Air pollution accountability research: Moving from a chain to a web.

Air pollution accountability studies examine the relationship(s) between an intervention, regulation, or event and the resulting downstream impacts, if any, on emissions, exposure, and/or health. The sequence of events has been schematically described as an accountability chain. Here, we update the existing framework to capture real-life complexities and to highlight important factors that fall outside the linear chain. This new "accountability web" is intended to convey the intricacies associated with conducting an accountability study to various audiences, including researchers, policy makers, and stakeholders. We also identify data considerations for planning and completing a robust accountability study, including those relevant to novel and innovative air pollution and exposure data. Finally, we present a series of recommendations for the accountability research community that can serve as a guide for the next generation of accountability studies.

Polymeric gene delivery of ischemia-inducible VEGF significantly attenuates infarct size and apoptosis following myocardial infarct.

The development of clinically beneficial myocardial gene therapy has been slowed by reliance on the use of viral carriers and non-physiologic, constitutive gene expression. To specifically address these issues, we have developed a non-viral gene carrier, water-soluble lipopolymer (WSLP), and an ischemia-inducible plasmid construct expressing vascular endothelial growth factor (VEGF), pRTP801-VEGF, to treat myocardial ischemia and infarction. Rabbits underwent ligation of the circumflex artery followed by injection of (a) an ischemia-inducible VEGF gene construct in a WSLP carrier; (b) a constitutively expressed, or unregulated, SV-VEGF gene construct in a WSLP carrier; (c) WSLP carrier alone; or (d) no injection therapy. Following 4 weeks treatment, ligation alone resulted in infarction of 48+/-7% of the left ventricle. With injection of WSLP carrier alone, 49+/-6% of the left ventricle was infarcted (P=NS). The constitutively expressed gene construct, SV-VEGF, reduced the infarct size to 32+/-7% of the left ventricle (P=0.007). The ischemia-inducible gene construct, RTP801-VEGF, further reduced the infarct size to 13+/-4% of the left ventricle (P<0.001). The use of a non-viral carrier to deliver an ischemia-inducible VEGF construct is effective in the treatment of acutely ischemic myocardium.

Identification of a unique epigenetic sub-microenvironment in prostate cancer.

The glutathione S-transferase P1 (GSTP1) gene promoter is methylated in tumour cells in more than 90% of prostate carcinomas. Recently, GSTP1 promoter methylation was identified in tumour-associated stromal cells in addition to the tumour epithelium. To define the extent and location of stromal methylation, epigenetic mapping using pyrosequencing quantification of GSTP1 promoter methylation and an anatomical three-dimensional reconstruction of an entire human prostate specimen with cancer were performed. Normal epithelium and stroma, tumour epithelium, and tumour-associated stromal cells were laser capture-microdissected from multiple locations throughout the gland. As expected, the GSTP1 promoter in both normal epithelium and normal stromal cells distant from the tumour was not methylated and the tumour epithelium showed consistently high levels of promoter methylation throughout. However, tumour-associated stromal cells were found to be methylated only in a localized and distinct anatomical sub-field of the tumour, revealing the presence of an epigenetically unique microenvironment within the cancer. Morphologically, the sub-field consisted of typical, non-reactive stroma, representing a genomic alteration in cells that appeared otherwise histologically normal. Similar epigenetic anatomical mapping of a control prostate gland without cancer showed low background methylation levels in all cell types throughout the specimen. These data suggest that stromal cell methylation can occur in a distinct sub-region of prostate cancer and may have implications for understanding tumour biology and clinical intervention.

Effects of a specific endothelin-1A antagonist on exercise-induced pulmonary haemorrhage (EIPH) in thoroughbred horses.

