Increased Sociability in Mice Lacking Intergenic Dlx Enhancers.

The Dlx homeodomain transcription factors play important roles in the differentiation and migration of GABAergic interneuron precursors. The mouse and human genomes each have six Dlx genes organized into three convergently transcribed bigene clusters (Dlx1/2, Dlx3/4, and Dlx5/6) with cis-regulatory elements (CREs) located in the intergenic region of each cluster. Amongst these, the I56i and I12b enhancers from the Dlx1/2 and Dlx5/6 locus, respectively, are active in the developing forebrain. I56i is also a binding site for GTF2I, a transcription factor whose function is associated with increased sociability and Williams-Beuren syndrome. In determining the regulatory roles of these CREs on forebrain development, we have generated mutant mouse-lines where Dlx forebrain intergenic enhancers have been deleted (I56i(-/-), I12b(-/-)). Loss of Dlx intergenic enhancers impairs expression of Dlx genes as well as some of their downstream targets or associated genes including Gad2 and Evf2. The loss of the I56i enhancer resulted in a transient decrease in GABA+ cells in the developing forebrain. The intergenic enhancer mutants also demonstrate increased sociability and learning deficits in a fear conditioning test. Characterizing mice with mutated Dlx intergenic enhancers will help us to further enhance our understanding of the role of these Dlx genes in forebrain development.

Extraglottic Airway Device Misplacement: A Novel Classification System and Findings in Postmortem Computed Tomography.

STUDY OBJECTIVE: Extraglottic airway devices are frequently used during cardiac arrest resuscitations and for failed intubation attempts. Recent literature suggests that many extraglottic airway devices are misplaced. The aim of this study is to create a classification system for extraglottic airway device misplacement and describe its frequency in a cohort of decedents who died with an extraglottic airway device in situ. METHODS: We assembled a cohort of all decedents who died with an extraglottic airway device in situ and underwent postmortem computed tomographic (CT) imaging at the state medical examiner's office during a 6-year period, using retrospective data. An expert panel developed a novel extraglottic airway device misplacement classification system. We then applied the schema in reviewing postmortem CT for extraglottic airway device position and potential complications. RESULTS: We identified 341 eligible decedents. The median age was 47.0 years (interquartile range 32 to 59 years). Out-of-hospital personnel placed extraglottic airway devices in 265 patients (77.7%) who subsequently died out of hospital; the remainder died inhospital. The classification system consisted of 6 components: depth, size, rotation, device kinking, mechanical blockage of ventilation opening, and injury. Under the system, extraglottic airway devices were found to be misplaced in 49 cases (14.4%), including 5 (1.5%) that resulted in severe injuries. CONCLUSION: We created a novel extraglottic airway device misplacement classification system. Misplacement occurred in greater than 14% of cases. Severe traumatic complications occurred rarely. Quality improvement activities should include review of extraglottic airway device placement when CT images are available and use the classification system to describe misplacements.

Postmortem Computed Tomography in Firearm Homicides: A Retrospective Case Series.

Postmortem computed tomography (PMCT) is integrated into the evaluation of decedents in several American medical examiner offices and medicolegal death investigative centers in many other countries. We retrospectively investigated the value of PMCT in a series of firearm homicide cases from a statewide centralized medical examiner's office that occurred during 2016. Autopsies were performed or supervised by board-certified forensic pathologists who reviewed the PMCT scans prior to autopsy. PMCT scans were re-evaluated by a forensic radiologist blinded to the autopsy findings and scored by body region (head-neck, thoracoabdominal, and extremities). Injury discrepancies were scored using a modified Goldman classification and analyzed with McNemar's test. We included 60 males and 20 females (median age 31 years, range 3-73). Based on PMCT, 56 (79.1%) cases had injuries relevant to the cause of death in a single body region (24 head-neck region, 32 thoracoabdominal region). Out of these 56 cases, 9 had a missed major diagnosis by PMCT outside that region, including 6 extremity injuries visible during standard external examination. Yet all had evident lethal firearm injury. We showed that PMCT identifies major firearm injuries in homicide victims and excludes injuries related to the cause of death in other regions when a single body region is injured. Although PMCT has a known limited sensitivity for soft tissue and vascular pathology, it can be combined with external examination to potentially reduce or focus dissections in some of these cases depending on the circumstances and medicolegal needs.

