Dyspnea Postpulmonary Embolism From Physiological Dead Space Proportion and Stroke Volume Defects During Exercise.

BACKGROUND: Many patients with pulmonary embolism (PE) report dyspnea on exertion following long-term treatment. Increased physiological dead space proportion (VD/VT) and decreased cardiac stroke volume reserve may distinguish persistent effects of PE itself from symptoms reflecting comorbid conditions or deconditioning. METHODS: This retrospective study analyzed a consecutive series of incremental symptom-limited cardiopulmonary exercise tests that had been ordered to evaluate persistent dyspnea on exertion following long-term treatment for acute PE. Physiological VD/VT was determined at anaerobic threshold from exhaled CO2 and transcutaneous Pco2 (validated against Paco2 measurements). Cardiac stroke volume reserve was estimated at rest and at anaerobic threshold by using oxygen consumption/pulse and previously validated estimates of the arteriovenous oxygen content difference. RESULTS: Cardiopulmonary exercise tests were performed on 40 patients with post-PE dyspnea. In 65.0% (95% CI, 50.2-79.8), VD/VT at anaerobic threshold was abnormally elevated, stroke volume reserve was decreased, or both defects occurred. VD/VT at anaerobic threshold was abnormally elevated (≥ 0.27) in 35.0% (95% CI, 20.2-49.8). VD/VT at anaerobic threshold significantly correlated with the extent of unmatched perfusion defects on subsequent ventilation-perfusion scans (P = .0085). In 55.0% (95% CI, 39.6-70.4), stroke volume reserve at anaerobic threshold was abnormally decreased (≤ 128% of the resting value). Both defects were present in 25.0% (95% CI, 11.6-38.4). CONCLUSIONS: Increased VD/VT at anaerobic threshold and decreased stroke volume reserve during exercise are common among patients with dyspnea on exertion after long-term treatment of PE. The defects can be disclosed noninvasively by using cardiopulmonary exercise testing.

Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients.

INTRODUCTION: Tumour burden is a prognostic biomarker in metastatic melanoma. However, tumour burden is difficult to measure and there are currently no reliable surrogate biomarkers to easily and reliably determine it. The aim of this study was to assess the potential of plasma total cell free DNA as biomarker of tumour burden and prognosis in metastatic melanoma patients. MATERIALS AND METHODS: A prospective biomarker cohort study for total plasma circulating cell-free DNA (cfDNA) concentration was performed in 43 metastatic melanoma patients. For 38 patients, paired blood collections and scan assessments were available before treatment and at first response evaluation. Tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients. RESULTS: Baseline cfDNA concentration correlated with pre-treatment tumour burden (ρ = 0.52, P < 0.001). Baseline cfDNA levels correlated significantly with hazard of death and overall survival, and a cut off value of 89 pg/µl identified two distinct prognostic groups (HR = 2.22 for high cfDNA, P = 0.004). Patients with cfDNA ≥89 pg/µl had shorter OS (10.0 versus 22.7 months, P = 0.009; HR = 2.22 for high cfDNA, P = 0.004) and the significance was maintained when compared with lactic dehydrogenase (LDH) in a multivariate analysis. We also found a correlation between the changes of cfDNA and treatment-related changes in tumour burden (ρ = 0.49, P = 0.002). In addition, the ratio between baseline cfDNA and tumour burden was prognostic (HR = 2.7 for cfDNA/tumour volume ≥8 pg/(µl*cm3), P = 0.024). CONCLUSIONS: We have demonstrated that cfDNA is a surrogate marker of tumour burden in metastatic melanoma patients, and that it is prognostic for overall survival.

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice.

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 µL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a ß-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.

Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA.