REASONS FOR PERFORMING STUDY: During high intensity exercise, the very high pulmonary artery pressure (Ppa) experienced by Thoroughbred horses is considered a major factor in the aetiology of exercise-induced pulmonary haemorrhage (EIPH). Recently, endothelin-1 (ET-1), a potent vasoconstrictive hormone, has been found to increase Ppa in horses at rest via binding to its ET-1A receptor subtype. In addition, plasma concentrations of ET-1 are increased in horses during and after high intensity exercise. HYPOTHESIS: If ET-1 increases Ppa during exercise in the horse, administration of a specific ET-1A antagonist would decrease Ppa and therefore EIPH. METHODS: Saline (CON) or an ET-1A receptor antagonist, TBC3214 (3 mg/kg bwt i.v.; ANTAG) was administered to horses 1 h prior to maximal incremental exercise on a high-speed treadmill. Gas exchange measurements were made breath-by-breath and blood samples collected during each 1 min stage to determine blood gases, acid-base status and cardiac output. EIPH was determined via bronchoalveolar lavage (BAL) approximately 30 min after exercise. RESULTS: The time to fatigue, gas exchange and cardiovascular responses were not different between groups (P>0.05). Resting and peak Ppa did not differ significantly between treatments. Most importantly, ANTAG did not decrease EIPH. CONCLUSIONS: These results do not support a deterministic role for ET-1 in the increased Ppa and therefore EIPH, during maximal exercise in the equine athlete. POTENTIAL RELEVANCE: Treatment with an ET-1A receptor antagonist does not appear to be a viable therapeutic intervention in the prevention of EIPH.

Exercise-induced pulmonary haemorrhage during submaximal exercise.

REASONS FOR PERFORMING STUDY: Maximally exercising horses achieve mean pulmonary artery pressures (Ppa(mean)) that exceed the minimum threshold (75 mmHg) estimated for pulmonary capillary rupture and exercise-induced pulmonary haemorrhage (EIPH). EIPH is not expected to occur during moderate submaximal exercise (i.e. 40-60% VO2max) since Ppa(mean) remains well below this threshold. HYPOTHESIS: Prolonged submaximal exercise (trotting) would precipitate locomotory respiratory uncoupling and cause EIPH. This would be present as a result of the most negative intrapleural pressures (as estimated by the minimum oesophageal pressure; Poes(min)) occurring simultaneously with the most positive Ppa (Ppa(peak)) to produce estimated maximal pulmonary artery transmural pressures (PATMPmax) that surpass the EIPH threshold. METHODS: Five Thoroughbred horses trotted to fatigue (approximately 25 min) at 5 m/sec on a 10% incline. Ventilation (V(E)), Poes, and Ppa were measured at 5 min intervals, and bronchoalveolar lavage (BAL) red blood cells (RBCs) were quantified 45 min post exercise. RESULTS: BAL revealed an increased EIPH (rest: 2.0 +/- 1 x 10(5), exercise: 17 +/- 10 x 10(5) RBCs/ml BALF; P<0.05), despite the highest Ppamean reaching only mean +/- s.e. 55 +/- 3 mmHg, while V(E), tidal volume and Poes(min) approached 70-80% of the values achieved at maximal running speeds (10% incline: 12-13 m/sec) by these same horses. The resulting PATMPmax was well above the level considered causative of EIPH. CONCLUSIONS: The finding of significant EIPH during submaximal exercise broadens the spectrum of performance horses susceptible to EIPH and supports studies that suggest that extravascular factors are of primary importance in the aetiology of EIPH. POTENTIAL RELEVANCE: Consideration of strategies such as the equine nasal strip for reducing negative extravascular pressures is warranted even for exercise at moderate intensities.

Ambulatory instrumentation suitable for long-term monitoring of cattle health.

The benefits of real-time health diagnoses of cattle are potentially tremendous. Early detection of transmissible disease, whether from natural or terrorist events, could help to avoid huge financial losses in the agriculture industry while also improving meat quality. This work discusses physiological and behavioral parameters relevant to cattle state-of-health assessment. These parameters, along with a potentially harsh monitoring environment, drive a set of design considerations that must be addressed when building systems to acquire long-term, real-time measurements in the field. A prototype system is presented that supports the measurement of suitable physiologic parameters and begins to address the design constraints for continuous state-of-health determination in free-roaming cattle.

Inclined running increases pulmonary haemorrhage in the Thoroughbred horse.