The Role of Postmortem Computed Tomography in the Evaluation of Strangulation Deaths.

We explored the value of postmortem computed tomography (PMCT) to augment autopsy in evaluating strangulation fatalities. A literature search identified 16 studies describing autopsy findings in 576 deaths and two studies describing autopsy and PMCT findings in six deaths. Similar cases were identified from our institution, yielding 130 deaths with autopsy findings and 14 deaths with both autopsy and PMCT findings. The presence of laryngohyoid fracture and soft tissue hemorrhage was compared from autopsy and autopsy+PMCT cases. The detection rates of fractures in autopsy and autopsy+PMCT cases were not significantly different. PMCT identified all fractures observed at autopsy and five fractures not identified. While PMCT may not detect soft tissue injuries in decomposed remains or subtle internal hemorrhages in neck injury, it is equally able to detect bony injuries as autopsy and might surpass autopsy in detecting subtle fractures. We conclude PMCT is useful to supplement autopsy in strangulation cases.

Comparative radiologic identification with standardized single CT images of the paranasal sinuses-Evaluation of inter-rater reliability.

The aim of this study was to assess the reproducibility of a standardized image for personal identification (SIPI), used in the comparative analysis of paranasal sinuses, and test the effect of inaccurate reformation of the SIPI on suitability for comparative identification. Five raters with different professional backgrounds independently reformatted SIPIs from ten post-mortem head CTs. Inter-rater, intra-rater agreement as well angular deviations between reformatted SIPI images and reference SIPI images were calculated. Second, raters assessed the suitability of 70 accurately and inaccurately reformatted SIPIs for identification with a 4-point Likert scale. Inter-rater agreement as well as levels of significance regarding image suitability were calculated. Inter-rater agreement regarding reproducibility of SIPI reformation was excellent (inter-rater correlation coefficient (ICC) 0.9995, intra-rater ICC 0.9983). Deviation between the angular dimensions of the reformatted SIPIs and the reference SIPIs was ≤1° in 94% of all 300 measurements. Inter-rater agreement regarding the effect of inaccurate SIPI reformation on suitability for comparative identification was fair (ICC 0.6809). There was no statistically significant difference between raters' evaluation of image suitability (p=0.9755). This study shows that the standardized image for personal identification can be accurately reformatted by different raters with varying professional backgrounds. In addition, raters agree that inaccurately reformatted SIPIs are still suitable for comparative identification in the majority of cases.

Neonatal rat age, sex and strain modify acute antioxidant response to ozone.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US and its impact continues to increase in women. Oxidant insults during critical periods of early life appear to increase risk of COPD through-out the life course. To better understand susceptibility to early life exposure to oxidant air pollutants we used Fisher (F344), Sprague-Dawley (SD) and Wistar (WIS) male and female neonatal rat pups to assess: (A) if strain (i.e. genetics), sex, or stage of early life development affected baseline lung antioxidant or redox enzyme levels and (B) if these same factors modulated antioxidant responsiveness to acute ozone exposure (1 ppm × 2 h) on post-natal day (PND) 14, 21, or 28. In air-exposed pups from PND14-28, some parameters were unchanged (e.g. uric acid), some decreased (e.g. superoxide dismutase), while others increased (e.g. glutathione recycling enzymes) especially post-weaning. Lung total glutathione levels decreased in F344 and SD pups, but were relatively unchanged in WIS pups. Post-ozone exposure, data suggest that: (1) the youngest (PND14) pups were the most adversely affected; (2) neonatal SD and WIS pups, especially females, were more prone to ozone effects than males of the same age and (3) F344 neonates (females and males) were less susceptible to oxidative lung insult, not unlike F344 adults. Differences in antioxidant levels and responsiveness between sexes and strains and at different periods of development may provide a basis for assessing later life health outcomes - with implications for humans with analogous genetic or dietary-based lung antioxidant deficits.

Accuracy of non-contrast PMCT for determining cause of death.

