air was used for less than 10 min shift−1. The mixed-effect model showed that working with open machines and grinding as cutting task were important determinants of exposure to inhalable aerosol. It is important to keep occupational exposure to aerosols low with the help of good ventilation systems, enclosed machines, and organization of work.

INTRODUCTION:Particles in exhaled air (PEx) provide samples of respiratory tract lining fluid from small airways containing, for example, Surfactant protein A (SP-A) and albumin, potential biomarkers of small airway disease. We hypothesized that there are differences between morning, noon, and afternoon measurements and that the variability of repeated measurements is larger between days than within days. METHODS:PEx was obtained in sixteen healthy non-smoking adults on 11 occasions, within one day and between days. SP-A and albumin were quantified by ELISA. The coefficient of repeatability (CR), intraclass correlation coefficient (ICC), and coefficient of variation (CV) were used to assess the variation of repeated measurements. RESULTS:SP-A and albumin increased significantly from morning towards the noon and afternoon by 13% and 25% on average, respectively, whereas PEx number concentration and particle mean mass did not differ significantly between the morning, noon and afternoon. Between-day CRs were not larger than within-day CRs. CONCLUSIONS:Time of the day influences the contents of SP-A and albumin in exhaled particles. The variation of repeated measurements was rather high but was not influenced by the time intervals between measurements.

Surfactant protein A (SP-A) is considered a potential biomarker of small airway inflammation. In the present study we aimed to assess within- and between-day variation of SP-A and albumin in exhaled particles (PEx) and define the expected maximal normal variation in healthy individuals. 16 healthy non-smoking subjects were examined with PExA®, in the morning, noon and afternoon during three non-consecutive days. SP-A and albumin were quantified by ELISA and expressed as weight % in PEx (wt%). The differences on the same time points between different days were assessed and the coefficient of repeatability (CR) and 95% CI of limits of agreement (LoA) was calculated. Diurnal variation was tested with repeated two-way ANOVA. The variation of SP-A between days was low and non-significant (p = 0.75), whereas albumin in PEx varied more (p Our findings show that there is a rather small diurnal variation of SP-A in PEx. We also describe the maximal normal variation of these biomarkers over time, which may be of importance in clinical settings, when assessing the effect of exposure challenge and medical intervention.

Background Total paper dust exposure has been associated with respiratory problems among workers in the soft tissue paper industry. However, a comprehensive job exposure matrix (JEM) has not been developed for application to this industry. Our study was intended to address this need and to support further studies of mortality and morbidity in a cohort of Swedish workers from this industry. Methods We evaluated four participating soft tissue paper mills in Sweden. We combined information on process and equipment status from the mills with knowledge of the mills obtained through research efforts and paper dust measurements made at all four mills to develop a semi-quantitative JEM with seven dust exposure levels. The JEM was targeted at workers enrolled into a soft tissue paper mill cohort and working any time between 1960 and 2009. Results The JEM includes a total of 14 421 cells, with each cell corresponding to the exposure for a job title, department, or work location for a one-year period. Exposure levels in the JEM were estimated to decline at three of the four mills from 1971 to 2009, but overexposures (ie, exceedances of the relevant occupational exposure limits) remained common at the end of the period. Conclusions The JEM results highlight the need for ongoing exposure control efforts in the soft tissue paper industry, and will inform ongoing epidemiological studies of the health effects of exposure to paper dust in Sweden. It is freely available for use by other researchers.

BACKGROUND Noise exposures are associated with a host of adverse health effects, yet these exposures remain inadequately characterized in many industrial operations, including paper mills. We assessed noise at four paper mills using three measures: (i) personal noise dosimetry, (ii) area noise measurements, and (iii) questionnaire items addressing several different aspects of perceived noise exposure. METHODS We assessed exposures to noise characterized using the three measures and compared the relationships between them. We also estimated the validity of each of the three measures using a novel application of the Method of Triads, which does not appear to have been used previously in the occupational health literature. RESULTS We collected 209 valid dosimetry measurements and collected perceived noise exposure survey items from 170 workers, along with 100 area measurements. We identified exposures in excess of 85 dBA at all mills. The dosimetry and area noise measurements assigned to individual subjects generally showed good agreement, but for some operations within mill, large differences between the two measures were observed, and a substantial fraction of paired measures differed by >5 dB. Perceived noise exposures varied greatly between the mills, particularly for an item related to difficulty speaking in noise. One perceived noise exposure item related to difficulty hearing due to noise showed strong and significant correlations with both dosimetry and area measurements. The Method of Triads analysis showed that dosimetry measures had the highest estimated validity coefficient (0.70), and that the best performing perceived exposure measure had validity that exceeded that of area measurements (0.48 versus 0.40, respectively). CONCLUSIONS Workers in Swedish pulp mills have the potential for exposures to high levels of noise. Our results suggest that, while dosimetry remains the preferred approach to exposure assessment, perceived noise exposures can be used to evaluate potential exposures to noise in epidemiological studies.

The protection of children in motor vehicle crashes has improved since the introduction of child restraint systems. However, motor vehicle crashes remain one of the top leading causes of death for children. Today, computer-aided engineering is an essential part of vehicle development and it is anticipated that safety assessments will increasingly rely on simulations. Therefore, this study presents a review of important biomechanical aspects for the safety of children in cars, including child human body models, for scenarios ranging from on-road driving, emergency maneuvers, and pre-crash events to crash loading. The review is divided into four parts: Crash safety, On-road driving for forward facing children, Numerical whole body models, and Discussion and future outlook.The first two parts provide ample references and a state-of-the-art description of important biomechanical aspects for the safety of children in cars. That children are not small adults has been known for decades and has been considered during the development of current restraints that protect the child in the crash phase. The head, neck, thorax, and pelvis are body areas where development with age changes the biomechanics and the interaction with restraint systems. The rear facing child seat distributes the crash load over a large area of the body and has proved to be a very efficient means of reducing child injuries and fatalities. Children up to age 4. years need to be seated rearward facing for optimal protection, mainly because of the proportionally large head, neck anthropometry and cartilaginous pelvis. Children aged 4 up to 12. years should use a belt positioning booster together with the vehicle seat belt to ensure good protection, as the pelvis is not fully developed and because of the smaller size of these children compared to adults. On-road driving studies have illustrated that children frequently change seated posture and may choose slouched positions that are poor for lap belt interaction if seated directly on the rear seat. Emergency maneuvers with volunteers illustrate that pre-crash loading forces forward-facing children into involuntary postures with large head displacements, having potential influence on the risk of head impact. Children, similar to adults, benefit from the safety systems offered in the vehicle. By providing child adaptability of the vehicle, such as integrated booster cushions, the child-restraint interaction can be further optimized. An example of this is the significant reduction of lap belt misuse when using integrated boosters, due to the simplified and natural positioning of the lap belt in close contact with the pelvis. The research presented in this review illustrates that there is a need for enhanced tools, such as child human body models, to take into account the requirements of children of different ages and sizes in the development of countermeasures.To study how children interact with restraints during on-road driving and during pre- and in-crash events, numerical child models implementing age-specific anthropometric features will be essential. The review of human whole body models covers multi body models (age 1.5 to 15. years) and finite element models (ages 3, 6, and 10. years). All reviewed child models are developed for crash scenarios. The only finite element models to implement age dependent anthropometry details for the spine and pelvis were a 3. year-old model and an upcoming 10. year-old model. One ongoing project is implementing active muscles response in a 6. year-old multi body model to study pre-crash scenarios. These active models are suitable for the next important step in providing the automotive industry with adequate tools for development and assessment of future restraint systems in the full sequence of events from pre- to in-crash.