BACKGROUND: The application of precision medicine in oncology requires in-depth characterisation of a patient's tumours and the dynamics of their responses to treatment. PATIENTS AND METHODS: We used next-generation sequencing of circulating cell-free DNA (cfDNA) to monitor the response of a KIT p.L576P-mutant metastatic vaginal mucosal melanoma to sequential targeted, immuno- and chemotherapy. RESULTS: Despite a KIT mutation, the response to imatinib was mixed. Unfortunately, tumours were not accessible for molecular analysis. To study the mechanism underlying the mixed clinical response, we carried out whole-exome sequencing and targeted longitudinal analysis of cfDNA. This revealed two tumour subclones; one with a KIT mutation that responded to imatinib and a second KIT-wild-type subclone that did not respond to imatinib. Notably, the subclones also responded differently to immunotherapy. However, both subclones responded to carboplatin/paclitaxel, and although the KIT-wild-type subclone progressed after chemotherapy, it responded to subsequent re-administration of paclitaxel. CONCLUSION: We show that cfDNA can reveal tumour evolution and subclonal responses to therapy even when biopsies are not available.

Design, development and evaluation of rK28-based point-of-care tests for improving rapid diagnosis of visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) is diagnosed by microscopic confirmation of the parasite in bone marrow, spleen or lymph node aspirates. These procedures are unsuitable for rapid diagnosis of VL in field settings. The development of rK39-based rapid diagnostic tests (RDT) revolutionized diagnosis of VL by offering high sensitivity and specificity in detecting disease in the Indian subcontinent; however, these tests have been less reliable in the African subcontinent (sensitivity range of 75-85%, specificity of 70-92%). We have addressed limitations of the rK39 with a new synthetic polyprotein, rK28, followed by development and evaluation of two new rK28-based RDT prototype platforms. METHODOLOGY/PRINCIPAL FINDINGS: Evaluation of 62 VL-confirmed sera from Sudan provided sensitivities of 96.8% and 93.6% (95% CI = K28: 88.83-99.61%; K39: 84.30-98.21%) and specificities of 96.2% and 92.4% (95% CI = K28: 90.53-98.95%; K39: 85.54-96.65%) for rK28 and rK39, respectively. Of greater interest was the observation that individual VL sera with low rK39 reactivity often had much higher rK28 reactivity. This characteristic of the fusion protein was exploited in the development of rK28 rapid tests, which may prove to be crucial in detecting VL among patients with low rK39 antibody levels. Evaluation of two prototype lateral flow-based rK28 rapid tests on 53 VL patients in Sudan and 73 VL patients in Bangladesh provided promisingly high sensitivities (95.9% [95% CI = 88.46-99.1 in Sudan and 98.1% [95% CI = 89.93-99.95%] in Bangladesh) compared to the rK39 RDT (sensitivities of 86.3% [95% CI = 76.25-93.23%] in Sudan and 88.7% [95% CI = 76.97-95.73%] in Bangladesh). CONCLUSIONS/SIGNIFICANCE: Our study compares the diagnostic accuracy of rK39 and rK28 in detecting active VL cases and our findings indicate that rK28 polyprotein has great potential as a serodiagnostic tool. A new rK28-based RDT will prove to be a valuable asset in simplifying VL disease confirmation at the point-of-care.

A cross-sectional survey of Australian chicken farms to identify risk factors associated with seropositivity to Newcastle-disease virus.

Several outbreaks of virulent Newcastle-disease occurred in Australia in 1998-2000. We conducted a cross-sectional survey of 753 Australian chicken farms to identify risk factors associated with the seroprevalence of chicken flocks with Newcastle-disease virus (NDV). We had a 99.7% response rate to the survey and the overall prevalence of NDV seropositive farms was 39.8%. Associations were analysed for the layer, chicken-meat and breeder production sectors in sector-specific logistic-regression models using 187, 198 and 146 farms, respectively. In the layer sector, increased risk of seroprevalence was associated with increasing age of the chickens, and decreased risk when the nearest-neighbour poultry farm was >10 km distant (odds ratio (OR)=0.30). In the chicken-meat sector, increased risk of seroprevalence was associated with location in the Sydney basin (OR=13.67), eastern Victoria (OR=26.10) or western Victoria (OR=5.43), and decreased risk when the nearest-neighbour poultry farm was greater than 0.5 km distant (OR=0.34). In the breeder sector, increased risk of seroprevalence was associated with increasing age of the chickens, the presence of wild birds on the farm (OR=5.28) and location in eastern Victoria (OR=16.19). A conditional logistic-regression for 112 pairs of farms matched for age, survey region and production sector identified a distance of >1.0 km to the nearest-neighbour poultry farm (OR=0.24) and ownership by owner 2 (OR=0.02), owner 5 (OR=0.11) or owner 9 (OR=0.25) as significant in reducing the risk of NDV seroprevalence. Our survey found that high levels of biosecurity and hygiene practices had been adopted by most farms.