The aim of this study was (1) to compare levels of accuracy regarding the categorization of causes of death between non-contrast post-mortem computed tomography (PMCT) and the final forensic report as well as between autopsy and the final forensic report, and (2) to assess levels of confidence regarding the categorization of causes of death after non-contrast PMCT and after autopsy. This prospective study was conducted over a 5 month period during which 221 cases were admitted to our institute for forensic investigations. Whole-body PMCT and forensic autopsy were performed in every case. Of these, 101 cases were included in the final study population. Inclusion criteria were: (1) age > 18 years, (2) presence of at least one of the two principal investigators at the time of admission. One radiologist and one forensic pathologist independently read all PMCT datasets using a report template. Cause of death category and confidence levels were determined by consensus. Forensic autopsy was performed by two forensic pathologists; both unblinded to imaging results. Both post-imaging and post-autopsy cause of death categorization were compared against the final cause of death, as stated in the forensic expert report, which included findings from histology and/or toxicology. Accuracy of post-imaging cause of death categorization in reference to the final cause of death category was substantial (82%, 83/101 cases, Kappa 0.752). Accuracy of post-autopsy cause of death categorization in reference to the final cause of death category was near perfect (89%, 90/101 cases, Kappa 0.852). Post-imaging sensitivity and specificity regarding the categorization of causes of death were 82% and 97%, respectively. Post-autopsy sensitivity and specificity regarding the categorization of causes of death were 89% and 98%, respectively. There was a high consistency between the accuracy of post-imaging cause of death categorization and post-imaging levels of confidence. There was less consistency between accuracy of post-autopsy cause of death categorization and post-autopsy levels of confidence. In this study categorization of causes of death based on non-contrast enhanced PMCT alone, and on PMCT and macroscopic autopsy together, proved to be consistent with the final cause of death-category as determined based on all available information including PMCT, autopsy, and (if available) histology and/or toxicology in more than 82% and 89% of all cases, respectively. There was higher consistency between levels of confidence and accuracy of causes of death categorization was higher post-imaging than post-autopsy. These results underline the fact that the diagnostic potential of PMCT goes beyond the assessment of trauma cases.

Antemortem identification by fusion of MR and CT of the paranasal sinuses.

Radiologic forensic identification is usually performed by comparing antemortem and postmortem radiographs. While computed tomography (CT) has become a valuable addition to radiologic identification, magnetic resonance (MR) imaging has only rarely been used for this purpose. In our case, identification was accomplished using fused MR- and CT images in a survivor of a gunshot injury to the head. This case supports and highlights the possibility to perform intermodality radiologic identification comparing preexisting MR imaging to subsequently aquired CT data in living (or deceased) humans as long as manual modifications of windowing, color and contrast enable differentiation of the two modalities in the fused image.

A new method for estimating patient body weight using CT dose modulation data.

BACKGROUND: Body weight (BW) is a relevant metric in emergency care. However, visual/physical methods to estimate BW are unreliable. We have developed a method for estimating BW based on effective mAs (mAseff) from computed tomography (CT) dose modulation. METHODS: The mAseff of CT examinations was correlated with the BW of 329 decedents. Linear regression analysis was used to calculate an equation for BW estimation based on the results of decedents with a postmortem interval (PMI) < 4 days (n = 240). The equation was applied to a validation group of 125 decedents. Pearson correlation and t-test statistics were used. RESULTS: We found an overall strong correlation between mAseff and BW (r = 0.931); r values ranged from 0.854 for decedents with PMI ≥ 4 days to 0.966 for those with PMI < 4 days; among the latter group, r was 0.974 for females and 0.960 for males and 0.969 in the presence and 0.966 in the absence of metallic implants (all correlations with p values < 0.001). The estimated BW was equal to 3.732 + (0.422 × mAseff) - (3.108 × sex index), where the sex index is 0 for males and 1 for females. The validation group showed a strong correlation (r = 0.969) between measured BW and the predicted BW, without significant differences overall (p = 0.119) as well as in female (p = 0.394) and in male decedents (p = 0.196). No outliers were observed. CONCLUSIONS: CT dose modulation is a rapid and reliable method for BW estimation with potential use in clinical practice, in particular in emergency settings.

Comparative Evaluation of Potentially Radiolucent Projectile Components by Radiographs and Computed Tomography.

