Publications by authors named "Ben J Lee"

24 Publications

  • Page 1 of 1

Thermoregulation is not impaired in breast cancer survivors during moderate-intensity exercise performed in warm and hot environments.

Physiol Rep 2021 Jul;9(14):e14968

Environmental Extremes Laboratory, University of Brighton, Eastbourne, UK.

This study aimed to assess how female breast cancer survivors (BCS) respond physiologically, hematologically, and perceptually to exercise under heat stress compared to females with no history of breast cancer (CON). Twenty-one females (9 BCS and 12 CON [age; 54 ± 7 years, stature; 167 ± 6 cm, body mass; 68.1 ± 7.62 kg, and body fat; 30.9 ± 3.8%]) completed a warm (25℃, 50% relative humidity, RH) and hot (35℃, 50%RH) trial in a repeated-measures crossover design. Trials consisted of 30 min of rest, 30 min of walking at 4 metabolic equivalents, and a 6-minute walk test (6MWT). Physiological measurements (core temperature (T ), skin temperature (T ), heart rate (HR), and sweat analysis) and perceptual rating scales (ratings of perceived exertion, thermal sensation [whole body and localized], and thermal comfort) were taken at 5- and 10-min intervals throughout, respectively. Venous blood samples were taken before and after to assess; IL-6, IL-10, CRP, IFN-γ, and TGF-β . All physiological markers were higher during the 35 versus 25℃ trial; T (~0.25℃, p = 0.002), T (~3.8℃, p < 0.001), HR (~12 beats·min , p = 0.023), and whole-body sweat rate (~0.4 L·hr , p < 0.001), with no difference observed between groups in either condition (p > 0.05). Both groups covered a greater 6MWT distance in 25 versus 35℃ (by ~200 m; p = 0.003). Nevertheless, the control group covered more distance than BCS, regardless of environmental temperature (by ~400 m, p = 0.03). Thermoregulation was not disadvantaged in BCS compared to controls during moderate-intensity exercise under heat stress. However, self-paced exercise performance was reduced for BCS regardless of environmental temperature.
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http://dx.doi.org/10.14814/phy2.14968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295682PMC
July 2021

One night of sleep fragmentation does not affect exercise-induced leukocyte trafficking or mitogen-stimulated leukocyte oxidative burst in healthy men.

Physiol Behav 2021 Oct 23;239:113506. Epub 2021 Jun 23.

University of Bath, Department for Health, Claverton Down, Bath, UK. Electronic address:

Purpose: This study examined whether one night of sleep fragmentation alters circulating leukocyte counts and mitogen-stimulated oxidative burst by leukocytes at rest and in response to an acute bout of vigorous exercise.

Methods: In a randomised crossover design, nine healthy men (mean ± SD: age 22 ± 2 years; BMI 24.9 ± 1.9 kg/m) were exposed to one night of fragmented or uninterrupted sleep before cycling for 45 min at 71% ± 4% V̇O. Finger-tip blood samples were collected at rest, immediately post-exercise and one-hour post-exercise. Total leukocytes, lymphocytes, monocytes and neutrophils were counted. Leukocyte oxidative burst was assessed in whole blood by measuring Reactive Oxygen Species (ROS) production with luminol-amplified chemiluminescence after stimulation with phorbol 12-myristate 13-acetate (PMA).

Results: Exercise elicited the expected trafficking pattern of leukocytes, lymphocytes, monocytes and neutrophils. Compared to rest, PMA-stimulated ROS production was increased one-hour post-exercise (+73% ± 65%; p = 0.019; data combined for fragmented and uninterrupted sleep). There were no statistically significant effects of fragmented sleep on leukocyte, lymphocyte, monocyte, and neutrophil counts or on ROS production at rest, immediately post-exercise or one-hour post-exercise (p > 0.05). However, with fragmented sleep, there was a +10% greater lymphocytosis immediately post-exercise (fragmented +40% ± 37%; uninterrupted +30% ± 35%; p = 0.51) and a -19% smaller neutrophilia by one-hour post-exercise (fragmented +103% ± 88%; uninterrupted +122% ± 131%; p = 0.72).

Conclusion: Fragmented sleep did not substantially alter the magnitude or pattern of exercise-induced leukocyte trafficking or mitogen-stimulated oxidative burst by leukocytes.
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http://dx.doi.org/10.1016/j.physbeh.2021.113506DOI Listing
October 2021

Field validation of The Heat Strain Decision Aid during military load carriage.

Comput Biol Med 2021 07 20;134:104506. Epub 2021 May 20.

Biophysics and Biomedical Modeling Division, U.S. Army Research Institute of Environmental Medicine, 10 General Greene Avenue, Bldg 42, Natick, MA, 01760, USA.

Objectives: We aimed to determine the agreement between actual and predicted core body temperature, using the Heat Strain Decision Aid (HSDA), in non-Ground Close Combat (GCC) personnel wearing multi terrain pattern clothing during two stages of load carriage in temperate conditions.

Design: Cross-sectional.

Methods: Sixty participants (men = 49, women = 11, age 31 ± 8 years; height 171.1 ± 9.0 cm; body mass 78.1 ± 11.5 kg) completed two stages of load carriage, of increasing metabolic rate, as part of the development of new British Army physical employment standards (PES). An ingestible gastrointestinal sensor was used to measure core temperature. Testing was completed in wet bulb globe temperature conditions; 1.2-12.6 °C. Predictive accuracy and precision were analysed using individual and group mean inputs. Assessments were evaluated by bias, limits of agreement (LoA), mean absolute error (MAE), and root mean square error (RMSE). Accuracy was evaluated using a prediction bias of ±0.27 °C and by comparing predictions to the standard deviation of the actual core temperature.