Introduction Nurses with long durations of rotating night shifts appear to be at increased risk for coronary heart disease. But industrial shift work among women has not been well-studied. Methods Women employed more than one year from cohorts of soft paper mills (n=3354) and pulp and paper mills (n=2278) were evaluated in this study. Mill-specific job exposure matrices for shift work and noise exposure were developed at department and job levels. Every worked year was classified for shift work as probable, possible (including shift with no nights) or not likely. Similarly, noise was classified into seven 5 dBA bins 100 dBA. Here we present the cohort mortality from myocardial infarction for 1969–2013 as standardised mortality ratios (SMRs) with 95% confidence intervals (CI) compared to the general population in Sweden. Results Fatal myocardial infarctions (n=186) were increased in the cohort of women (SMR 123, 95% CI 106–142). Those classified as probable shift work (n=2399) had an SMR of 129 (98-166) and possible shift work (n=2060) had an SMR of 118 (93-146). Women with >10 years exposure to noise >90 dBA (n=602) had an SMR of 142 (103-192). SMRs declined with lower noise and shorter duration of noise, similar for both types of paper mills. Conclusions Shift working women in paper mills have an increased mortality from myocardial infarction, but the effect of shift work is difficult to separate from noise exposure. Probable shift work and shorter durations of noise exposure >85 dBA were still associated with elevated SMRs.

Preclinical data, and an increasing list of clinical investigations, show anti-inflammatory agents to favourably influence the biology of colorectal tumor. We have earlier reported on re-expression of activated immune cells after three days preoperative treatment of patients with colorectal carcinoma, randomized to receive oral NSAID (indomethacin or celebrex). Antisecretory prophylaxis (esomeprasol) was provided to all patients and served as sham treatment. Concomittant to MHC locus activation, Prominin1/CD133, a marker associated with stemness and poor prognosis in several solid tumors, was downregulated. The aim of the present study was to evaluate expression of additional regulators belonging to the stem cell niche, OCT4, SOX2 and BMP7, as well as some microRNAs, reported to act as tumor suppressors or oncomiRs. Peroperative tumor biopsies were analyzed by microarrays, quantitative real-time PCR and immunohistochemistry (IHC). The stem cell master regulator SOX2 was increased by NSAIDs (p

Background: Sporadic colorectal tumors probably carry genetic alterations that may be related to familiar clusters according to risk loci visualized by SNP arrays on normal tissues. The aim of the present study was therefore to search for DNA regions (copy number variations, CNVs) as biomarkers associated to genetic susceptibility for early risk predictions of colorectal cancer. Such sequence alterations could provide additional information on phenotypic grouping of patients. Material and Methods: High resolution 105K oligonucleotide microarrays were used in search for CNV loci in DNA from tumor-free colon mucosa at primary operations for colon cancer in 60 unselected patients in comparison to DNA in buffy coat cells from 44 confirmed tumor-free and healthy blood donors. Array-detected CNVs were confirmed by Multiplex ligation-dependent probe amplification (MLPA). Results: A total number of 205 potential CNVs were present in DNA from colon mucosa. 184 (90%) of the 205 potential CNVs had been identified earlier in mucosa DNA from healthy individuals as reported to the Database of Genomic Variants. Remaining 21 (10%) CNVs were potentially novel sites. Two CNVs (3q23 and 10q21.1) were significantly related to colon cancer, but not confirmed in buffy coat DNA from the cancer patients. Conclusion: Our study reveals two CNVs that indicate increased risk for colon cancer; these DNA alterations may have been acquired by colon stem cells with subsequent appearance among epithelial mucosa cells. Impact: Certain mucosa CNV alterations may indicate individual susceptibility for malignant transformation in relationship to intestinal toxins and bacterial growth.

Objective: One objective of this study is to evaluate the head kinematics of the Q3 model. Another objective is to evaluate the effect on head kinematics of increased thoracic spine flexibility; more humanlike mass distribution; and more humanlike body geometry in the Q3 model. The evaluations were based on the head kinematics of children deduced from real crashes and on new data of mass distribution and updated body dimensions for 3-year-olds. Methods: The head kinematics of the Q3 model was evaluated by comparing the Q3 model's head displacement response with the deduced response of 3-year-old children in real crashes. To do so, data from crashes were collected. The data were used to develop the mathematical vehicle and restraint system models (MADYMO, TASS, the Netherlands). Three crashes involving 3-year-old children in frontal impacts were reconstructed. The models were run 35 times each (one model per crash), each time with a different setting to each of the variables for which the exact value was not known. Examples of those variables include crash pulse, initial dummy position, and initial seat belt position. Two versions of the Q3 model were used: one that correlated with the Q3 ATD and one that was modified regarding anthropometry and thoracic flexibility. The basis for the updated anthropometry was new data regarding characteristic dimensions and mass distribution collected at a Swedish hospital. Results: In the anthropometry study, 26 children were measured. The main differences between the average of the measured children and the Q3 model were found in the mass distribution of the torso and thighs: the Q3's pelvis was too heavy and the thorax, abdomen/lumbar spine, and thighs were too light. Another difference was identified in the buttock-knee length. Two of the 3 reconstructed crashes had confirmed head impacts. The Q3 model responded with head kinematics that reflected the deduced courses of events for the real children in one of 3 crashes (the one without head impact). The modified Q3 model reflected the real children in 2 of 3 crashes. Conclusions: In high-severity, straight frontal crashes, the Q3 model predicted non-head impact adequately. However, in oblique frontal crashes, the Q3 model did not sufficiently predict the head impacts. The modified Q3 model predicted the head impacts better than the Q3 model did. Greater flexibility of the thorax and redistributed mass made a positive difference regarding the head kinematics.

Background : In asthma, clinical measures like FEV1 are a poor surrogate for detection of sub-groups and disease progression, and more efficient screening methods are needed. The PExA method (particles in exhaled air) non-invasively counts and collects exhaled particles, which reflect the composition of the fluid lining the peripheral airways and contains possible information about lung status. Collected particle mass is in the ng range and currently only single protein analysis is carried out routinely. The pilot study aimed to investigate Multiplex technology for simultaneous analysis of acute-phase proteins in exhaled particles. Method : Particles were collected during 30 minutes from two groups: Population 1: 12 healthy volunteers. Population 2: 6 subjects with asthma and 6 age-matched controls. Albumin, fibronectin, fibrinogen, SAP, A2M, CRP, haptoglobin and AGP were analysed using two Multiplex-kits (MILLIPLEX® SKINMAG-50K, MILLIPLEX® HCVD3-67K). Results: Albumin, fibrinogen, fibronectin, haptoglobin and AGP could be quantified in the majority of samples (>60%) and A2M in 7 samples, all regardless of subject9s health status. CRP and SAP were detected in one asthmatic and one control, respectively. Median particle mass collected in Population 1 was 340 ng (143-739). In Population 2, sampled mass in asthmatics (median 295 ng, 116-430) was significantly decreased compared to controls (median 535 ng, 234-1060) (P Conclusion : Multiplex technology can improve analysis possibilities by facilitating simultaneous quantification of multiple proteins in exhaled particles. Asthma may lead to a decrease in exhaled particles and less sample material.