Projectile components that are traditionally radiolucent can be of considerable importance in determination of weapon type and caliber, but they are often missed on evaluation of postmortem radiographs. We hypothesized that these components would be significantly better visualized by evaluation of computed tomography (CT) scans compared to the practice standard of radiography alone. In this project, potentially radiolucent projectile components were both pulled apart and fired, and the radiolucent components were recovered. These components were embedded in blocks of ballistics gelatin and were imaged using both radiography and CT. The scans were evaluated by three blinded, board-certified radiologists for the presence/absence of projectile components and true-negative regions in each block. If a radiologist indicated visualization of a projectile component, they were further requested to describe their observation. It was found that traditionally radiolucent projectile components are not significantly more often identified on CT scans than radiography (p < 0.05).

Distal tibia allograft for glenohumeral instability: does radius of curvature match?

BACKGROUND: A distal tibia osteochondral allograft is a potential graft option for glenoid reconstruction because the distal tibia may have a similar radius of curvature (ROC) as the glenoid. This study evaluated ROC mismatch as measured on computed tomography (CT) scans between the glenoid, distal tibia, and humeral head. METHODS: Bilateral CT images were formatted for 10 decedents from the Office of the Medical Investigator database, giving 20 specimens per anatomic location. The ROCs of the glenoid, distal tibia, and humeral head were measured. A statistical model was generated to assess ROC mismatch of randomly paired distal tibias and glenoids. RESULTS: The mean ± standard deviation ROC was 2.9 ± 0.25 cm for the glenoid, 2.3 ± 0.21 cm for the distal tibia, and 2.5 ± 0.12 cm for the humeral head. No differences were found in laterality, intraobserver, or interobserver measurements. The least-squares difference in the ROC between the glenoid and tibia was 0.57 cm, glenoid and humerus was 0.40 cm, and humerus and tibia was 0.17 cm. Only 22% of randomly paired distal tibias and glenoids had a difference in ROC of 0.3 cm or less. CONCLUSION: CT measurement of the ROC of the glenoid, distal tibia, and humeral head is reliable and reproducible. The probability of obtaining a random distal tibia allograft with a similar ROC to the glenoid is low. Obtaining ROC measurements of the injured glenoid and the distal tibia allograft specimen before use for glenoid reconstruction may be useful.

Strain differences in antioxidants in rat models of cardiovascular disease exposed to ozone.

We examined the hypothesis that antioxidant substances and enzymes in lung, heart and in bronchoalveolar lavage fluid (BALF) are altered in response to O3 in cardiovascular disease and/or metabolic syndrome (CVD)-prone rat models. CVD strains [spontaneously hypertensive (SH), SH stroke-prone (SHSP), SHHF/Mcc heart failure obese (SHHF), insulin-resistant JCR:LA-cp obese (JCR) and Fawn-Hooded hypertensive (FHH)] were compared with normal strains [Wistar, Sprague-Dawley (SD) and Wistar Kyoto (WKY)]. Total glutathione (GSH + GSSG or GSx), reduced ascorbate (AH2), uric acid (UA) and antioxidant enzymes were determined in lung, heart and BALF immediately (0 h) or 20-h post 4-h nose-only exposure to 0.0, 0.25, 0.5 and 1.0 ppm O3. Basal- and O3-induced antioxidant substances in tissues varied widely among strains. Wistar rats had a robust O3-induced increase in GSx and AH2 in the lung. Two CVD strains (JCR and SHHF) had high basal levels of AH2 and GSx in BALF as well as high basal lung UA. Across all strains, high BALF GSx was only observed when high BALF AH2 was present. CVD rats tended to respond less to O3 than normal. High-basal BALF AH2 levels were associated with decreased O3 toxicity. In summary, large differences were observed between both normal and CVD rat strains in low-molecular weight antioxidant concentrations in lung, BALF and heart tissue. Wistar (normal) and JCR and SHHF (CVD) rats appeared to stand out as peculiar in terms of basal- or O3-induced changes. Results elucidate interactions among antioxidants and air pollutants that could enhance understanding of cardiopulmonary disease.

Postmortem magnetic resonance imaging: Reproducing typical autopsy heart measurements.