Serosurvey of selected zoonotic agents in polar bears (Ursus maritimus).

Between 1982 and 1999 blood samples were collected from 500 polar bears (Ursus maritimus) captured in the Beaufort and Chukchi seas, to determine the seroprevalence of Brucella species, Toxoplasma gondii, and Trichinella species infections. The bears were classified into four age groups, cubs, yearlings, subadults and adults. Brucella and Toxoplasma antibodies were detected by agglutination (a buffered acidified card antigen and rapid automated presumptive test for brucellosis and a commercial latex agglutination test for toxoplasmosis); an ELISA was used to detect Trichinella antibodies. The overall seroprevalence of Brucella species was 5 per cent, and subadults and yearlings were 2-62 times (95 per cent confidence interval 1.02 to 6.82) more likely to be seropositive for Brucella species than adults and their cubs. The antibody prevalence for Toxoplasma gondii was 6 per cent, and for Trichinella species 55.6 per cent. The prevalence of antibodies to Trichinella species increased with age (P<0.001).

Geographical distribution of organochlorine pesticides (OCPs) in polar bears (Ursus maritimus) in the Norwegian and Russian Arctic.

Geographical variation of organochlorine pesticides (OCPs) was studied in blood samples from 90 adult female polar bear (Ursus maritimus) from Svalbard, Franz Josef Land, Kara Sea, East-Siberian Sea and Chukchi Sea. In all regions, oxychlordane was the dominant OCP. Regional differences in mean levels of HCB, oxychlordane, trans-nonachlor, alpha-HCH, beta-HCH and p,p'-DDE were found. The highest levels of oxychlordane, trans-nonachlor and DDE were found in polar bears from Franz Josef Land and Kara Sea. HCB level was lowest in polar bears from Svalbard. Polar bears from Chukchi Sea had the highest level of alpha- and beta-HCH. The lowest alpha-HCH concentration was found in bears from Kara Sea. In all the bears, summation operator HCHs was dominated by beta-HCH. The geographical variation in OCP levels and pattern may suggest regional differences in pollution sources and different feeding habits in the different regions. Polar bears from the Western Russian Arctic were exposed to higher levels of chlordanes and p,p'-DDE than polar bears from locations westwards and eastwards from this region. This may imply the presence of a significant pollution source in the Russian Arctic area. The study suggests that the western Russian Arctic is the most contaminated region of the Arctic and warrants further research.

Are alpha-gliadins glycosylated?

Alpha-gliadins isolated by carboxymethylcellulose chromatography contain noncovalently bound glucose probably due to contaminating proteoglycans and to material shed from the column. Traces of carbohydrate remain strongly bound to alpha-gliadins even after harsh denaturation, but our results indicate alpha-gliadins are not glycoproteins. Suggestions that gliadins are glycoproteins are probably due to contamination with this glucose and the presence of these proteoglycans.

Thermoregulatory ability of beef heifers following intake of endophyte-infected tall fescue during controlled heat challenge.