Non-invasive and quantitative sampling of particles in exhaled air may provide valuable information on the status of the peripheral airways. Studies of particles have confirmed the presence of pulmonary surfactant, including both important phospholipids and surfactant proteins. The aim of this study was to develop a reliable method for the quantification of the major phosphatidylcholines (PCs) in exhaled particles. Methods: Exhaled particles were counted and collected on membranes using the PExA method. The particles were extracted with methanol:chloroform prior to flow injection into an electrospray ionization tandem mass spectrometer. A quantitative method was developed for dipalmitoylphosphatidylcholine (DPPC) and palmitoyl-oleoylphosphatidylcholine (POPC) using selected reaction monitoring. The method was validated by investigating within- and between-batch precision, accuracy, carry-over, stock stability, short-term and long-term stability, freeze-thaw stability and processed sample integrity. The validated method was used to investigate the inter-individual variation in exhaled particles from 6 healthy volunteers. Results: The method had a linear response for DPPC and POPC with a limit of quantification of 0.005 µM. The DPPC and POPC concentration, expressed as weight % in exhaled particles, were 13.9 (median, range 8.8-16%) and 4.1 (median, range 2.4-4.9%), respectively, of the total particle sample. Conclusion: We have developed a reliable method for the quantification of surfactant PCs in exhaled particles using a simple sample extraction and mass spectrometry. The method can readily be applied in clinical studies to investigate the role of PCs in different lung related diseases.

The objective of this study was to define the crash characteristics of near-side impact crashes in which children seated in the rear rows are injured. The crash characteristics included the direction of force, heading angle, horizontal impact location, vertical impact location, extent of deformation and intrusion at the child occupant's seating position. Cases from in-depth crash investigation databases of the NASS-CDS (National Automotive Sampling System-Crashworthiness Data System), CIREN (Crash Injury Research and Engineering Network) and Chalmers University of Technology were reviewed. The principal direction of force was most frequently between 60° and 75°. The heading angle of the bullet vehicle was most commonly between 61° and 90°. The bullet vehicle hit the passenger compartment of the target vehicle, particularly the rear door. Often, one or both of the adjacent pillars to the rear door were involved, most commonly the B pillar. In 11 of 16 crashes, the car sill was not engaged. Most commonly, the deformation extent was into Zone 3 or more – about 40 cm – and the intrusion at the child's seating position was in the range 20–30 cm. This review of the crashes revealed differences between the current side impact test procedures and the actual side impact crashes in which children were injured.

Particulate air pollution affects cardiovascular and pulmonary disease and mortality. A main hypothesis about the mechanisms involved is that particles induce inflammation in lower airways, systemic inflammation and oxidative stress.To examine whether short-term exposure to wood smoke in healthy subjects affects markers of pulmonary inflammation and oxidative stress.13 subjects were exposed first to clean air and then to wood smoke in a chamber during 4-hour sessions, 1 week apart. The mass concentrations of fine particles at wood smoke exposure were 240-280 mug/m(3), and number concentrations were 95 000-180 000/cm(3), about half of the particles being ultrafine (100 nm). Blood and breath samples were taken before and at various intervals after exposure to wood smoke and clean air and examined for exhaled nitric oxide and Clara cell protein in serum and urine, and malondialdehyde in exhaled breath condensate.Exposure to wood smoke increased alveolar nitric oxide 3 hours post-exposure while malondialdehyde levels in breath condensate were higher both immediately after and 20 hours after exposure. Serum Clara cell protein was increased 20 hours after exposure.Wood smoke at levels that can be found in smoky indoor environments caused an inflammatory response and signs of increased oxidative stress in the respiratory tract, especially in the lower airways.

The importance of prostaglandins in tumor growth and progression is well recognized, including antineoplastic activities by cyclooxygenase (COX) inhibitors. Variation in treatment response to COX inhibition has questioned differences in expression of cell surface and nuclear membrane receptors among tumors with different disease progression. The purpose of this study was to evaluate whether EP(1-4) subtype, PPAR gamma receptor and COX-1/COX-2 expression in colorectal cancer are related to tumor-specific mortality. Reverse transcription-polymerase chain reaction and immunohistochemistry were used to demonstrate expression and protein appearance in tumor tissue compared with normal colon tissue. EP(1) and EP(2) subtype receptor protein was highly present in tumor cells, EP(3) occurred occasionally and EP(4) was not visible. PPAR gamma, EP(2) and EP(4) mRNA were significantly higher in normal colon tissue compared with tumor tissue, without any distinct relationship to Dukes A-D tumor stage. Multivariate analyses indicated that increased tumor tissue EP(2) and COX-2 expression predicted poor survival (p

Alterations in eicosanoid metabolism is well established in a variety of malignant tumors, particularly colorectal carcinoma. Recent studies in our laboratory have emphasized a role for EP subtype receptors in progression of colorectal cancer and disease specific mortality. Therefore, the aim of the present study was to extend our knowledge to include additional receptor expression (DP1, DP2, FP, IP, TP) for prostanoids (PGD2, TXA2, PGF2alpha, PGI2) in relationship to tumor stage, differentiation and progression of colorectal cancer.Total RNA from 62 tumors and adjacent normal colon tissue (n = 48) was extracted. Quantification of receptor expression was performed by realtime PCR and related to the expression of an appropriate housekeeping gene (GAPDH). Tumors were assessed according to Dukes A-D (stage I-IV).DP1, DP2, FP and IP receptor subtypes displayed significantly reduced overall expression in tumor tissue compared to normal colon tissue, while the TP receptor subtype showed significantly higher expression in tumor tissue. Overall expression of the prostanoid receptors in tumor tissue was not related to clinical indexes as tumor stage and tumor cell differentiation evaluated by multivariate analyses. Cultured colorectal cancer cell lines with low (HT-29) and high (HCA-7) intrinsic PGE2 production at confluent state did not express DP1 and IP receptor subtypes, but displayed low expression of DP2, FP and TP receptor subtypes.The results in the present study indicate imbalanced expression of prostanoid receptors in colorectal cancer compared to normal colon tissue without clear cut relationship to disease progression. Therefore, future studies should be performed on defined cells within the tumor tissue compartment determining whether any prostanoid receptor(s) is useful as a molecular target in treatment or prevention of colorectal cancer.

Exhaled breath aerosol particles are a promising matrix for the analysis of both endogenous and exogenous compounds, since the collection is easy to perform and non-invasive. We compared two collection devises based on different collection principles, filtration (SensAbuse®) and impaction (PExA®), with methadone as model substance for an exogenous compound. We collected exhaled particles from 13 subjects undergoing methadone maintenance treatment with both devises and analyzed the samples by liquid chromatography-mass spectrometry. The result showed that methadone was present in the samples from both devises. However, the method based on filtration collected 60 times more per liter exhaled air than the method based on impaction. The difference can to a large extent be explained by the design of the collecting devises, as the filtration method, in principle, collects all particles, independent of dimension. The method based on impaction just collects particles in the interval of 0.3 to 7 μm, according to the cut-off limits of the impactor. Complementary experiments showed that the contribution from the oral cavity can be substantial in a random fashion with a large inter-individual variation. We also showed that breathing pattern and design of the collecting devises are crucial for the efficiency of collecting exhaled particles. Large particles can contribute hugely to the analyzed mass, but are also easily separated by gravitation and impaction in the collection device. We have shown that exogenous compounds can be collected and analyzed in exhaled breath aerosol particles. However, the breathing pattern and the design of the collecting devise can influence the result by magnitudes.

The aims of the present study were to determine whether exposure to high peaks of ozone resulted in an increased prevalence of asthma or respiratory symptoms among bleachery workers and whether nitric oxide (NO) was elevated in the exhaled air of these workers. Bleachery workers (n=228) from three Swedish pulp mills who had been exposed to ozone, together with 63 unexposed control subjects, were investigated by means of spirometry, Phadiatop, exhaled and nasal NO and answers to a questionnaire concerning respiratory symptoms and exposure. Exposure to an ozone peak that gave rise to respiratory symptoms was defined as a "gassing". Bleachery workers reporting four or more gassings involving ozone had an increased prevalence of adult-onset asthma, wheeze, and current asthma symptoms. They also had a higher median concentration of exhaled NO in comparison with those who reported no such gassings (19.2 versus 15.7 parts per billion). No such associations were found in respect of nasal NO. The results from this study show that bleachery workers who have been repeatedly exposed to ozone gassings have an increased prevalence of adult-onset asthma. The results also indicate exhaled nitric oxide may be a marker of airway inflammation in bleachery workers who have been exposed to high peaks of ozone.

PURPOSE: To compare p53 alterations in survivors and nonsurvivors after surgery for colorectal cancer. PATIENTS AND METHODS: Twenty-nine potentially cured patients with colorectal carcinoma, without recurrent disease for more than 6 years after their primary surgery, were selected to match a group of 41 colorectal cancer patients with early metastatic spread to the liver. All patients were screened for mutations in the p53 gene, exons 5 to 9, by denaturing gradient gel electrophoresis and subsequent sequencing. RESULTS: The frequency of p53 mutations was significantly different in cured patients (60%) compared with patients with early relapse (41%, P < .05). A significant difference was found in the distribution of mutations, indicating that potentially cured patients had a different proportion of mutations in conserved regions of p53 (P = .02). This difference was explained by a significantly different frequency of mutations in exon 8 (40% v 15%, P = .03), which is part of the conserved region V. All mutations in region V were codon 273 mutations in cured patients, whereas three of four mutations were located in codon 273 in patients with metastatic disease. Allelic loss of p53 (loss of heterozygosity [LOH]) was demonstrated in 26% of the cured patients and in 39% of patients with metastatic disease (P = .36). The combination of mutation and LOH of p53 was the same (17%) in both groups. CONCLUSION: A large number of p53 mutations in colorectal cancer do not promote disease progression. Some mutations, particularly within conserved regions, may even counteract negative functional effects of other p53 structural alterations. A complete loss of p53 function was not related to survival or progression after curative operation of colorectal carcinoma.

Results from tests on the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) reference chemicals 31–50 in 67 different in vitro toxicity assays are presented in this paper as a prerequisite to in vitro/in vivo comparisons for all MEIC in vitro toxicity data in forthcoming papers, i.e. the final MEIC evaluation of the relevance of the tests. With the aim of increasing knowledge about the relative significance of some in vitro methodological factors, the strategies and methods of the preceding parts in the MEIC series (Parts II and III) were again employed to enable comparative cytotoxicity analysis of the new in vitro results presented in this paper. A principal components analysis (PCA) of the results from tests of the 20 chemicals in 67 assays demonstrated a dominating first component describing as much as 74% of the variance in the toxicity data, indicating a similar ranking of the cytotoxicities of the chemicals in most of the tests. The influence on the general variability of the results of a few, key methodological factors was also evaluated by using linear regression comparisons of the results of all pairs of methods available in the study, i.e. methods which were similar in all respects except for the factor being analysed. Results from this “random probe” analysis were: a) the cytotoxicities of 11 of the 20 chemicals increased considerably with exposure time (> 10 times over 4–168 hours); b) in general, human cell line toxicity was well predicted by cytotoxicity in animal cells; c) prediction of human cell line toxicity by most ecotoxicological tests was only fairly good; d) 14 comparisons of similar assays with different cell lines showed similar toxicities (mean R2 = 0.83); e) nine comparisons of similar assays employing different primary cultures and cell lines shared similar toxicities (mean R2 = 0.71); and f) 16 comparisons of similar assays with different growth/viability endpoints showed similar toxicities (mean R2 = 0.71). Results b, d, e and f must contribute to the PCA-documented high general similarity of the in vitro toxicity data. Results a and c, together with factors which were not analysed, such as different protocols and inter-laboratory variability of tests, could explain the 26% dissimilarity. To provide background information to the planned final MEIC evaluation of the relevance of the 61 methods in which all 50 chemicals have been tested, an additional PCA was made of the 50 chemical-61 assay in vitro database (from Parts II and III and the present paper). This supplementary PCA demonstrated an 80% similarity of results. Compared with the previous analysis of the tests of the first 30 MEIC reference chemicals (MEIC Part III), the present analysis of the tests of the last 20 MEIC chemicals indicates a somewhat higher variation in the results. Correspondingly, some deviating endpoint measurements and cell line responses were demonstrated by the pairwise comparisons in the present study. As a result, the analysis revealed a high correlation (R2 = 0.73) between the average human cell line toxicity and the results from a new protein denaturation test. These preliminary results suggest that intracellular protein denaturation may be a frequently occurring mechanism in basal cytotoxicity.

During 1988-1992, 684 scats were collected throughout the year in the territory of the only reproducing family group (mean five individuals) of wolves Canis lupus in Scandinavia. Moose Alces alces, roe deer Capreolus capreolus, and badger Meles meles constituted the three most important prey species, and hair from them was found in 52%, 50%, and 19% of scats, respectively. When compensating for different area/volume ratios in prey species of different size, these three species were estimated to constitute 97% of the biomass ingested. The proportions of moose, roe deer, and badger were 66%, 27%, and 8% by mass, and 25%, 52%, and 23% by number, respectively. Young-of-the-year dominated two samples of dead moose (51% of 65 killed by wolves; 43% of 155 killed by hunters), but no significant differences between the samples were found in any age class. Wolves killed significantly more female moose (76%) than hunters (53%), and among wolf-predated moose, no male was older than two years. Mean winter density of moose and roe deer in the wolf territory (523 km^2), estimated by fecal pellet group counts, was 1.5 moose and 0.4 roe deer/km^2. Moose density decreased slightly at the end of the study, but it was estimated that wolves killed only about 5% of the moose population each year and that this could be compensated for by a decrease of about 10-20% in the hunter kill. In spite of a high predation pressure from wolves, in addition to predation from an increasing lynx Lynx lynx population, the density of roe deer increased threefold. It is concluded that the future predation pressure on moose may be more pronounced if the density of wolves increases, and roe deer may be more affected by predation when the present favourable ecological conditions cease.

The multicentre evaluation of in vitro cytotoxicity (MEIC) study is a programme designed to evaluate the relevance of in vitro toxicity tests for predicting human toxicity, and is organised by the Scandinavian Society for Cell Toxicology. The project started in 1989 and is scheduled to be finished by June 1996. MEIC is a voluntary effort by international laboratories to test the same 50 reference chemicals in their own in vitro toxicity systems. At present, 31 laboratories have submitted results for the first 30 reference chemicals from a total of 68 in vitro cytotoxicity tests. In the definitive evaluation of the MEIC programme, these in vitro results will be compared with human lethal blood concentrations and other relevant acute systemic toxicity data, and the results will be published as a series of articles. This paper, which is the first article in this series, describes and analyses the methodologies used in the 68 tests. The origins and purities of the test chemicals, the biological systems and the toxicity endpoints are also discussed. Since MEIC is not centrally directed, the selection of tests was entirely dependent on the preferences of the individual laboratories. Thus, the collection of tests is not representative of the full range of existing in vitro toxicity tests. In our study, basal cytotoxicity tests and ecotoxicological tests are prevalent, while tests for toxicity to primary cultures of differentiated cells, measured by organotypic toxicity endpoints, are clearly under-represented.

Results from tests of the first 30 MEIC reference chemicals in 68 different toxicity assays are presented as a prerequisite to subsequent in vitro/in vivo comparisons of acute toxicity data. A comparative cytotoxicity study was also carried out. Firstly, the variability of all of the results was analysed by using principal components analysis (PCA), analyses of variance (ANOVAs) and pairwise comparisons of means according to Tukey's method. The first PCA component described 80% of the variance of all of the cytotoxicity data. Tukey's ANOVA indicated a similar sensitivity for the assays, of approximately 80%. Secondly, the influence of five major methodological components on the general variability of the results was evaluated by linear regression and ANOVA linear contrast analyses. The findings were that: a) the toxicity of many chemicals increased with exposure time; b) in general, human cytotoxicity was predicted well by animal cytotoxicity tests; c) this prediction was poor for two chemicals; d) the prediction of human cytotoxicity by the ecotoxicological tests was only fairly good; e) one organotypic endpoint used, i.e. contractility of muscle cells, gave different results to those obtained according to viability/growth toxicity criteria; f) twelve comparisons of similar test systems involving different cell types (including highly differentiated cells) showed similar toxicities regardless of cell type; and g) nine out often comparisons of test systems with identical cell types and exposure times revealed similar toxicities, regardless of the viability or growth endpoint measurement used. Factors b, f and g must be the main causes of the remarkable similarity between the total results, while factors a, c, d and e, together with other minor factors that were not analysed, contributed to the 20% dissimilarity. The findings strongly support the basal cytotoxicity concept, and will facilitate future in vitro toxicity testing.

The objective of this study is to investigate the effects of crash-related car parameters on head and chest injury measures for 3- and 12-year-old children in near-side impacts.The evaluation was made using a model of a complete passenger car that was impacted laterally by a barrier. The car model was validated in 2 crash conditions: the Insurance Institute for Highway Safety (IIHS) and the US New Car Assessment Program (NCAP) side impact tests. The Small Side Impact Dummy (SID-IIs) and the human body model 3 (HBM3) (Total HUman Model for Safety [THUMS] 3-year-old) finite element models were used for the parametric investigation (HBM3 on a booster). The car parameters were as follows: vehicle mass, side impact structure stiffness, a head air bag, a thorax-pelvis air bag, and a seat belt with pretensioner. The studied dependent variables were as follows: resultant head linear acceleration, resultant head rotational acceleration, chest viscous criterion, rib deflection, and relative velocity at head impact. The chest measurements were only considered for the SID-IIs.The head air bag had the greatest effect on the head measurements for both of the occupant models. On average, it reduced the peak head linear acceleration by 54 g for the HBM3 and 78 g for the SID-IIs. The seat belt had the second greatest effect on the head measurements; the peak head linear accelerations were reduced on average by 39 g (HBM3) and 44 g (SID-IIs). The high stiffness side structure increased the SID-IIs' head acceleration, whereas it had marginal effect on the HBM3. The vehicle mass had a marginal effect on SID-IIs' head accelerations, whereas the lower vehicle mass caused 18 g higher head acceleration for HBM3 and the greatest rotational acceleration. The thorax-pelvis air bag, vehicle mass, and seat belt pretensioner affected the chest measurements the most. The presence of a thorax-pelvis air bag, high vehicle mass, and a seat belt pretensioner all reduced the chest viscous criterion (VC) and peak rib deflection in the SID-IIs.The head and thorax-pelvis air bags have the potential to reduce injury measurements for both the SID-IIs and the HBM3, provided that the air bag properties are designed to consider these occupant sizes also. The seat belt pretensioner is also effective, provided that the lateral translation of the torso is managed by other features. The importance of lateral movement management is greater the smaller the occupant is. Light vehicles require interior restraint systems of higher performance than heavy vehicles do to achieve the same level of injury measures for a given side structure.

Expression of Prominin 1/CD133 is associated with poor prognosis and chemoresistance in several types of solid tumors. The aim of the present study was, therefore, to evaluate Prominin 1/CD133 expression in colorectal carcinoma after short-term preoperative treatment by non-steroidal anti-inflammatory drugs (NSAIDs). Patients aimed at curative operation for colon cancer were randomized to receive NSAIDs (indomethacin 50 mg x2 or celecoxib 100 mg x2) three days preoperatively. Antisecretory prophylaxis (esomeprasol 40 mg x1) was provided to all patients and served as sham intake. CD133 expression in tumor tissue was also assessed in tumors from Dukes' B patients selected for either long or short postoperative survival. No patients received perioperative chemoradiotherapy. Tumor tissue was collected at surgery for quantification of mRNAs (Prom1 and Wnt4) by qPCR. Immunohistochemistry stained for CD133, Ep-CAM, CD34 and CD45. PGE2 content in tumor tissue was determined. Transcript of CD133 in tumor tissue was lower in patients treated with NSAIDs (0.28 ± 0.07 vs. 0.51 ± 0.08; p

The ability of 15 sesquiterpenoids containing an unsaturated dialdehyde functionality to damage cellular membranes, measured as the induction of ATP leakage in ELD cells, has been investigated. In spite of the structural similarities of this class of compounds, large differences in potency were noted. The results from the ATP leakage assay were correlated (by the multivariate PLS method) with a number of structural descriptors, such as intra-atomic distances and angles, atomic charges, and lipophilicity, to obtain quantitative structure-activity relationships. A component consisting of the distance and the dihedral angle between the two aldehyde groups, the dipole moment, the lipophilicity, and the chemical stability, was found to be correlated with the activity in the ATP leakage assay. This indicates that the activity of the unsaturated dialdehydes depends on their accumulation in lipophilic parts of a cell (e.g. the membranes), the way they orient themselves in a membrane, and their chemical reactivity towards proteins associated with a membrane.

The purpose of this naturalistic study was to investigate the effect of booster seat design on the choice of children’s seating positions during naturalistic riding. Data was collected through observations of children during in-vehicle riding by means of a film camera. The children were positioned in high back boosters in the rear seat while a parent drove the car. The study included two different booster designs: one with large head and torso side supports, and one with small head side supports and no torso side supports. Six children between three and six years of age participated in the study. Each child was observed in both boosters. The duration of the seating positions that each child assumed was quantified. The design with large side head supports resulted more often in seating positions without head and shoulder contact with the booster’s back. There was shoulder-to-booster back contact during an average of 45% of riding time in the seat with the large head side supports compared to 75% in the seat with the small head supports. The children in the study were seated with the head in front of the front edge of the head side supports more than half the time, in both boosters. Laterally, the children were almost constantly positioned between the side supports of the booster in both seats. The observed seating positions probably reduce the desired protective effect by the side supports in side impact, and may increase the probability of head impact with the vehicle interior in frontal impact.

Non-steroidal anti-inflammatory drugs (NSAIDs) attenuate tumor net growth in clinical and experimental cancer. Evaluations in cell culture experiments have implied involvement of growth factor and G-protein related signaling pathways to explain decreased proliferation, angiogenesis, increased cell adhesion and apoptosis. Sparse information is however available from studies on growing tumors in vivo. The aim of the present study was to map alterations in selected signal proteins in relation to heterogeneous tissue expression of COX-2 in tumors during COX inhibition. MCG 101 cells were exposed to indomethacin treatment both in vivo and in vitro to reduce PGE 2 production. Tumor tissue specimens were taken for immunohistochemical analyses and qPCR determinations. Protein markers were selected to reflect cell proliferation and cell cycling, angiogenesis and metastasis in relationship to COX-2 staining in tumor tissue. Indomethacin did not change overall COX-2 staining in tumor tissue, but altered its distribution towards increased staining in cell nuclei/ nucleoli and decreased COX-2 staining heterogeneity in tumor tissue. P53 staining was decreased, while PCNA and TGFβ3 staining were increased by indomethacin in tumor areas with high presence of COX-2, which correlated to staining of BAX, TUNEL, Bcl-2, c-jun, p21, p27, p53 and NM23. Net tumor growth was predicted by EGF-R, p21 and p27 proteins in tumor tissue during indomethacin treatment (multivariate analysis). RNA transcript analyses showed decreased EGF-R and KRas expression in vivo, following indomethacin treatment, which also included KRas, PI3K, JAK1, STAT3 and c-jun, mRNAs in cultured tumor cells. In conclusion, our results extend earlier studies on cell culture experiments and demonstrate that EGF-R and downstream KRas pathways communicate effects of increased prostaglandin activity in tumor tissue in vivo.

This study shows that the Artemia assay, which is usually performed by incubation for a 24-hour period in artificial sea water, can also be performed in phosphate buffered saline (PBS) at pH 7.2, or in a cell culture medium, both of which are used in toxicity assays employing mammalian cells. Thus, by using the equivalent media for incubation, toxic effects in the Artemia assay and toxic mechanisms can more accurately be compared with results obtained in mammalian cell toxicity. Survival of control animals is good for 72 hours, provided that infection can be avoided. A pH greater than 6 is essential for good survival of Artemia Salina, and a pH greater than 10.5 should be avoided. Because of the risk of infection at low saline concentrations, a decreased incubation time of 16 or 12 hours is recommended. The lethal concentrations (LC10 and LC50) in the 24-hour Artemia assay of the first ten chemicals in the MEIC programme were measured in PBS, and the results compared with those from a previous study of the effects in a 10-minute acute ATP leakage assay, specifically indicating lesions in the cell membrane. The EC10 and EC50 values for the alcohols in the Artemia assay were 35–75% of the corresponding values in the ATP leakage assay. For paracetamol and amitriptyline, the EC10 and EC50 values in the Artemia assay were 2–16% of the corresponding values in the ATP-leakage assay. The greatly increased toxicity of the two compounds in the animals may be related to systemic effects. FeSO4 was very toxic to Artemia salina at a concentration of lug/ml, but it did not cause leakage of ATP from cultured cells, even at a concentration of 8,000μg/ml, showing that widely different mechanisms of interaction with FeSO4 are measured by the two assays. A lower toxicity of polygodial, a sesquiterpenoid unsaturated dialdehyde, in cell culture medium, was obvious in the Artemia assay. Thus, some factors in cell culture medium must, by interacting with the toxic molecule, protect the animals against the toxicity of polygodial, as was previously found in cultured cells.

The lipophilicity, expressed as log P, has been estimated for 19 sesquiterpenoid unsaturated dialdehydes by the determination of the 1-octanol/water partition coefficient for 12 of the compounds, and the reversed-phase HPLC retention data for all the compounds. The log P values obtained varied between 0.5 and 3.02. The stability of 17 unsaturated dialdehydes in three common in vitro assay media at 37°C was investigated by HPLC analysis of samples dissolved in the media. All compounds investigated were essentially stable for up to 24 hours in a phosphate buffered saline, while several of them disappeared rapidly in media containing amino acids and fetal calf serum. This chemical instability must be considered when results from biological assays of unsaturated dialdehydes are compared.

The neutral red absorption assay, complemented by protein determination, was used to measure the cytotoxic activity of 22 sesquiterpenoid unsaturated dialdehydes towards hamster fibroblasts in monolayer culture (cell line V79). No significant differences between the activities determined by the two assays were found for the compounds assayed, indicating that their inhibitory effects on cell growth are related to general cytotoxicity. In spite of the apparent similarity between the chemical structures of the assayed compounds, the most active was approximately 200 times more cytotoxic than the least active. Isovelleral and its derivatives are the most cytotoxic substances, but the ranking in order of general cytotoxicity within this subgroup differed from that found in a previous study of mutagenic activity. Marasmic acid and its derivatives are also potent cytotoxic agents, but two of the most cytotoxic compounds in this group have been reported to be the least mutagenic. Such differences between cytotoxic and mutagenic activities indicate that the molecular targets for these two activities differ. The cytotoxicity of polygodial found in this study is lower than that previously reported from other cytotoxicity assays, which suggests that different tests of general cytotoxicity may have different molecular targets. Because of the diverse cytotoxic activities of the unsaturated dialdehydes, it should be possible to study them in a quantitative structure-activity relationship (QSAR) multivariate analysis.

Trace element levels of liver samples obtained from necropsied dogs suffering from hepatitis and/or liver cirrhosis were determined by PIXE. Two different techniques for preparation of the samples were compared: the pellet press method and wet digestion. Both methods gave similar results, but the pellet press method was chosen for the subsequent routine analyses because of its simplicity due to few preparation steps and little risk of contamination. Preliminary results indicate elevated levels of Cu in chronic hepatitis and cirrhosis. In hereditary copper-induced hepatitis (Bedlington hepatitis) Fe and Br levels were increased as well.

1. 1. Diadenosine tetraphosphate (Ap4A) levels were determined in HL-60 cells differentiating into granulocytes or monocytes after treatment for 0–7 days with retinoic acid (RA) or 4-β-phorbol-12-myristate-13-acetate (PMA) respectively. 2. 2. The levels increased significantly compared to untreated control cells within 2 days and then declined again. 3. 3. In RA treated cells the levels finally decreased far below those of untreated HL-60 cells and became equal to those found in human granulocytes. 4. 4. PMA treatment had no effect on Ap4A levels in human granulocytes. 5. 5. A possible interaction between Ap4A and ADP-ribosyl transferase is discussed.

Previous studies in our laboratory have demonstrated that prostaglandin (PG) E2 is involved in anorexia/cachexia development in MCG 101 tumor-bearing mice. In the present study, we investigate the role of PGE receptor subtype EP2 in the development of anorexia after MCG 101 implantation in wild-type (EP2 (+/+)) or EP2-receptor knockout (EP2(-/-)) mice. Our results showed that host absence of EP2 receptors attenuated tumor growth and development of anorexia in tumor-bearing EP2 knockout mice compared to tumor-bearing wild-type animals. Microarray profiling of the hypothalamus revealed a relative twofold change in expression of around 35 genes including mRNA transcripts coding for Phospholipase A2 and Prostaglandin D2 synthase (Ptgds) in EP2 receptor knockout mice compared to wild-type mice. Prostaglandin D2 synthase levels were increased significantly in EP2 receptor knockouts, suggesting that improved food intake may depend on altered balance of prostaglandin production in hypothalamus since PGE2 and PGD2 display opposing effects in feeding control.

Ghrelin is a novel brain-gut peptide that stimulates food intake and may secondarily increase body weight via a growth hormone secretagogue receptor (GHS-R). Tumor-bearing mice (MCG101), characterized by anorexia, fat loss and muscle wasting due to increased concentration of PGE2 and proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha), were provided ghrelin i.p. at a low (20 microg/day) and high dose (40 microg/day) to examine the ability of ghrelin to counteract tumor-induced anorexia. Immunohistochemical staining and Western blot analyses were used to identify GHS-R expression in the brain as well as its relationship to NPY expression in hypothalamic neurons. GHS-R mRNA in hypothalamus and ghrelin mRNA in gastric fundus were quantified by RT-PCR. Body composition was determined by carcass extractions. GHS-R expression in hypothalamus and plasma ghrelin levels were significantly increased in freely-fed tumor-bearing mice, while gastric fundus expression of ghrelin was unaltered compared to non-tumor-bearing mice (controls). Ghrelin treatment increased food intake, body weight and whole body fat at both low and high doses of ghrelin in normal controls, while tumor-bearing mice showed improved intake and body composition at the high dose of ghrelin only. Exogenous ghrelin normalized the GHS-R expression in hypothalamus from tumor-bearing mice without alterations in the gastric fundus expression of ghrelin. Tumor growth was not altered by exogenous ghrelin. Our results indicate that MCG 101-bearing mice became ghrelin resistant despite upregulation of hypothalamic GHS-R expression, which confirms similar indirect observations in cancer patients. Thus, other factors downstream of the ghrelin-GHS-R system appear to be more important than ghrelin to explain cancer-induced anorexia.

Earlier observations on cyclo-oxygenase inhibitors (NSAIDs) restricting tumor growth were re-evaluated by comparing the effects of non-selective, preferential selective and selective derivatives of COX-inhibitors on tumor growth in mouse models with either prostaglandin-sensitive (MCG-101, human tumors) and -insensitive transplants (K1735-M2). Tumor growth, with and without provision of a classical cyclo-oxygenase inhibitor (indomethacin), was related to tumor content of COX-1/COX-2 protein as well as to EP1-EP4 and prostacyclin receptor expression. Mouse serum amyloid protein (SAP) was measured as an indicator of systemic inflammation, which relates to pro-inflammatory cytokines. Indomethacin inhibited tumor growth and prolonged the survival of mice bearing MCG-101 tumors, which display a high production of PGE2, while K1735-M2 tumors with insignificant amounts of PGE2 did not respond to indomethacin at all. However, the effects of various NSAIDs on tumor growth were highly variable in combination with the fact that most preferential selective and selective COX-2 inhibitors attenuated poorly systemic inflammation evaluated by plasma concentrations of mouse SAP. The ability of NSAIDs to attenuate tumor growth was not related to the tumor content of COX-2 protein as expected. Multivariate analysis suggests that significant COX-inhibition of tumor growth may be related to tumor expression of subtype EP2, EP3 (p

Preclinical and clinical studies in our laboratory have suggested that prostaglandin (PG) E2 is involved in anorexia and cachexia development, although the role of COX pathways on the pathogenesis of cancer cachexia remains to be clarified. Expressions of PGE (EP1, EP2, EP3alpha,beta,gamma and EP4) and PGI (IP) receptors in the central nervous system (brain cortex, hypothalamus and brain stem), in peripheral (liver, white adipose tissue and skeletal muscle) and tumor tissue from MCG-101-bearing mice with and without indomethacin treatment were investigated by RT-PCR and immunohistochemistry. Expression of EP1 in the liver and EP4 receptor in white adipose tissue were upregulated and responded to indomethacin treatment, while downregulated expression of EP3 in skeletal muscle from tumor-bearing mice was unresponsive to indomethacin treatment despite improved carcass weight. Expression of EP and IP receptors in brain and tumor tissue from tumor-bearing mice were neither related nor responsive to systemic PGE2 levels including increased IL-1beta, IL-6 and TNF-alpha host activities. The expression IP receptor in CNS, peripheral tissue and tumor tissue was unchanged by cachexia development. Our results suggest that transcription of EP receptors in liver, fat and skeletal muscle tissue may be a control level for host metabolic alterations during tumor progression, while overall EP and IP receptor expression in CNS did not indicate an important control level for appetite regulation in MCG 101-bearing mice despite prostanoid related anorexia.

We studied by microphysiometry functional effects of two different signalling molecules in the murine tumor cell lines, MCG 101 and K1735-M2, namely norepinephrine (NE) and prostaglandin E2 (PGE2). This methodology implies estimation of intracellular metabolism by measurements of extracellular acidification rate (ECAR). MCG 101 (an undifferentiated, epithelial-like tumor), in contrast to K1735-M2 (a melanoma), has been found to produce great amounts of PGE2. Challenge of MCG 101 cells with PGE2 (0.284 and 2.84 microM for 9 min) elicited an increase in ECAR by about 10 and 41% above basal level, respectively. Pretreatment with indomethacin (0.5 microM) reduced the response to the two PGE2 concentrations by about 70 and 25%, respectively. In contrast, PGE2 caused virtually no response in K1735-M2 cells. Moreover, NE caused increases in ECAR in both cell types, possibly via beta3-adrenoceptors, as investigated pharmacologically in MCG 101, and by immunocytochemistry in both cell lines. The results obtained strongly suggest functional receptors for PGE2 in MCG 101, but not K1735-M2 tumor cells. Functional receptors for NE were demonstrated in both cell lines. There is possibly an autocrine loop in the MCG 101 cells, in which PGE2 activates cyclooxygenase.

Mutations in p53 is a most common genetic alteration in human cancer and has been related to outcome, also in colorectal cancer. However, published results are not unanimous on this point without clear-cut explanations. Different analytical methods have been applied which could explain some discrepancies. Another factor of importance may be the source of nucleic acids used for identification of sequence alterations. Therefore we compared mutations in exons 7 and 8 of p53 found in genomic DNA, with mutations in cDNA from the same patients and evaluated to what extent LOH and mutations in p53 occurred simultaneously, which are prerequisites for a complete loss of p53 function according to the classic concept. cDNA and genomic DNA from tumor tissue from 123 patients were used. Thirty-four mutations were found in both tumor cDNA and genomic DNA. Twenty missense mutations were the same in both cDNA and genomic DNA. Two missense mutations, one 1 bp deletion and one nonsense mutation were only found in genomic DNA, while nine missense mutations and one 9 bp deletion were found in cDNA only. Statistical analysis showed no significant difference among mutations in cDNA and genomic DNA (p=1.00). Only 2 patients (5%) had both LOH and mutations in exons 2-11 of p53. One patient had LOH without p53 mutation, while 18 patients (44%) showed p53 mutation without LOH and 20 patients (49%) had wt p53 without LOH. Forty-four patients (52%) were non-informative for LOH of the entire cohort consisting of 85 patients. Our findings suggest that different results reported on the role mutated p53 may have in colorectal cancer are probably not explained by the source of nucleic acids (RNA vs DNA) for sequence determinations. Rather, unknown information about simultaneous but different presence of p53 LOH in previously published reports is more likely. However, an unexpectedly infrequent occurrence (5-10%) of complete and isolated p53 ablation (LOH + mutation) would demand considerably larger patient populations (1500-2000 patients) than ever published for relating p53 function to survival in patients with colorectal cancer. Therefore, the role of p53 dysfunction in progression of colorectal cancer remains uncertain.

It is well-recognized that cycloogygenase inhibitors attenuate tumor growth in tumor models, although underlying mechanisms are unclear. In the present study we report that indomethacin retards MCG-101 tumor growth on mice by induction of apoptosis/necrosis and inhibits telomere elongation. The inhibition of telomerase activity by NSAIDs (indomethacin, mobic, sulindac sulfone, suramin) was, however, not a universal finding, since a mouse melanoma (K1735-M2) did not respond. By contrast, a human cell line of colon carcinoma origin (HT-29), responded by both retarded growth and telomerase activity despite a low intrinsic production of prostaglandins, mainly PGE 2 . Therefore, it is not likely that indomethacin inhibition of tumor growth and telomere elongation is directly related to Cox-1/Cox-2 activities in tumor cells. Also, NSAIDs at 25 μM (sulindac sulfone) decreased growth and telomerase activity in MCG-101 cells, without any effects on PGE 2 production, while ibuprofen reduced PGE 2 production but had no effect on growth or telomerase activity. Our results demonstrate that cyclooxygenase inhibitors can retard tumor growth both in murine tumors and in human tumor cells by inhibition of telomerase activity in addition to previously recognized mechanisms as induction of apoptosis, inhibition of cell proliferation, influence on the expression of growth factors around growing tumors and attenuation of neoangiogenesis.

BACKGROUND The appearance of p53 mutations in colorectal carcinoma was determined, independent of differentiation and tumor stage of the primary tumors, in relation to the survival of patients who were scheduled to undergo liver resection. METHODS Tumor material was analyzed for p53 mutations in primary colorectal tumors and subsequent liver metastases from 41 consecutive patients who were scheduled to undergo surgical liver resection. DNA sequencing and immunohistochemical staining of p53 protein within tumor nuclei were performed. RESULTS Primary tumors displayed p53 mutations within exons 5–9 in 41% of patients. No mutations were found in exons 4, 10, or 11. Forty-one percent of metastatic lesions had the same single mutation that was found in the primary tumor, whereas 11% of metastatic lesions had one additional mutation within exons 5–9; 22% had mutations only in their liver metastases, whereas corresponding primary tumors displayed wild-type p53. None of the patients had mutated p53 in their primary tumor and wild type in their metastases. Survival after undergoing liver resection was correlated negatively (P 0.7–0.8 mm) of resected liver metastases. CONCLUSIONS The presence of p53 mutations in patients with metastatic lesions was related significantly (P < 0.003) to better survival after the patients underwent liver resection compared with patients with wild type p53 in their metastatic lesions. This finding was not related to covariates, such as Duke classification, tumor differentiation, type of liver metastasis, or metastatic radicality during resections. Explanations for this unexpected finding remain unclear, although the authors speculate that occult tumor cells with p53 mutations may be less responsive to growth factor(s) exposure during hepatic regeneration after resection. Cancer 2001;91:727–36. © 2001 American Cancer Society.

The role of amino acids, glucose and lipids in improving amino acid balance in peripheral tissues was evaluated. Primed constant infusion of L-[ring-2H5]phenylalanine in combination with flux measurements of glucose, free fatty acids (FFA) and amino acids across arm and leg tissues were applied in male volunteers after an overnight fast with subsequent primed constant infusions of amino acids (0.2 g N kg-1 body weight day-1), long-chain triglycerides (0.98-1.079 g kg-1 day-1) and glucose (3.13-3.62 g kg-1 day-1). Amino acids and phenylalanine tracer infusion continued for 6 h; the lipid infusion was provided during 2-6 h from the start, and glucose infusion was provided between 4 and 6 h. Flux measurements were performed at steady state before the next infusion started. Arterial concentrations of infused substrates increased during provision, but remained constant thereafter. Plasma insulin increased when glucose was provided, whereas insulin-like growth factor (IGF) I was unchanged during all infusions. Blood flow was unchanged in arm tissue during all infusions, while leg blood flow increased during fat and glucose infusion. FFA and glucose balance were unchanged during amino acid infusion but improved during lipid and glucose infusions. Amino acid balance was negative across arm and leg tissues in the fasted state, but reached balance during amino acid infusion. This effect was equally dependent on protein synthesis and protein degradation without any contribution from lipids and glucose. 3-Methylhistidine release from tissues was not influenced by any substrate. Our results suggest that extracellular amino acid concentrations determine amino acid balance across peripheral tissues independently of non-protein calories, insulin and IGF-I.

The intact plasma membrane of animal cells in culture is considered to be almost impermeable to ATP. Thus, leakage of intracellular ATP to the extracellular medium is indicative of membrane damage. We have developed a rapid and simple method for determination of ATP leakage, using L-1210 and ELD cells incubated with velleral or isovelleral (two terpenes). ATP was determined by a bioluminescent firefly luciferin-luciferase reaction in a two-step assay in the same sample. Extracellular ATP (= leakage) was measured directly in cell suspensions, and total ATP was determined in cell lysate suspensions after addition of a detergent. ATP leakage (% of total ATP) increased after incubation with both terpenes, in a dose-dependent manner.