Results: Modelling individual predictions provided an acceptable level of accuracy based on bias criterion; where the total of all trials bias ± LoA was 0.08 ± 0.82 °C. Predicted values were in close agreement with the actual data: MAE 0.37 °C and RMSE 0.46 °C for the collective data. Modelling using group mean inputs were less accurate than using individual inputs, but within the mean observed.

Conclusion: The HSDA acceptably predicts core temperature during load carriage to the new British Army non-GCC PES, in temperate conditions.
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http://dx.doi.org/10.1016/j.compbiomed.2021.104506DOI Listing
July 2021

Plasma uptake of selected phenolic acids following New Zealand blackcurrant extract supplementation in humans.

J Diet Suppl 2021 May 5:1-16. Epub 2021 May 5.

Institute of Sport, University of Chichester, Chichester, UK.

New Zealand blackcurrant (NZBC) extract is a rich source of anthocyanins and in order to exert physiological effects, the anthocyanin-derived metabolites need to be bioavailable . We examined the plasma uptake of selected phenolic acids following NZBC extract supplementation alongside maintaining a habitual diet (i.e. not restricting habitual polyphenol intake). Twenty healthy volunteers (nine females, age: 28±7years, height 1.73±0.09 m, body mass 73±11kg) consumed a 300mg NZBC extract capsule (CurraNZ; anthocyanin content 105mg) following an overnight fast. Venous blood samples were taken pre and 1, 1.5, 2, 3, 4, 5, and 6h post-ingestion of the capsule. Reversed-phase high-performance liquid chromatography (HPLC) was used for analysis of two dihydroxybenzoic acids [i.e. vanillic acid (VA) and protocatechuic acid (PCA)] and one trihydroxybenzoic acid [i.e. gallic acid (GA)] in plasma following NZBC extract supplementation. Habitual anthocyanin intake was 168 (95%CI:68-404) mg⋅day and no associations were observed between this and VA, PCA, and GA plasma uptake by the NZBC extract intake. Plasma time-concentration curves revealed that GA, and PCA were most abundant at 4, and 1.5h post-ingestion, representing a 261% and 320% increase above baseline, respectively, with VA remaining unchanged. This is the first study to demonstrate that an NZBC extract supplement increases the plasma uptake of phenolic acids GA, and PCA even when a habitual diet is followed in the days preceding the experimental trial, although inter-individual variability is apparent.
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http://dx.doi.org/10.1080/19390211.2021.1914802DOI Listing
May 2021

No Effects of Different Doses of New Zealand Blackcurrant Extract on Cardiovascular Responses During Rest and Submaximal Exercise Across a Week in Trained Male Cyclists.

Int J Sport Nutr Exerc Metab 2020 Nov 12;31(1):66-72. Epub 2020 Nov 12.

University of Chichester.

Supplementation with anthocyanin-rich blackcurrant increases blood flow, cardiac output, and stroke volume at rest. It is not known whether cardiovascular responses can be replicated over longer timeframes in fed trained cyclists. In a randomized, double-blind, crossover design, 13 male trained cyclists (age 39 ± 10 years, V˙O2max 55.3 ± 6.7 ml·kg-1·min-1) consumed two doses of New Zealand blackcurrant (NZBC) extract (300 and 600 mg/day for 1 week). Cardiovascular parameters were measured during rest and submaximal cycling (65% V˙O2max) on day 1 (D1), D4, and D7. Data were analyzed with an RM ANOVA using dose (placebo vs. 300 vs. 600 mg/day) by time point (D1, D4, and D7). Outcomes from placebo were averaged to determine the coefficient of variation within our experimental model, and 95% confidence interval (CI) was examined for differences between placebo and NZBC. There were no differences in cardiovascular responses at rest between conditions and between days. During submaximal exercise, no positive changes were observed on D1 and D4 after consuming NZBC extract. On D7, intake of 600 mg increased stroke volume (3.08 ml, 95% CI [-2.08, 8.26]; d = 0.16, p = .21), cardiac output (0.39 L/min, 95% CI [-1.39, .60]; d = 0.14, p = .40) (both +2.5%), and lowered total peripheral resistance by 6.5% (-0.46 mmHg·min/ml, 95% CI [-1.80, .89]; d = 0.18, p = .46). However, these changes were trivial and fell within the coefficient of variation of our study design. Therefore, we can conclude that NZBC extract was not effective in enhancing cardiovascular function during rest and submaximal exercise in endurance-trained fed cyclists.
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http://dx.doi.org/10.1123/ijsnem.2020-0164DOI Listing
November 2020

No Effects of New Zealand Blackcurrant Extract on Physiological and Performance Responses in Trained Male Cyclists Undertaking Repeated Testing across a Week Period.

Sports (Basel) 2020 Aug 13;8(8). Epub 2020 Aug 13.

Institute of Sport, University of Chichester, College Lane, Chichester PO19 6PE, UK.

Anthocyanin supplements are receiving attention due to purported benefits to physiological, metabolic, and exercise responses in trained individuals. However, the efficacy of anthocyanin intake over multiple testing days is not known. We compared a placebo and two doses of anthocyanin-rich New Zealand blackcurrant (NZBC) extract (300 and 600 mg·day) on plasma lactate, substrate oxidation, and 16.1 km time trial (TT) performance on three occasions over 7-days in a fed state (day 1 (D1), D4, and D7). Thirteen male cyclists participated in a randomized, crossover, placebo-controlled double-blind design. There was no difference in plasma lactate and substrate oxidation between conditions and between days. A time difference was observed between D1 (1701 ± 163 s) and D4 (1682 ± 162 s) for 600 mg ( = 0.05), with an increment in average speed (D1 = 34.3 ± 3.4 vs. D4 = 34.8 ± 3.4 km·h, = 0.04). However, there was no difference between the other days and between conditions. Overall, one week of intake of NZBC extract did not affect physiological and metabolic responses. Intake of 600 mg of NZBC extract showed inconsistent benefits in improving 16.1 km time trial performance over a week period in trained fed cyclists.
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http://dx.doi.org/10.3390/sports8080114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466623PMC
August 2020

No Effect of New Zealand Blackcurrant Extract on Recovery of Muscle Damage Following Running a Half-Marathon.

Int J Sport Nutr Exerc Metab 2020 May 29;30(4):287-294. Epub 2020 May 29.

University of Chichester.

New Zealand blackcurrant (NZBC) contains anthocyanins, known to moderate blood flow and display anti-inflammatory properties that may improve recovery from exercise-induced muscle damage. The authors examined whether NZBC extract supplementation enhances recovery from exercise-induced muscle damage after a half-marathon race. Following a randomized, double-blind, independent groups design, 20 (eight women) recreational runners (age 30 ± 6 years, height 1.73 ± 0.74 m, body mass 68.5 ± 7.8 kg, half-marathon finishing time 1:56:33 ± 0:18:08 hr:min:s) ingested either two 300-mg/day capsules of NZBC extract (CurraNZ™) or a visually matched placebo, for 7 days prior to and 2 days following a half-marathon. Countermovement jump performance variables, urine interleukin-6, and perceived muscle soreness and fatigue were measured pre, post, and at 24 and 48 hr after the half-marathon and analyzed using a mixed linear model with statistical significance set a priori at p < .05. The countermovement jump performance variables were reduced immediately post-half-marathon (p < .05), with all returning to pre-half-marathon levels by 48 hr, except the concentric and eccentric peak force and eccentric duration, with no difference in response between groups (p > .05). Urine interleukin-6 increased 48-hr post-half-marathon in the NZBC group only (p < .01) and remained unchanged compared with pre-half-marathon levels in the placebo group (p > .05). Perceived muscle soreness and fatigue increased immediately post-half-marathon (p < .01) and returned to pre-half-marathon levels by 48 hr, with no difference between groups (p > .05). Supplementation with NZBC extract had no effect on the recovery of countermovement jump variables and perceptions of muscle soreness or fatigue following a half-marathon in recreational runners.
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http://dx.doi.org/10.1123/ijsnem.2019-0312DOI Listing
May 2020

Accuracy of Metabolic Cost Predictive Equations During Military Load Carriage.

J Strength Cond Res 2020 May 8. Epub 2020 May 8.

Occupational Performance Research Group, Institute of Sport, University of Chichester, Chichester, United Kingdom.

Vine, CA, Coakley, SL, Blacker, SD, Doherty, J, Hale, B, Walker, EF, Rue, CA, Lee, BJ, Flood, TR, Knapik, JJ, Jackson, S, Greeves, JP, and Myers, SD. Accuracy of metabolic cost predictive equations during military load carriage. J Strength Cond Res XX(X): 000-000, 2020-To quantify the accuracy of 5 equations to predict the metabolic cost of load carriage under ecologically valid military speed and load combinations. Thirty-nine male serving infantry soldiers completed thirteen 20-minute bouts of overground load carriage comprising 2 speeds (2.5 and 4.8 km·h) and 6 carried equipment load combinations (25, 30, 40, 50, 60, and 70 kg), with 22 also completing a bout at 5.5 km·h carrying 40 kg. For each speed-load combination, the metabolic cost was measured using the Douglas bag technique and compared with the metabolic cost predicted from 5 equations; Givoni and Goldman, 1971 (GG), Pandolf et al. 1997 (PAN), Santee et al. 2001 (SAN), American College of Sports Medicine 2013 (ACSM), and the Minimum-Mechanics Model (MMM) by Ludlow and Weyand, 2017. Comparisons between measured and predicted metabolic cost were made using repeated-measures analysis of variance and limits of agreement. All predictive equations, except for PAN, underpredicted the metabolic cost for all speed-load combinations (p < 0.001). The PAN equation accurately predicted metabolic cost for 40 and 50 kg at 4.8 km·h (p > 0.05), underpredicted metabolic cost for all 2.5 km·h speed-load combinations as well as 25 and 30 kg at 4.8 km·h, and overpredicted metabolic cost for 60 and 70 kg at 4.8 km·h (p < 0.001). Most equations (GG, SAN, ACSM, and MMM) underpredicted metabolic cost while one (PAN) accurately predicted at moderate loads and speeds, but overpredicted or underpredicted at other speed-load combinations. Our findings indicate that caution should be applied when using these predictive equations to model military load carriage tasks.
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http://dx.doi.org/10.1519/JSC.0000000000003644DOI Listing
May 2020

Addition of pectin-alginate to a carbohydrate beverage does not maintain gastrointestinal barrier function during exercise in hot-humid conditions better than carbohydrate ingestion alone.

Appl Physiol Nutr Metab 2020 Oct 4;45(10):1145-1155. Epub 2020 May 4.

Institute of Sport, Occupational Performance Research Group, University of Chichester, West Sussex, PO10 6PE, UK.

The objective of this study was to compare the effects of consuming a 16% maltodextrin+fructose+pectin-alginate (MAL+FRU+PEC+ALG) drink against a nutrient-matched maltodextrin+fructose (MAL+FRU) drink on enterocyte damage and gastrointestinal permeability after cycling in hot and humid conditions. Fourteen recreational cyclists (7 men) completed 3 experimental trials in a randomized placebo-controlled design. Participants cycled for 90 min (45% maximal aerobic capacity) and completed a 15-min time-trial in hot (32 °C) humid (70% relative humidity) conditions. Every 15 min, cyclists consumed 143 mL of either () water; () MAL+FRU+PEC+ALG (90 g·h CHO/16% ); or () a ratio-matched MAL+FRU drink (90 g·h CHO/16% ). Blood was sampled before and after exercise and gastrointestinal (GI) permeability, which was determined by serum measurements of intestinal fatty acid binding protein (I-FABP) and the percent ratio of lactulose (5 g) to rhamnose (2 g) recovered in postexercise urine. Compared with water, I-FABP decreased by 349 ± 67pg·mL with MAL+FRU+PEC+ALG ( = 0.007) and by 427 ± 56 pg·mL with MAL+FRU ( = 0.02). GI permeability was reduced in both the MAL+FRU+PEC+ALG (by 0.019 ± 0.01, = 0.0003) and MAL+FRU (by 0.014 ± 0.01, = 0.002) conditions relative to water. In conclusion, both CHO beverages attenuated GI barrier damage to a similar extent relative to water. No metabolic, cardiovascular, thermoregulatory, or performance differences were observed between the CHO beverages. Consumption of multiple-transportable CHO, with or without hydrogel properties, preserves GI barrier integrity and reduces enterocyte damage during prolonged cycling in hot-humid conditions.
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http://dx.doi.org/10.1139/apnm-2020-0118DOI Listing
October 2020

On the climbing performance enhancing effects of New Zealand blackcurrant extract.

Eur J Appl Physiol 2020 06 20;120(6):1471-1472. Epub 2020 Apr 20.

Institute of Sport, University of Chichester, Chichester, UK.

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http://dx.doi.org/10.1007/s00421-020-04376-8DOI Listing
June 2020

Dietary supplementation with New Zealand blackcurrant extract enhances fat oxidation during submaximal exercise in the heat.

J Sci Med Sport 2020 Oct 12;23(10):908-912. Epub 2020 Mar 12.

Institute of Sport, University of Chichester, UK.

Objectives: This study investigated the effect of 7 days' supplementation with New Zealand blackcurrant extract on thermoregulation and substrate metabolism during running in the heat.

Design: Randomized, double-blind, cross-over study.

Methods: Twelve men and six women (mean±SD: Age 27±6 years, height 1.76±0.10m, mass 74±12kg, V̇O 53.4±7.0mLkgmin) completed one assessment of maximal aerobic capacity and one familiarisation trial (18°C, 40% relative humidity, RH), before ingesting 2×300mgday capsules of CurraNZ™ (each containing 105mg anthocyanin) or a visually matched placebo (2×300mg microcrystalline cellulose M102) for 7 days (washout 14 days). On day 7 of each supplementation period, participants completed 60min of fasted running at 65% V̇O in hot ambient conditions (34°C and 40% relative humidity).

Results: Carbohydrate oxidation was decreased in the NZBC trial [by 0.24gmin (95% CI: 0.21-0.27gmin)] compared to placebo (p= 0.014, d=0.46), and fat oxidation was increased in the NZBC trial [by 0.12gmin (95% CI: 0.10 to 0.15gmin)], compared to placebo (p=0.008, d=0.57). NZBC did not influence heart rate (p=0.963), rectal temperature (p=0.380), skin temperature (p=0.955), body temperature (p=0.214) or physiological strain index (p=0.705) during exercise.

Conclusions: Seven-days intake of 600mg NZBC extract increased fat oxidation without influencing cardiorespiratory or thermoregulatory variables during prolonged moderate intensity running in hot conditions.
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http://dx.doi.org/10.1016/j.jsams.2020.02.017DOI Listing
October 2020

Verteporfin-Loaded Anisotropic Poly(Beta-Amino Ester)-Based Micelles Demonstrate Brain Cancer-Selective Cytotoxicity and Enhanced Pharmacokinetics.

Int J Nanomedicine 2019 23;14:10047-10060. Epub 2019 Dec 23.

Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21231, USA.

Background: Nanomedicine can improve traditional therapies by enhancing the controlled release of drugs at targeted tissues in the body. However, there still exists disease- and therapy-specific barriers that limit the efficacy of such treatments. A major challenge in developing effective therapies for one of the most aggressive brain tumors, glioblastoma (GBM), is affecting brain cancer cells while avoiding damage to the surrounding healthy brain parenchyma. Here, we developed poly(ethylene glycol) (PEG)-poly(beta-amino ester) (PBAE) (PEG-PBAE)-based micelles encapsulating verteporfin (VP) to increase tumor-specific targeting.

Methods: Biodegradable, pH-sensitive micelles of different shapes were synthesized via nanoprecipitation using two different triblock PEG-PBAE-PEG copolymers varying in their relative hydrophobicity. The anti-tumor efficacy of verteporfin loaded in these anisotropic and spherical micelles was evaluated in vitro using patient-derived primary GBM cells.

Results: For anisotropic micelles, uptake efficiency was ~100% in GBM cells (GBM1A and JHGBM612) while only 46% in normal human astrocytes (NHA) at 15.6 nM VP (p ≤ 0.0001). Cell killing of GBM1A and JHGBM612 vs NHA was 52% and 77% vs 29%, respectively, at 24 hrs post-treatment of 125 nM VP-encapsulated in anisotropic micelles (p ≤ 0.0001), demonstrating the tumor cell-specific selectivity of VP. Moreover, anisotropic micelles showed an approximately fivefold longer half-life in blood circulation than the analogous spherical micelles in a GBM xenograft model in mice. In this model, micelle accumulation to tumors was significantly greater for anisotropic micelle-treated mice compared to spherical micelle-treated mice at both 8 hrs (~1.8-fold greater, p ≤ 0.001) and 24 hrs (~2.1-fold greater, p ≤ 0.0001).

Conclusion: Overall, this work highlights the promise of a biodegradable anisotropic micelle system to overcome multiple drug delivery challenges and enhance efficacy and safety for the treatment of brain cancer.
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http://dx.doi.org/10.2147/IJN.S231167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935022PMC
March 2020

Reduced inflammatory and phagocytotic responses following normobaric hypoxia exercise despite evidence supporting greater immune challenge.

Appl Physiol Nutr Metab 2020 Jun 21;45(6):628-640. Epub 2019 Nov 21.

Department of Exercise Science, High Point University, High Point, NC 27268, USA.

This study examined changes in immune markers following sustained treadmill exercise in normobaric hypoxia. Ten subjects performed 1 h of treadmill exercise (65% maximal oxygen uptake) under normoxic (NORM: fraction of inspired oxygen (FO) = 20.9%) and normobaric hypoxic (HYP: FO = 13.5%) conditions. Blood samples, collected before, after (Post), 1 h after (1-Post), and 4 h after (4-Post) exercise, were assayed for plasma cytokines (interleukin (IL)-1RA/IL-1β/IL-8/tumor necrosis factor alpha (TNF-α)) and markers of leukocyte activation (macrophage inflammatory protein-1β (MIP-1β)/myeloperoxidase (MPO)/soluble intercellular adhesion molecule-1 (sICAM-1)) using ELISA. Pro- to anti-inflammatory cytokine ratios (TNF-α/IL-1RA; IL-1β/IL-1RA) were calculated. Peripheral blood mononuclear cells (PBMC) were analyzed for changes in inflammatory status (phosphorylated nuclear factor kappa B/nuclear factor kappa B) using Western Blot. Data were analyzed with 2-way (condition × time) repeated-measure ANOVAs with Newman-Keuls post hoc tests. MIP-1β was elevated at 1-Post HYP exercise (+11%; < 0.01) but did not increase following exercise in NORM. TNF-α/IL-1RA and IL-1β/IL-1RA ratios were both reduced ( < 0.05) following HYP exercise (-16% and -52%, respectively, at 1-Post and -7% and -32%, respectively, at 4-Post). IL-8 increased ( < 0.05) at Post and 1-Post NORM (+33% and +57%, respectively) and HYP (+60% and +83%, respectively) exercise, but was not different between conditions ( > 0.05). Interestingly, plasma sICAM-1 did not increase ( > 0.05) following NORM exercise but was increased ( < 0.05) at Post (+17%), 1-Post (+16%), and 4-Post (+14%) HYP exercise. There was also a delayed peak in plasma MPO concentrations following HYP exercise and PBMC exhibited a reduced ( < 0.05) inflammatory capacity at Post (-38%) and 1-Post (-49%). Following HYP exercise, participants exhibited () circulatory bias towards anti-inflammation; () elevated sICAM; () delayed peak in plasma MPO; and () diminished inflammatory response in PBMC. Collectively, these data suggest immunosuppression. This is undesirable, given that elevated MIP-1β (reported here) and elevated intestinal fatty acid binding protein (reported previously) both suggest higher lipopolysaccharide concentrations following HYP exercise.
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http://dx.doi.org/10.1139/apnm-2019-0657DOI Listing
June 2020

Prolonged treadmill running in normobaric hypoxia causes gastrointestinal barrier permeability and elevates circulating levels of pro- and anti-inflammatory cytokines.

Appl Physiol Nutr Metab 2020 Apr;45(4):376-386

Department of Exercise Science, High Point University, High Point, NC 27268, USA.

This study examined the impact of treadmill running in normobaric hypoxia on gastrointestinal barrier permeability and the systemic inflammatory response. Ten recreationally active participants completed two 1-h bouts of matched-workload treadmill exercise (65% normoxic maximal oxygen consumption) in counterbalanced order. One bout was performed in normoxia (NORM: fraction of inspired oxygen (FO) = 20.9%) and the other in normobaric hypoxia (HYP: FO = 13.5%). Minute ventilation, respiratory rate (), tidal volume (), oxygen consumption, carbon dioxide production, respiratory exchange ratio (RER), and heart rate (HR) were measured with a metabolic cart. Peripheral oxygen saturation (SpO) was measured with pulse oximetry. Absolute tissue saturation (StO) was measured with near-infrared spectroscopy. Fatty acid-binding protein (I-FABP) and circulating cytokine concentrations (interleukin (IL)-1Ra, IL-6, IL-10) were assayed from plasma samples that were collected pre-exercise, postexercise, 1 h-postexercise, and 4 h-postexercise. Data were analyzed with 2-way (condition × time) repeated-measures ANOVAs. Newman-Keuls post hoc tests were run where appropriate ( < 0.05). As compared with NORM, 1 h of treadmill exercise in HYP caused greater ( < 0.05) changes in minute ventilation (+30%), (+16%), (+10%), carbon dioxide production (+18%), RER (+16%), HR (+4%), SpO (-16%), and StO (-10%). Gut barrier permeability and circulating cytokine concentrations were also greater ( < 0.05) following HYP exercise, where I-FABP was shown increased at postexercise (+68%) and IL-1Ra at 1 h-postexercise (+266%). I-FABP and IL-1Ra did not change ( > 0.05) following NORM exercise. IL-6 and IL-10 increased with exercise in both study conditions but were increased more ( < 0.05) following HYP at postexercise (+705% and +127%, respectively) and 1 h-postexercise (+400% and +128%, respectively). Normobaric hypoxia caused significant desaturation and increased most cardiopulmonary responses by 10%-30%. Significant gut barrier permeability and increased pro- and anti-inflammatory cytokine concentrations could promote an "open window" in the hours following HYP exercise.
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http://dx.doi.org/10.1139/apnm-2019-0378DOI Listing
April 2020

Verteporfin-Loaded Poly(ethylene glycol)-Poly(beta-amino ester)-Poly(ethylene glycol) Triblock Micelles for Cancer Therapy.

Biomacromolecules 2018 08 23;19(8):3361-3370. Epub 2018 Jul 23.

Department of Ophthalmology, Department of Neurosurgery, Department of Materials Science and Engineering, and Department of Chemical and Biomolecular Engineering , Johns Hopkins University , Baltimore , Maryland 21218 , United States.

Amphiphilic polymers can be used to form micelles to deliver water-insoluble drugs. A biodegradable poly(ethylene glycol) (PEG)-poly(beta-amino ester) (PBAE)-PEG triblock copolymer was developed that is useful for drug delivery. It was shown to successfully encapsulate and pH-dependently release a water-insoluble, small molecule anticancer drug, verteporfin. PEG-PBAE-PEG micelle morphology was also controlled through variations to the hydrophobicity of the central PBAE block of the copolymer in order to evade macrophage uptake. Spherical micelles were 50 nm in diameter, while filamentous micelles were 31 nm in width with an average aspect ratio of 20. When delivered to RAW 264.7 mouse macrophages, filamentous micelles exhibited a 89% drop in cellular uptake percentage and a 5.6-fold drop in normalized geometric mean cellular uptake compared to spherical micelles. This demonstrates the potential of high-aspect-ratio, anisotropically shaped PEG-PBAE-PEG micelles to evade macrophage-mediated clearance. Both spherical and filamentous micelles also showed therapeutic efficacy in human triple-negative breast cancer and small cell lung cancer cells without requiring photodynamic therapy to achieve an anticancer effect. Both spherical and filamentous micelles were more effective in killing lung cancer cells than breast cancer cells at equivalent verteporfin concentrations, while spherical micelles were shown to be more effective than filamentous micelles against both cancer cells. Spherical and filamentous micelles at 5 and 10 μM respective verteporfin concentration resulted in 100% cell killing of lung cancer cells, but both micelles required a higher verteporfin concentration of 20 μM to kill breast cancer cells at the levels of 80% and 50% respectively. This work demonstrates the potential of PEG-PBAE-PEG as a biodegradable, anisotropic drug delivery system as well as the in vitro use of verteporfin-loaded micelles for cancer therapy.
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http://dx.doi.org/10.1021/acs.biomac.8b00640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249031PMC
August 2018

Heat and Hypoxic Acclimation Increase Monocyte Heat Shock Protein 72 but Do Not Attenuate Inflammation following Hypoxic Exercise.

Front Physiol 2017 16;8:811. Epub 2017 Oct 16.

Occupational Performance Research Group, Department of Sport and Exercise Sciences, University of Chichester, Chichester, United Kingdom.

Acclimation to heat or hypoxic stress activates the heat shock response and accumulation of cytoprotective heat shock proteins (HSPs). By inhibiting the NF-κB pathway HSP72 can preserve epithelial function and reduce systemic inflammation. The aim of this study was to determine the time course of mHSP72 accumulation during acclimation, and to assess intestinal barrier damage and systemic inflammation following hypoxic exercise. Three groups completed 10 × 60-min acclimation sessions (50% normoxic VOpeak) in control ( = 7; 18°C, 35% RH), hypoxic ( = 7; FO = 0.14, 18°C, 35% RH), or hot ( = 7; 40°C, 25% RH) conditions. Tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 10 (IL-10), and intestinal fatty acid binding protein (I-FABP) were determined at rest and following a cycling normoxic stress test (NST; ~2 weeks before acclimation), pre-acclimation hypoxic stress test (HST1; FO = 0.14, both at 50% normoxic VOpeak; ~1 week before acclimation) and post-acclimation HST (48 h; HST2). Monocyte HSP72 (mHSP72) was determined before and after exercise on day 1, 3, 5, 6, and 10 of acclimation. Accumulation of basal mHSP72 was evident from day 5 ( < 0.05) of heat acclimation and increased further on day 6 ( < 0.01), and day 10 ( < 0.01). In contrast, basal mHSP72 was elevated on the final day of hypoxic acclimation ( < 0.05). Following the NST, plasma TNF-α (-0.11 ± 0.27 ngmL), IL-6 (+0.62 ± 0.67 ngmL) IL-10 (+1.09 ± 9.06 ngmL) and I-FABP (+37.6 ± 112.8 pgmL) exhibited minimal change. After HST1, IL-6 (+3.87 ± 2.56 ngmL), IL-10 (+26.15 ± 26.06 ngmL) and I-FABP (+183.7 ± 182.1 pgmL) were elevated ( < 0.01), whereas TNF-α was unaltered (+0.08 ± 1.27; > 0.05). A similar trend was observed after HST2, with IL-6 (+3.09 ± 1.30 ngmL), IL-10 (+23.22 ± 21.67 ngmL) and I-FABP (+145.9 ±123.2 pgmL) increased from rest. Heat acclimation induces mHSP72 accumulation earlier and at a greater magnitude compared to matched work hypoxic acclimation, however neither acclimation regime attenuated the systemic cytokine response or intestinal damage following acute exercise in hypoxia.
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http://dx.doi.org/10.3389/fphys.2017.00811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650636PMC
October 2017

Whole body precooling attenuates the extracellular HSP72, IL-6 and IL-10 responses after an acute bout of running in the heat.

J Sports Sci 2018 Feb 5;36(4):414-421. Epub 2017 Apr 5.

b Centre for Applied Biological and Exercise Sciences , Coventry University , Coventry , UK.

The impact of whole-body precooling on the extracellular heat shock protein 72 (eHSP72) and cytokine responses to running in the heat is undefined. The aim of this study was to determine whether precooling would attenuate post-exercise eHSP72 and cytokine responses. Eight male recreational runners completed two 90-minute bouts of running at 65% [Formula: see text]Omax in 32 ± 0.9°C and 47 ± 6 % relative humidity (RH) preceded by either 60-minutes of precooling in 20.3 ± 0.3°C water (COOL) or 60 min rest in an air-conditioned laboratory (20.2 ± 1.7°C, 60 ± 3% RH; CON). eHSP72, TNF-α, IL-6, IL-10 IL-1ra were determined before and immediately after exercise. The elevation in post-exercise eHSP72 was attenuated after COOL (+0.04 ± 0.10 ng.mL) compared to CON (+ 0.29 ± 0.26 ng.mL;P < 0.001). No changes in TNF-α were observed at any stage. COOL reduced the absolute post-exercise change in IL-6 (P = 0.011) and IL-10 (P = 0.03) compared to CON. IL-1ra followed this trend (P = 0.063). A precooling-induced attenuation of eHSP72 and proinflammatory cytokines may aid recovery during multi-day sporting events, but could be counterproductive if a training response or adaptation to environmental stress is a desired outcome.
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http://dx.doi.org/10.1080/02640414.2017.1313441DOI Listing
February 2018

Physiological Responses to Treadmill Running With Body Weight Support in Hypoxia Compared With Normoxia.

J Sport Rehabil 2018 May 1;27(3):224-229. Epub 2018 Jun 1.

Context: Anecdotal reports suggest elite sports clubs combine lower-body positive-pressure rehabilitation with a hypoxic stimulus to maintain or increase physiological and metabolic strain, which are reduced during lower-body positive pressure. However, the effects of hypoxia on cardiovascular and metabolic response during lower-body positive-pressure rehabilitation are unknown.

Objective: Evaluate the use of normobaric hypoxia as a means to increase physiological strain during body-weight-supported (BWS) running.

Design: Crossover study.

Setting: Controlled laboratory.

Participants: Seven familiarized males (mean (SD): age, 20 (1) y; height, 1.77 (0.05) m; mass, 69.4 (5.1) kg; hemoglobin, 15.2 (0.8) g·dL) completed a normoxic and hypoxic (fraction of inspired oxygen [O] = 0.14) trial, during which they ran at 8 km·h on an AlterG™ treadmill with 0%, 30%, and 60% BWS in a randomized order for 10 minutes interspersed with 5 minutes of recovery.

Main Outcome Measures: Arterial O saturation, heart rate, O delivery, and measurements of metabolic strain via indirect calorimetry.

Results: Hypoxic exercise reduced hemoglobin O saturation and elevated heart rate at each level of BWS compared with normoxia. However, the reduction in hemoglobin O saturation was attenuated at 60% BWS compared with 0% and 30%, and consequently, O delivery was better maintained at 60% BWS.

Conclusion: Hypoxia is a practically useful means of increasing physiological strain during BWS rehabilitation. In light of the maintenance of hemoglobin O saturation and O delivery at increasing levels of BWS, fixed hemoglobin saturations rather than a fixed altitude are recommended to maintain an aerobic stimulus.
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http://dx.doi.org/10.1123/jsr.2016-0210DOI Listing
May 2018

Effective microorganism - X attenuates circulating superoxide dismutase following an acute bout of intermittent running in hot, humid conditions.

Res Sports Med 2016 Apr-Jun;24(2):130-44. Epub 2016 Mar 31.

f Muscle Cellular and Molecular Physiology (MCMP) & Applied Sport and Exercise Science(ASEP) Research Groups, Institute of Sport and Physical Activity Research (ISPAR), Department of Sport and Exercise Sciences , University of Bedfordshire , Bedford , UK.

This study determined the effectiveness of antioxidant supplementation on high-intensity exercise-heat stress. Six males completed a high-intensity running protocol twice in temperate conditions (TEMP; 20.4°C), and twice in hot conditions (HOT; 34.7°C). Trials were completed following7 days supplementation with 70 ml·day(-1) effective microorganism-X (EM-X; TEMPEMX or HOTEMX) or placebo (TEMPPLA or HOTPLA). Plasma extracellular Hsp72 (eHsp72) and superoxide dismutase (SOD) were measured by ELISA. eHsp72 and SOD increased pre-post exercise (p < 0.001), with greater eHsp72 (p < 0.001) increases observed in HOT (+1.5 ng·ml(-1)) compared to TEMP (+0.8 ng·ml(-1)). EM-X did not influence eHsp72 (p > 0.05). Greater (p < 0.001) SOD increases were observed in HOT (+0.22 U·ml(-1)) versus TEMP (+0.10 U·ml(-1)) with SOD reduced in HOTEMX versus HOTPLA (p = 0.001). Physiological and perceptual responses were all greater (p < 0.001) in HOT versus TEMP conditions, with no difference followed EM-X (p > 0.05). EM-X supplementation attenuated the SOD increases following HOT, potentiating its application as an ergogenic aid to ameliorate oxidative stress.
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http://dx.doi.org/10.1080/15438627.2015.1126279DOI Listing
March 2017

Cross Acclimation between Heat and Hypoxia: Heat Acclimation Improves Cellular Tolerance and Exercise Performance in Acute Normobaric Hypoxia.

Front Physiol 2016 8;7:78. Epub 2016 Mar 8.

Centre for Applied Biological and Exercise Sciences, Coventry University Coventry, UK.

Background: The potential for cross acclimation between environmental stressors is not well understood. Thus, the aim of this investigation was to determine the effect of fixed-workload heat or hypoxic acclimation on cellular, physiological, and performance responses during post acclimation hypoxic exercise in humans.

Method: Twenty-one males (age 22 ± 5 years; stature 1.76 ± 0.07 m; mass 71.8 ± 7.9 kg; [Formula: see text]O2 peak 51 ± 7 mL(.)kg(-1.)min(-1)) completed a cycling hypoxic stress test (HST) and self-paced 16.1 km time trial (TT) before (HST1, TT1), and after (HST2, TT2) a series of 10 daily 60 min training sessions (50% N [Formula: see text]O2 peak) in control (CON, n = 7; 18°C, 35% RH), hypoxic (HYP, n = 7; fraction of inspired oxygen = 0.14, 18°C, 35% RH), or hot (HOT, n = 7; 40°C, 25% RH) conditions.

Results: TT performance in hypoxia was improved following both acclimation treatments, HYP (-3:16 ± 3:10 min:s; p = 0.0006) and HOT (-2:02 ± 1:02 min:s; p = 0.005), but unchanged after CON (+0:31 ± 1:42 min:s). Resting monocyte heat shock protein 72 (mHSP72) increased prior to HST2 in HOT (62 ± 46%) and HYP (58 ± 52%), but was unchanged after CON (9 ± 46%), leading to an attenuated mHSP72 response to hypoxic exercise in HOT and HYP HST2 compared to HST1 (p < 0.01). Changes in extracellular hypoxia-inducible factor 1-α followed a similar pattern to those of mHSP72. Physiological strain index (PSI) was attenuated in HOT (HST1 = 4.12 ± 0.58, HST2 = 3.60 ± 0.42; p = 0.007) as a result of a reduced HR (HST1 = 140 ± 14 b.min(-1); HST2 131 ± 9 b.min(-1) p = 0.0006) and Trectal (HST1 = 37.55 ± 0.18°C; HST2 37.45 ± 0.14°C; p = 0.018) during exercise. Whereas PSI did not change in HYP (HST1 = 4.82 ± 0.64, HST2 4.83 ± 0.63).

Conclusion: Heat acclimation improved cellular and systemic physiological tolerance to steady state exercise in moderate hypoxia. Additionally we show, for the first time, that heat acclimation improved cycling time trial performance to a magnitude similar to that achieved by hypoxic acclimation.
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http://dx.doi.org/10.3389/fphys.2016.00078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4781846PMC
March 2016

Human monocyte heat shock protein 72 responses to acute hypoxic exercise after 3 days of exercise heat acclimation.

Biomed Res Int 2015 22;2015:849809. Epub 2015 Mar 22.

Exercise Science Applied Research Group, Coventry University, Priory Street, Coventry CV1 5FB, UK.

The aim of this study was to determine whether short-term heat acclimation (STHA) could confer increased cellular tolerance to acute hypoxic exercise in humans as determined via monocyte HSP72 (mHSP72) expression. Sixteen males were separated into two matched groups. The STHA group completed 3 days of exercise heat acclimation; 60 minutes cycling at 50% V̇O2peak in 40°C 20% relative humidity (RH). The control group (CON) completed 3 days of exercise training in 20°C, 40% RH. Each group completed a hypoxic stress test (HST) one week before and 48 hours following the final day of CON or STHA. Percentage changes in HSP72 concentrations were similar between STHA and CON following HST1 (P = 0.97). STHA induced an increase in basal HSP72 (P = 0.03) with no change observed in CON (P = 0.218). Basal mHSP72 remained elevated before HST2 for the STHA group (P < 0.05) and was unchanged from HST1 in CON (P > 0.05). Percent change in mHSP72 was lower after HST2 in STHA compared to CON (P = 0.02). The mHSP72 response to hypoxic exercise was attenuated following 3 days of heat acclimation. This is indicative of improved tolerance and ability to cope with the hypoxic insult, potentially mediated in part by increased basal reserves of HSP72.
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http://dx.doi.org/10.1155/2015/849809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385626PMC
December 2015

The impact of submaximal exercise during heat and/or hypoxia on the cardiovascular and monocyte HSP72 responses to subsequent (post 24 h) exercise in hypoxia.

Extrem Physiol Med 2014 29;3:15. Epub 2014 Sep 29.

Sport and Exercise Science Applied Research Group, Coventry University, Coventry, UK.

Background: The aims of this study were to describe the cellular stress response to prolonged endurance exercise in acute heat, hypoxia and the combination of heat and hypoxia and to determine whether prior acute exposure to these stressors improved cellular tolerance to a subsequent exercise bout in hypoxia 24 h later.

Methods: Twelve males (age 22 ± 4 years, height 1.77 ± 0.05 m, mass 79 ± 12.9 kg, VO2 max 3.57 ± 0.7 L · min(-1)) completed four trials (30-min rest, 90-min cycling at 50% normoxic VO2 max) in normothermic normoxia (NORM; 18°C, FIO2 = 0.21), heat (HEAT; 40°C, 20% RH), hypoxia (HYP; FIO2 = 0.14) or a combination of heat and hypoxia (COM; 40°C, 20% RH, FIO2 = 0.14) separated by at least 7 days. Twenty-four hours after each trial, participants completed a hypoxic stress test (HST; 15-min rest, 60-min cycling at 50% normoxic VO2 max, FIO2 = 0.14). Monocyte heat shock protein 72 (mHSP72) was assessed immediately before and after each exercise bout.

Results: mHSP72 increased post exercise in NORM (107% ± 5.5%, p > 0.05), HYP (126% ± 16%, p < 0.01), HEAT (153% ± 14%, p < 0.01) and COM (161% ± 32%, p < 0.01). mHSP72 had returned to near-resting values 24 h after NORM (97% ± 8.6%) but was elevated after HEAT (130% ± 19%), HYP (118% ± 17%) and COM (131% ± 19%) (p < 0.05). mHSP72 increased from baseline after HSTNORM (118% ± 12%, p < 0.05), but did not increase further in HSTHEAT, HSTHYP and HSTCOM.

Conclusions: The prior induction of mHSP72 as a result of COM, HEAT and HYP attenuated further mHSP72 induction after HST and was indicative of conferred cellular tolerance.
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http://dx.doi.org/10.1186/2046-7648-3-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4179935PMC
October 2014

Bioelectric characterization of epithelia from neonatal CFTR knockout ferrets.

Am J Respir Cell Mol Biol 2013 Nov;49(5):837-44

Departments of 1 Anatomy & Cell Biology.

Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.
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http://dx.doi.org/10.1165/rcmb.2012-0433OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931095PMC
November 2013

Comparative processing and function of human and ferret cystic fibrosis transmembrane conductance regulator.

J Biol Chem 2012 Jun 8;287(26):21673-85. Epub 2012 May 8.

Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242, USA.

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR(ΔF508/ΔF508) animal model may be useful.
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http://dx.doi.org/10.1074/jbc.M111.336537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381131PMC
June 2012
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