PURPOSE: The aim of this study was to evaluate the utility of cardiac postmortem magnetic resonance (PMMR) to perform routine measurements of the ventricular wall thicknesses and the heart valves and to assess if imaging measurements are consistent with traditional autopsy measurements. METHODS: In this retrospective study, 25 cases with cardiac PMMR and subsequent autopsy were included. The thicknesses of the myocardial walls as well as the circumferences of all heart valves were measured on cardiac PMMR and compared to autopsy measurements. Paired samples T-test and the Wilcoxon-Signed rank test, were used to compare autopsy and cardiac PMMR measurements. For exploring correlations, the Pearson's Correlation coefficient and the Spearman's Rho test were used. RESULTS: Cardiac PMMR measurements of the aortic and pulmonary valve circumferences showed no significant differences from autopsy measurements. The mitral and tricuspid valves circumferences differed significantly from autopsy measurements. Left myocardial and right myocardial wall thickness also differed significantly from autopsy measurements. Left and right myocardial wall thickness, and tricuspid valve circumference measurements on cardiac PMMR and autopsy, correlated strongly and significantly. CONCLUSION: Several PMMR measurements of cardiac parameters differ significantly from corresponding autopsy measurements. However, there is a strong correlation between cardiac PMMR measurements and autopsy measurements in the majority of these parameters. It is important to note that myocardial walls are thicker when measured in situ on cardiac PMMR than when measured at autopsy. Investigators using post-mortem MR should be aware of these differences in order to avoid false diagnoses of cardiac pathology based on cardiac PMMR.

Biomarkers of oxidative stress study VI. Endogenous plasma antioxidants fail as useful biomarkers of endotoxin-induced oxidative stress.

This is the newest report in a series of publications aiming to identify a blood-based antioxidant biomarker that could serve as an in vivo indicator of oxidative stress. The goal of the study was to test whether acutely exposing Göttingen mini pigs to the endotoxin lipopolysaccharide (LPS) results in a loss of antioxidants from plasma. We set as a criterion that a significant effect should be measured in plasma and seen at both doses and at more than one time point. Animals were injected with two doses of LPS at 2.5 and 5 µg/kg iv. Control plasma was collected from each animal before the LPS injection. After the LPS injection, plasma samples were collected at 2, 16, 48, and 72 h. Compared with the controls at the same time point, statistically significant losses were not found for either dose at multiple time points in any of the following potential markers: ascorbic acid, tocopherols (α, δ, γ), ratios of GSH/GSSG and cysteine/cystine, mixed disulfides, and total antioxidant capacity. However, uric acid, total GSH, and total Cys were significantly increased, probably because LPS had a harmful effect on the liver. The leakage of substances from damaged cells into the plasma may have increased plasma antioxidant concentrations, making changes difficult to interpret. Although this study used a mini-pig animal model of LPS-induced oxidative stress, it confirmed our previous findings in different rat models that measurement of antioxidants in plasma is not useful for the assessment of oxidative damage in vivo.

Progress in assessing air pollutant risks from in vitro exposures: matching ozone dose and effect in human airway cells.

In vitro exposures to air pollutants could, in theory, facilitate a rapid and detailed assessment of molecular mechanisms of toxicity. However, it is difficult to ensure that the dose of a gaseous pollutant to cells in tissue culture is similar to that of the same cells during in vivo exposure of a living person. The goal of the present study was to compare the dose and effect of O3 in airway cells of humans exposed in vivo to that of human cells exposed in vitro. Ten subjects breathed labeled O3 ((18)O3, 0.3 ppm, 2 h) while exercising intermittently. Bronchial brush biopsies and lung lavage fluids were collected 1 h post exposure for in vivo data whereas in vitro data were obtained from primary cultures of human bronchial epithelial cells exposed to 0.25-1.0 ppm (18)O3 for 2 h. The O3 dose to the cells was defined as the level of (18)O incorporation and the O3 effect as the fold increase in expression of inflammatory marker genes (IL-8 and COX-2). Dose and effect in cells removed from in vivo exposed subjects were lower than in cells exposed to the same (18)O3 concentration in vitro suggesting upper airway O3 scrubbing in vivo. Cells collected by lavage as well as previous studies in monkeys show that cells deeper in the lung receive a higher O3 dose than cells in the bronchus. We conclude that the methods used herein show promise for replicating and comparing the in vivo dose and effect of O3 in an in vitro system.