REASONS FOR PERFORMING STUDY: Capillary stress failure-induced (exercise-induced) pulmonary haemorrhage (EIPH) during intense running in horses is thought to involve both intravascular (i.e. mean pulmonary arterial pressure [Ppa] > 100 mmHg) and extravascular (e.g. negative inspiratory pressure swings) mechanisms. HYPOTHESIS: That inclined running would reduce breathing frequency (coupled to stride frequency) and increase tidal volume thus increasing lung volume changes and intrapleural pressure swings resulting in more pronounced EIPH. METHODS: Six Thoroughbred horses were run to volitional fatigue (incremental step test) on a level (L) and inclined (I; 10%) treadmill in random order. Pulmonary minute ventilation, arterial blood gases and mean Ppa were obtained during each run while EIPH severity was quantified via bronchoalveolar lavage (BAL) 30 mins post run. RESULTS: Time to fatigue did not differ between trials (P > 0.05). At end-exercise, breathing frequency was reduced (L, 127.8 +/- 3.0; I, 122.6 +/- 2.1 breaths/min; P < 0.05) and tidal volume increased (L, 11.5 +/- 0.6; I, 13.1 +/- 0.5 L; P < 0.05) during inclined running. No differences existed in end-exercise plasma [lactate] between trials (L, 24.5 +/- 2.9; I, 26.2 +/- 3.4 mmol/l, P > 0.05); however, the mean peak Ppa was reduced during the inclined run (L, 105+5; I, 96 +/- 4 mmHg, P < 0.05). In the face of reduced Ppa, EIPH severity was increased significantly (P < 0.05) during the inclined vs. level run (L, 37.0 +/- 11.7; I, 49.6 +/- 17.0 x 10(6) red blood cells/ml BAL fluid). CONCLUSIONS: Although inclined running lowered peak Ppa, EIPH severity was increased. It is likely that this effect resulted, in part, from an altered ventilatory pattern (i.e. increased tidal volumes and associated intrapleural pressure changes). POTENTIAL RELEVANCE: This conclusion supports an important role for extravascular factors in the aetiology of EIPH.

Movements of thoracic and abdominal compartments during ventilation at rest and during exercise.

The present investigation utilised simultaneous measurements of chest (Ch) and abdominal (Ab) circumferences and respiratory airflow to test the hypothesis that Ch circumferential expansion contributes proportionally little to tidal volume in the running Thoroughbred. During exercise, there were only small changes in Ch and Ab circumference and no increase with increasing tidal volume. At rest, walk and trot, the flow, Ch and Ab signals were in phase. However, during canter and gallop, the Ch and Ab changes were 180 degrees out of phase with each other and both were out of phase with airflow. In contrast to exercise, increase in ventilation at rest achieved by administration of lobeline resulted in a 4-6-fold increase in tidal volume; large excursions of the chest were always in phase with airflow. Furthermore, 3 horses showed an increase in chest circumference, demonstrating that chest stiffness per se does not preclude chest circumferential expansion. In conclusion, in the absence of significant increases in either Ch or Ab expansion during running, elongation of the thoracoabdominal segment may be the main determinant of tidal volume.

Structural and oxidative enzyme characteristics of the diaphragm.

During exercise, the horse can achieve oxygen uptakes and ventilations in excess of 200 ml/kg/min and 1800 l/min, respectively. Whether the diaphragm has the capacity to contribute substantially to inspiratory effort in the exercising horse is not known. To investigate the potential for the horse diaphragm to generate tension, lung displacement and sustain ventilatory function, we measured diaphragm thickness, muscle length and oxidative enzyme activity (citrate synthase) within the ventral, medial and dorsal costal and crural diaphragm. In the diaphragms of 6 mature horses (5 Thoroughbreds, one Quarter Horse; body mass (mean +/- s.e.) 475 +/- 14 kg, age 4 +/- 1 years), the mass of the freshly-excised diaphragm was 4.54 +/- 0.19 kg of which 79% was the costal diaphragm, 17% the crural diaphragm and 4% the central tendon. The medial costal region (2.1 +/- 0.1 cm) was significantly thicker (P<0.05) than either the ventral (1.4 +/- 0.1 cm) or dorsal (1.2 +/- 0.2 cm) costal regions and the crural diaphragm was significantly thicker (>3.2 +/- 0.3 cm, P<0.05) than any costal diaphragm region. With respect to the costal diaphragm, excised muscle length was greatest (P<0.05) in the medial costal (17.2 +/- 1.0 cm) than either the ventral costal (<12.6 +/- 1.5 cm) or dorsal costal (<13.9 +/- 1.8 cm) regions and therefore the medial region would be expected to exhibit the greatest absolute length change on inspiration. Citrate synthase activity was high throughout the diaphragm (40.8 +/- 113 to 55.3 +/- 9.7 micromol/g/min), but was not significantly different among regions. These structural characteristics and the oxidative potential of the horse diaphragm are consistent with the diaphragm providing a significant and substantial contribution to the inspiratory effort during exercise in the horse. Consequently, clinical and physiological investigations of exercise performance should not ignore the potentially crucial importance of the diaphragm.

Effect of body incline on cardiac performance.

Maximal cardiac performance is improved in man during upright compared to supine exercise. Whether cardiac performance in quadrupeds is dependent upon body position is unknown. Therefore, we undertook the present investigation to determine if peak cardiac output (Qpeak) would be influenced by body inclination in the Thoroughbred horse. To test the hypothesis, four Thoroughbred horses performed an incremental exercise protocol (speed increased by 1 m/s/min to fatigue) on both a level (L) and inclined (I: 6 degrees) treadmill. Specifically, we hypothesised that Qpeak would be increased on the incline, as this represents a progression towards upright exercise. Cardiac output was determined using the Fick relationship from continuous measurements of pulmonary VO2 and paired arterial (carotid artery or transverse facial) and mixed venous (pulmonary artery) samples. Qpeak was significantly increased on the incline (L: 279 +/- 20; I: 336 +/- 17 l/min; P<0.05), while CaO2 was not significantly different (L: 25.5 +/- 1.1; I: 25.4 +/- 1.9 ml/100 ml), and therefore, whole body O2 delivery (QO2) was significantly increased (L: 70.7 +/- 4.9; I: 84.4 +/- 3.1 l/min; P<0.05). In conclusion, within the scope of this investigation, these data suggest that cardiac performance, as judged by increased Qpeak and QO2, is enhanced in the inclined body position. Furthermore, these findings provide preliminary information that level and incline treadmill exercise tests may yield significantly different results in the Thoroughbred horse and consequently this factor should be considered when interpreting exercise testing and performance data.

Disulfide-mediated dimerization of L1 Ig domains.

The neural cell adhesion molecule L1 contains immunoglobulin-like (Ig) domains in its extracellular region that mediate homophilic binding, neurite outgrowth and other activities relevant to CNS development. To correlate conformations of these domains to biological function, several L1-Fc fusion proteins whose bioactivities were previously characterized were analyzed by rotary shadowing electron microscopy. We found that bioactive L1-Fcs containing Ig domains 1-4 or 1-6 exhibited extended, branched structures. In contrast, inactive L1-Fcs containing only the first two or three Ig domains assumed compact shapes that suggested interactions between the L1 arms of these proteins. Analysis of an untagged L1 fragment composed of Ig domains 1-3 demonstrated a mixture of monomeric and dimeric forms. Surprisingly, these dimers were stabilized by intermolecular disulfide bonds. Finally, cell surface L1-GFP fusion proteins containing only the first two or three Ig domains in the extracellular region also engaged in disulfide-mediated dimerization. These results suggest a novel mechanism by which mutations in L1 could interfere with its biological functioning.

XMAP215 is a long thin molecule that does not increase microtubule stiffness.

XMAP215 is a microtubule associated protein that speeds microtubule plus end growth by seven- to tenfold and protects these ends from destabilization by the Kin I kinesin, XKCM1. To understand the mechanisms responsible for these activities, it is necessary to know the structure of XMAP215. By unidirectional shadowing and electron microscopy, XMAP215 appeared as an elongate molecule of 60+/-18 nm, suggesting that XMAP215 could span up to seven to eight tubulin dimers along a protofilament. Most XMAP215 molecules were straight but a subset were bent suggesting that XMAP215 is flexible. Antibodies to the C terminus labeled one end of XMAP215 with no evidence for XMAP215 dimerization. Incubation of XMAP215 and tubulin at 4 degrees C resulted in assembly of curved protofilaments, which appeared to be incomplete tubulin rings. Measurements from rotary shadowed samples showed that tubulin/XMAP215 partial rings had an average width of 8.8+/-1.8 nm compared with 5.6+/-1.1 nm for rings assembled from tubulin dimers alone, suggesting that XMAP215 adds a width of approximately 3.2 nm to the curved tubulin protofilament. XMAP215 did not change the radius of curvature of these partial tubulin rings. Measurements of microtubule flexural rigidity by thermal fluctuations showed that XMAP215 did not change microtubule rigidity. Finally, sequence analysis shows that the N-terminal half of XMAP215 contains four repeats, each composed of multiple HEAT repeats.

The clinical significance of tenascin-C splice variant expression in chondrosarcoma.

OBJECTIVES: Tenascin-C (TNC) is an oligomeric glycoprotein of the extracellular matrix that is prominently expressed in malignant tumors. The purpose of this study was: (1) to determine the in vitro TNC splicing pattern in cultured human chondrocytes and chondrosarcoma cells, (2) to determine the in vivo TNC splicing pattern in clinical chondrosarcoma specimens, and (3) to perform survival analysis based on the TNC splicing pattern of the tumor specimens. METHODS: Human articular chondrocytes and chondrosarcoma cells (cell line JJ012) were grown in a three-dimensional alginate bead system and harvested at two time points. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the in vitro TNC splicing pattern for the two cell types. Clinical chondrosarcoma specimens were obtained intra-operatively and underwent RT-PCR to determine the in vivo TNC splicing pattern. Specific immunohistochemical staining for the large TNC splice variant was performed on the clinical specimens. Survival analysis was used to determine the association between the specific TNC splicing pattern and survival. RESULTS: The in vitro mRNA expression pattern of TNC in normal human articular chondrocytes was characterized by a high ratio of the small to the large splice variant (TNC(small):TNC(large)), whereas the in vitro mRNA expression pattern for cultured chondrosarcoma cells was characterized by a low TNC(small):TNC(large) ratio. Clinical chondrosarcoma specimens with a lower TNC(small):TNC(large) ratio showed a trend towards decreased survival. The TNC splicing pattern of these specimens was verified through specific immunohistochemical staining for the large TNC isoform. CONCLUSIONS: The specific TNC splicing pattern may have clinical significance in chondrosarcoma. TNC expression may therefore play a future role in objective tumor grading and novel therapeutic approaches to this malignancy.

NO inhalation reduces pulmonary arterial pressure but not hemorrhage in maximally exercising horses.

In horses, the exercise-induced elevation of pulmonary arterial pressure (Ppa) is thought to play a deterministic role in exercise-induced pulmonary hemorrhage (EIPH), and thus treatment designed to lower Ppa might reasonably be expected to reduce EIPH. Five Thoroughbred horses were run on a treadmill to volitional fatigue (incremental step test) under nitric oxide (NO; inhaled 80 ppm) and control (N(2), same flow rate as per NO run) conditions (2 wk between trials; order randomized) to test the hypothesis that NO inhalation would reduce maximal Ppa but that this reduction may not necessarily reduce EIPH. Before each investigation, a microtipped pressure transducer was placed in the pulmonary artery 8 cm past the pulmonic valve to monitor Ppa. EIPH severity was assessed via bronchoalveolar lavage (BAL) 30 min postrun. Exercise time did not differ between the two trials (P > 0.05). NO administration resulted in a small but consistent and significant reduction in peak Ppa (N(2), 102.3 +/- 4.4; NO, 98.6 +/- 4.3 mmHg, P < 0.05). In the face of lowered Ppa, EIPH severity was significantly higher in the NO trial (N(2), 22.4 +/- 6.8; NO, 42.6 +/- 15.4 x 10(6) red blood cells/ml BAL fluid, P < 0.05). These findings support the notion that extremely high Ppa may reflect, in part, an arteriolar vasoconstriction that serves to protect the capillary bed from the extraordinarily high Ppa evoked during maximal exercise in the Thoroughbred horse. Furthermore, these data suggest that exogenous NO treatment during exercise in horses may not only be poor prophylaxis but may actually exacerbate the severity of EIPH.