Consumption of endophyte-infected tall fescue during summer months can result in severe hyperthermia in cattle. Six heifers (296+/-8.3 kg BW) were used to determine changes in body temperature control that occur with consumption of an endophyte-infected tall fescue diet during controlled heat challenge. All animals were exposed in five separate periods to a step increase in ambient temperature (Ta) from 21 to 31 degrees C while fed E+ (5 microg ergovaline x kg(-1) x d(-1)) or endophyte-free (E-) diets. Core body temperature (Tcore) was monitored continuously using implanted, telemetric temperature transmitters. Heat production and heat loss were also measured at selected times to identify primary effects of E+ on thermal balance. Pretreatment Tcore exhibited a diurnal rhythm at a constant Ta of 21 degrees C, with high and low values at 2300 and 1300, respectively. An increase in daily averaged Tcore (P < 0.001) occurred with an increase in Ta from 21 to 31 degrees C. Likewise, all phases of the daily cycle increased equally during this challenge. This increase at 31 degrees C was associated with higher levels of respiration rate, skin temperature, respiratory vaporization, and skin vaporization (P < 0.05) and lower blood levels of thyroxine (P < 0.05). Intake of the E+ diet further elevated Tcore in heifers during the short-term heat challenge (2 d), and the effect was most pronounced at 0000 to 0300 and declined thereafter. The increase in Tcore during E+ treatment was associated with an increase in respiration rate (P < 0.05), whereas metabolic heat production, skin temperature, skin vaporization and respiratory vaporization were unaffected. These results show that consumption of an E+ diet during continuous heat challenge results in a marked increase in core body temperature, especially during nighttime exposure to heat stress, due primarily to a reduction in cutaneous heat transfer, with no effect on heat production or other measured avenues of heat loss.

Vacuum-assisted wound closure provides early fascial reapproximation in trauma patients with open abdomens.

BACKGROUND: Damage control and decompressive laparotomies salvage severely injured patients who would have previously died. Unfortunately, many of these patients develop open abdomens. A variety of management strategies exist. The end result in many cases, however, is a large ventral hernia that requires a complex repair 6 to 12 months after discharge. We instituted vacuum-assisted wound closure (VAWC) to achieve early fascial closure and eliminate the need for delayed procedures. METHODS: For 12 months ending June 2000, 14 of 698 trauma intensive care unit admissions developed open abdomens and were managed with VAWC dressing. This was changed every 48 hours in the operating room with serial fascial approximation until complete closure. RESULTS: Fascial closure was achieved in 13 patients (92%) in 9.9 +/- 1.9 days, and 2.8 +/- 0.6 VAWC dressing changes were performed. There were 2 wound infections, no eviscerations, and no enteric fistulas. CONCLUSIONS: Use of VAWC can safely achieve early fascial closure in more than 90% of trauma patients with open abdomens.

Ototoxicity after high-dose chemotherapy with cyclophosphamide, thiotepa and carboplatin followed by stem cell transplantation in patients with breast cancer.

Our purpose was to determine the risk of ototoxicity in breast cancer patients receiving a myeloablative regimen consisting of cyclophosphamide 6000 mg/m2, thiotepa 500 mg/m2 and carboplatin 800 mg/m2 (CTCb) followed by stem cell transplantation. Fourteen consecutive patients with breast cancer were treated with high dose chemotherapy consisting of the CTCb regimen followed by stem cell transplantation. A pretransplant complete hearing study was obtained which consisted of hearing case history, audiometry and tympanometry. In addition, DPOAE (Distortion Product Otoaccoustic Emissions) was done to evaluate measurable changes in the cochlear (outer hair cell) functioning. Pre-transplant, all patients had no clinical evidence of hearing impairment and hearing studies were normal. Eleven patients had hearing studies and a telephone interview posttransplant. One patient was lost to follow-up and two patients died. One of the 11 patients tested had an abnormal post-transplant hearing study but none of them had clinically detectable hearing impairment. In our prospective study of breast cancer patients treated with the CTCb regimen, we did not observe clinically detectable hearing impairment in any of the patients tested.

Phylogeography of mitochondrial DNA variation in brown bears and polar bears.

We analyzed 286 nucleotides of the middle portion of the mitochondrial cytochrome b gene of 61 brown bears from three locations in Alaska and 55 polar bears from Arctic Canada and Arctic Siberia to test our earlier observations of paraphyly between polar bears and brown bears as well as to test the extreme uniqueness of mitochondrial DNA types of brown bears on Admiralty, Baranof, and Chichagof (ABC) islands of southeastern Alaska. We also investigated the phylogeography of brown bears of Alaska's Kenai Peninsula in relation to other Alaskan brown bears because the former are being threatened by increased human development. We predicted that: (1) mtDNA paraphyly between brown bears and polar bears would be upheld, (2) the mtDNA uniqueness of brown bears of the ABC islands would be upheld, and (3) brown bears of the Kenai Peninsula would belong to either clade II or clade III of brown bears of our earlier studies of mtDNA. All of our predictions were upheld through the analysis of these additional samples.

Genetic structure of the world's polar bear populations.

We studied genetic structure in polar bear (Ursus maritimus) populations by typing a sample of 473 individuals spanning the species distribution at 16 highly variable microsatellite loci. No genetic discontinuities were found that would be consistent with evolutionarily significant periods of isolation between groups. Direct comparison of movement data and genetic data from the Canadian Arctic revealed a highly significant correlation. Genetic data generally supported existing population (management unit) designations, although there were two cases where genetic data failed to differentiate between pairs of populations previously resolved by movement data. A sharp contrast was found between the minimal genetic structure observed among populations surrounding the polar basin and the presence of several marked genetic discontinuities in the Canadian Arctic. The discontinuities in the Canadian Arctic caused the appearance of four genetic clusters of polar bear populations. These clusters vary in total estimated population size from 100 to over 10 000, and the smallest may merit a relatively conservative management strategy in consideration of its apparent isolation. We suggest that the observed pattern of genetic discontinuities has developed in response to differences in the seasonal distribution and pattern of sea ice habitat and the effects of these differences on the distribution and abundance of seals.

Subcutaneous implantation of satellite transmitters with percutaneous antennae into male polar bears (Ursus maritimus).

Male polar bears (Ursus maritimus) have not been successfully instrumented with satellite transmitters because they readily shed collar-mounted transmitters. Seven male polar bears were captured on the pack ice off the northern coast of Alaska and surgically implanted with satellite transmitters with percutaneous antennae into the subcutaneous space of the dorsal cervical region. Transmitters failed prematurely with lifetimes of 30-161 days (mean = 97 days). Efforts to relocate implanted bears after transmitters failed were not successful. The mean number of location solutions per transmitter was 204 (range 118-369). An average of 10% and 19% of the locations were accurate to <150 m and to 150-350 m, respectively. Our successful tracking of male polar bears, the high quality of locations obtained from transmitters with percutaneous antennae implanted in the subcutaneous space, and the low visibility of such units make further technical development worthwhile if the reason for premature failure of the transmitters can be determined.

Chlorinated hydrocarbon contaminants in polar bears from eastern Russia, North America, Greenland, and Svalbard: biomonitoring of Arctic pollution.

Adipose tissue samples from polar bears (Ursus maritimus) were obtained by necropsy or biopsy between the spring of 1989 to the spring of 1993 from Wrangel Island in Russia, most of the range of the bear in North America, eastern Greenland, and Svalbard. Samples were divided into 16 regions corresponding as much as possible to known stocks or management zones. Concentrations of dieldrin (DIEL), 4,4'-DDE (DDE), sum of 16 polychlorinated biphenyl congeners (sigma PCB), and sum of 11 chlordane-related compounds and metabolites (sigma CHL) were determined. In order to minimize the effect of age, only data for adults (320 bears age 5 years and older) was used to compare concentrations among regions. Concentrations of sigma PCB were 46% higher in adult males than females, and there was no significant trend with age. Concentrations of sigma CHL were 30% lower in adult males than females. Concentrations of sigma PCB, sigma CHL, and DDE in individual adult female bears were standardized to adult males using factors derived from the least-square means of each sex category, and geometric means of the standardized concentrations on a lipid weight basis were compared among regions. Median geometric mean standardized concentrations (lipid weight basis) and ranges among regions were as follows: sigma PCB, 5,942 (2,763-24,316) micrograms/kg; sigma CHL, 1,952 (727-4,632) micrograms/kg; DDE, 219 (52-560) micrograms/kg; DIEL, 157 (31-335) micrograms/kg. Geometric mean sigma PCB concentrations in bears from Svalbard, East Greenland, and the Arctic Ocean near Prince Patrick Island in Canada were similar (20,256-24,316 micrograms/kg) and significantly higher than most other areas. Atmospheric, oceanic, and ice transport, as well as ecological factors may contribute to these high concentrations of sigma PCB. sigma CHL was more uniformly distributed among regions than the other CHCs. Highest sigma CHL concentrations were found in southeastern Hudson Bay, which also had the highest DDE and DIEL concentrations. In general, concentrations of sigma CHL, DDE, and DIEL were higher in eastern than western regions, suggesting an influence of North American sources. Average sigma PCB concentrations in bears from the Canadian Arctic were similar to those in 1982-84, while average sigma CHL and DDE concentrations were 35-44% lower and DIEL was 90% lower. However, the significance of these temporal trends during the 1980s is not conclusive because of the problems of comparability of data.

An inhibitor of the sodium pump obtained from human placenta.

BACKGROUND: Much effort has been expended in the search for an endogenous inhibitor of the cellular sodium/potassium pump, a compound of major physiological importance, which has been implicated in the mechanism of essential hypertension. Others have suggested that ouabain or an isomer of ouabain may be the endogenous pump inhibitor. Neonatal cord serum contains an inhibitor of the sodium pump; we attempted to isolate and characterise this substance from human placentas. METHODS: Homogenised placentas were dialysed and the resulting solutes were trapped on octadecylsilyl silica and then separated by high-performance liquid chromatography. Measurement of the activity of the sodium pump of human leucocytes was used to test each fraction for the presence of the inhibitor. FINDINGS: An inhibitor of the sodium pump was obtained by this technique in a mass spectrometrically pure form with a mass of 370 Da, an empirical formula of C24H34O3 and only one hydroxyl group. The characteristic fragmentation pattern observed in negative-ion mass spectrometry was compared with those of various model compounds; this comparison suggested that the active material was a dihydropyrone-substituted steroid. INTERPRETATION: These results suggest that a dihydropyrone-substituted steroid is an endogenous regulator of the sodium pump in humans and, presumably, other mammals. Proof of the endogenous origin will require the demonstration of a previously unrecognised biosynthetic pathway.

Serological evidence of morbillivirus infection in polar bears (Ursus maritimus) from Alaska and Russia.

One-hundred-and-ninety-one samples of blood serum collected from 186 polar bears (Ursus maritimus) between 1987 and 1992 were analysed for morbillivirus antibodies. The samples were collected in the Bering, Chukchi and East Siberian seas. Sixty-eight samples (35.6 per cent) had morbillivirus antibody titres > 5; the percentage of positive samples ranged from 26.2 to 46.2 per cent from year to year. The proportions of adults, sub-adults and cubs which were seropositive were 43.9, 35.7 and 37.9 per cent respectively. Some seropositive dams had seronegative young and some that were seronegative had seropositive young. One litter of two cubs, in which the dam was seronegative, had one seropositive and one seronegative cub. Seropositive bears occurred in all the areas from which the samples were collected but there was a significantly greater incidence in the bears sampled in Russia. The high prevalence of seropositive bears over the period suggests that the bear morbillivirus is endemic in these regions of the Arctic, but its source is unknown.

Effects of environmental heat and intake of tall fescue seed infested with Acremonium coenophialum on the acid-base status of young bulls.

Effects of high environmental temperature and dietary intake of tall fescue (Festuca arundinacea) seed containing the endophyte Acremonium coenophialum on bovine acid-base status were studied using 3 groups of bull calves (2 Simmental, 1 Angus). Experimental animals were housed in controlled-climate chambers and subjected to gradual increases in environmental temperature, first while being fed an endophyte-free diet and then while being fed a diet containing 17% endophyte-infested fescue seed. Marked acid-base disturbances were not observed in any animals. In general, Pco2, HCO3-, base excess, and arterial blood pH values were reduced in response to heat stress, both with endophyte-free and endophyte-containing diets. In most individuals anion gap increased. These results reflected metabolic compensation for mild chronic alveolar hyperventilation and retention of organic acids. These findings suggest that, under conditions similar to those found during the summer in central Missouri, normal cattle should not be at great risk of developing respiratory alkalosis or other severe acid-base disturbances as a result of heat stress and/or intake of tall fescue endophyte.