West nile virus transmission in winter: the 2013 great salt lake bald eagle and eared grebes mortality event.

West Nile Virus (WNV) infection has been reported in over 300 species of birds and mammals. Raptors such as eagles, hawks and falcons are remarkably susceptible, but reports of WNV infection in Bald Eagles (Haliaeetus leucocephalus) are rare and reports of WNV infection in grebes (Podicipediformes) even rarer. We report an unusually large wild bird mortality event involving between 15,000-20,000 Eared Grebes (Podiceps nigricollis) and over 40 Bald Eagles around the Great Salt Lake, Utah, in November-December 2013. Mortality in grebes was first reported in early November during a period when the area was unseasonably warm and the grebes were beginning to gather and stage prior to migration. Ten out of ten Eared Grebes collected during this period were WNV RT-PCR and/or isolation positive. This is the first report of WNV infection in Eared Grebes and the associated mortality event is matched in scale only by the combined outbreaks in American White Pelican (Pelecanus erythrorhynchos) colonies in the north central states in 2002-2003. We cannot be sure that all of the grebes were infected by mosquito transmission; some may have become infected through contact with WNV shed orally or cloacally from other infected grebes. Beginning in early December, Bald Eagles in the Great Salt Lake area were observed to display neurological signs such as body tremors, limb paralysis and lethargy. At least 43 Bald Eagles had died by the end of the month. Nine of nine Bald Eagles examined were infected with WNV. To the best of our knowledge, this is the largest single raptor mortality event since WNV became endemic in the USA. Because the majority of the eagles affected were found after onset of below-freezing temperatures, we suggest at least some of the Bald Eagles were infected with WNV via consumption of infected Eared Grebes or horizontal transmission at roost sites.

Fate of pathologically bound oxygen resulting from inhalation of labeled ozone in rats.

Inhaled ozone (O3) reacts chemically with respiratory tract biomolecules where it forms covalently bound oxygen adducts. We investigated the fate of these adducts following inhalation exposure of rats to labeled ozone ((18)O3, 2 ppm, 6 hr or 5 ppm, 2 hr). Increased (18)O was detected in blood plasma at 7 hr post exposure and was continuously present in urine for 4 days. Total (18)O excreted was ~53% of the estimated amount of (18)O3 retained by the rats during (18)O3 exposure suggesting that only moderate recycling of the adduct material occurs. The time course of excretion, as well as properties of the excreted (18)O were determined to provide guidance to future searches for urinary oxidative stress markers. These results lend plausibility to published findings that O3 inhalation could exert influences outside the lung, such as enhancement of atherosclerotic plaques.

Biomarkers of Dose and Effect of Inhaled Ozone in Resting versus Exercising Human Subjects: Comparison with Resting Rats.

To determine the influence of exercise on pulmonary dose of inhaled pollutants, we compared biomarkers of inhaled ozone (O3) dose and toxic effect between exercise levels in humans, and between humans and rats. Resting human subjects were exposed to labeled O3 ((18)O3, 0.4 ppm, for 2 hours) and alveolar O3 dose measured as the concentration of excess (18)O in cells and extracellular material of nasal, bronchial, and bronchoalveolar lavage fluid (BALF). We related O3 dose to effects (changes in BALF protein, LDH, IL-6, and antioxidant substances) measurable in the BALF. A parallel study of resting subjects examined lung function (FEV1) changes following O3. Subjects exposed while resting had (18)O concentrations in BALF cells that were 1/5th of those of exercising subjects and directly proportional to the amount of O3 breathed during exposure. Quantitative measures of alveolar O3 dose and toxicity that were observed previously in exercising subjects were greatly reduced or non-observable in O3 exposed resting subjects. Resting rats and resting humans were found to have a similar alveolar O3 dose.

Biomarkers of oxidative stress study V: ozone exposure of rats and its effect on lipids, proteins, and DNA in plasma and urine.

Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins, and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F2-isoprostanes, protein carbonyls, methionine oxidation, and tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F2-isoprostanes were investigated with various techniques. The criterion used to recognize a marker in the model of ozone exposure was that a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and the control groups at either ozone dose and time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criteria of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies.