The effect of hyperbaric oxygen preconditioning on heat shock protein 72 expression following in vitro stress in human monocytes

Hyperbaric oxygen (HBO) is thought to confer protection to cells via a cellular response to free radicals. This process may involve increased expression of heat shock proteins, in particular the highly inducible heat shock protein 72 (Hsp72). Healthy male volunteers (n = 16) were subjected to HBO for 1 h at 2.8 ATA. Inducible Hsp72 expression was measured by flow cytometry pre-, post- and 4 h-post HBO. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood via density centrifugation pre-, post- and 4 h post-HBO. PBMC were then subjected to an in vitro heat shock at 40°C or hypoxia at 37°C (5% O₂) with a control at 37°C. Cells were then analysed for Hsp72 expression by flow cytometry. Monocytes showed no significant changes in Hsp72 expression following HBO. No detectable Hsp72 was seen in lymphocytes or neutrophils. Following in vitro hypoxic exposure, a significant increase in Hsp72 expression was observed in monocytes isolated immediately post- (p = 0.006) and 4 h post-HBO (p = 0.010) in comparison to control values. HBO does not induce Hsp72 expression in PBMC. The reported benefits of HBO in terms of pre-conditioning are not due to inducement of Hsp72 expression in circulating blood cells, but may involve an enhancement of the stress response.     

In vitro heat shock of human monocytes results in a proportional increase of inducible Hsp70 expression according to the basal content

Heat shock proteins play an important role as molecular chaperones of the cell. Inducible heat shock protein 70 is rapidly synthesised in response to numerous stressors and monocytes are sensitive to changes in core temperature resulting in a circadian variation of Hsp70 expression. Monocytes were isolated via density centrifugation from nine healthy male volunteers at 5 am, 1 pm and 9 pm, representing the nadir (5 am), peak (9 pm) and intermediate (1 pm) of Hsp70 expression in the 24-h cycle. Analysis of freshly isolated monocytes for Hsp70 expression confirmed Hsp70 levels at the three selected time points. Monocytes were subjected to in vitro heat shock at 40°C (±0.1) for 90 min with a 90 min 37°C (±0.1) exposure acting as a control. A significant increase in Hsp70 was observed at 5 am (p < 0.001) and 1 pm (p = 0.028) at 40°C when compared to 37°C but not at 9 pm (p = 0.19). A significant increase was also observed from the basal levels of Hsp70, measured on freshly isolated monocytes and the levels detected after heat shock at 40°C at 5 am (p < 0.001) and 1 pm (p = 0.001), which was not observed at 9 pm (p = 0.15). Furthermore, a significant correlation was observed in the heat shock response at 40°C and that obtained at 37°C (p < 0.001). In conclusion, the heat shock response in monocytes is directly proportional to the amount of Hsp70 present in the cells and the stress response may be much higher at different times of the day.     

The effect of 15 consecutive days of heat–exercise acclimation on heat shock protein 70

The purpose of this study was to investigate the alterations in serum heat shock protein (Hsp) 70 levels during a 15-consecutive-day intermittent heat–exercise protocol in a 29-year-old male ultra marathon runner. Heat acclimation, for the purpose of physical activities in elevated ambient temperatures, has numerous physiological benefits including mechanisms such as improved cardiac output, increased plasma volume and a decreased core temperature (T _c). In addition to the central adaptations, the role of Hsp during heat acclimation has received an increasing amount of attention. The acclimation protocol applied was designed to correspond with the athlete’s tapering period for the 2007 Marathon Des Sables. The subject (VO₂max = 50.7 ml·kg⁻¹·min⁻¹, peak power output [PPO] = 376 W) cycled daily for 90 min at a workload corresponding to 50% of VO₂max in a temperature-controlled room (average WBGT = 31.9 ± 0.9°C). Venous blood was sampled before and after each session for measurement of serum osmolality and serum Hsp70. In addition, T _c, heart rate (HR) and power output (PO) was measured throughout the 90 min to ensure that heat acclimation was achieved during the 15-day period. The results show that the subject was successfully heat acclimated as seen by the lowered HR at rest and during exercise, decreased resting and exercising T _c and an increased PO. The heat exercise resulted in an initial increase in Hsp70 concentrations, known as thermotolerance, and the increase in Hsp70 after exercise was inversely correlated to the resting values of Hsp70 (Spearman’s rank correlation = −0.81, p < 0.01). Furthermore, the 15-day heat–exercise protocol also increased the basal levels of Hsp70, a response different from that of thermotolerance. This is, as far as we are aware, the first report showing Hsp70 levels during consecutive days of intermittent heat exposure giving rise to heat acclimation. In conclusion, a relatively longer heat acclimation protocol is suggested to obtain maximum benefit of heat acclimation inclusive of both cellular and systemic adaptations.     

The time-profile of the PBMC HSP70 response to in vitro heat shock appears temperature-dependent

Summary. Heat shock proteins (HSPs) are synthesised by cells subsequent to a stress exposure and are known to confer protection to the cell in response to a second challenge. HSP induction and decay are correlated to thermotolerance and may therefore be used as a biomarker of thermal history. The current study tested the temperature-dependent nature of the heat shock response and characterised its time profile of induction. Whole blood from 6 healthy males (Age: 26 ± (SD) 2 yrs; Body mass 74.2 ± 3.8 kgs; VO_2max: 49.1 ± 4.0 ml·kg⁻¹·min⁻¹) were isolated and exposed to in vitro heat shock (HS) at 37, 38, 39, 40, and 41 °C for a period of 90 min. After HS the temperature was returned to 37 °C and intracellular HSP70 was quantified from the leukocytes at 0, 2, 4, and 6 h after heat treatment. The concentration of HSP70 was not different between temperatures (P > 0.05), but the time-profile of HSP70 synthesis appeared temperature-dependent. At control (37 °C) and lower temperatures (38–39 °C) the mean HSP70 concentration increased up to 4 h post HS (P < 0.05) and then returned towards baseline values by 6 h post HS. With in vitro hyperthermic conditions (40–41 °C), the time-profile was characterised by a sharp rise in HSP70 levels immediately after treatment (P < 0.05 for 40 °C at 0 h), followed by a progressive decline over time. The results suggest a temperature-dependent time-profile of HSP70 synthesis. In addition, the temperature at which HSP70 is inducted might be lower than 37 °C.     

The effect of temperature and substrate on the development of the cortisol stress response in the lake sturgeon, <Emphasis Type="Italic">Acipenser fulvescens,</Emphasis> Rafinesque (1817)

Lake sturgeon, Acipenser fulvescens, are considered threatened or endangered throughout most of their North American Range. Current hatchery rearing for re-stocking programs utilise conventional methods with little to no understanding of the relationship between rearing conditions and the development of the hypothalamic-pituitary-interrenal (HPI) stress axis. In the present study we examined the effects of substrate type and temperature on the development of the HPI stress axis in prolarval and larval lake sturgeon. Lake sturgeon raised over either gravel or no substrate did not consistently show an increase in whole body cortisol at the prolarval stage. However, after the onset of exogenous feeding a consistent increase in whole body cortisol following a stress was evident. Lake sturgeon larvae raised in gravel substrate demonstrated a sustained increase in whole body cortisol for at least 240 min post stress whereas whole body cortisol in larvae raised in no substrate returned to baseline within 240 min post stress. Lake sturgeon larvae raised at 9, 12 and 15°C exhibited markedly different cortisol responses with baseline whole body cortisol being, 38.6 ± 3, 5.67 ± 0.41 and 25.38 ± 2.84 ng.g⁻¹ respectively. Furthermore, the chase induced increases in whole body cortisol at the larval stage were significantly different for each temperature treatment. These experiments demonstrate that physical environment has a significant impact on the development of the HPI stress axis in lake sturgeon.     

Regulation of heat shock proteins in the apple maggot Rhagoletis pomonella during hot summer days and overwintering diapause

Abstract Developing larvae of the apple maggot Rhagoletis pomonella are frequently exposed to summertime apple temperatures that exceed 40 °C and, during their overwintering diapause, pupae are exposed to sub‐zero soil temperatures for prolonged periods. To investigate the potential involvement of heat shock proteins (Hsps) in response to these environmental extremes, the genes encoding Hsp70 and Hsp90 in R. pomonella are cloned and expression monitored during larval feeding within the apple and during overwintering pupal diapause. Larvae reared in the laboratory at constant temperatures of 25, 28 or 35 °C express Hsp90 but very little Hsp70. Larvae do not survive rearing at 40 °C. The temperature cycles to which larvae were exposed inside apples in the field, ranging 16–46.9 °C over a 24‐h period, elicit strong Hsp70 and Hsp90 expression, which begins at mid‐day and reaches a peak in late afternoon, coinciding with peak air and apple temperatures. Heat shock proteins are also expressed strongly by pupae during their overwintering diapause. Hsp70 is not expressed in nondiapausing pupae but is highly expressed throughout diapause. Hsp90 is constitutively expressed in both diapausing and nondiapausing pupae. Rhagoletis pomonella thus strongly expresses its Hsps during pupal diapause, presumably as a protection against low temperature injury, and during larval development to cope with natural temperature cycles prevailing in late summer.     

Variation in basal heat shock protein 70 is correlated to core temperature in human subjects

Heat shock proteins are highly conserved proteins and play an important chaperone role in aiding the folding of nascent proteins within cells. The heat shock protein response to various stressors, both in vitro and in vivo, is well characterised. However, basal levels of heat shock protein 70 (Hsp70) have not previously been investigated. Monocyte-expressed Hsp70 was determined every 4 h, over a 24 h time period, in 17 healthy male subjects (177 ± 6.4 cm, 75.7 ± 10.9 kg, 19.8 ± 4.3 years) within a temperature and activity controlled environment. Core temperature was measured at 5-min intervals during the 24 h period. Hsp70 showed significant diurnal variation (F = 7.4; p < 0.001), demonstrating peaks at 0900 and 2100 hours, and a nadir at 05.00. Core temperature followed a similar temporal trend (range = 35.96–38.10°C) and was significantly correlated with Hsp70 expression (r _s = 0.44; p < 0.001). These findings suggest a high responsiveness of Hsp70 expression in monocytes to slight variations in core temperature.     

Kinetics and characteristics of 70 °C, VFA-grown, UASB granular sludge

We studied in batch reactors the kinetics and characterization of 70 °C, volatile fatty acids (VFAs)-grown, upflow anaerobic sludge blanket granular sludge with 55 and 35 °C sludge as reference. The half-saturation constant (K _s), the inhibition constant (K _i), the maximum specific methane production rate (μ_CH4max), and the inhibition response coefficient (n) of the 70 °C sludge were 6.15 mM, 48.2 mM, 0.132 h⁻¹, and 2.48, respectively, while no inhibition occurred at 55 and 35 °C, where the K _s was 3.67 and 3.82 mM, respectively. At 70 °C, the highest initial specific methanogenic activity (ISMA, 0.311 gCH₄-COD per gram volatile solids per day) on VFAs was about 12–15% lower than that on acetate and three to four times less than the ISMA for the 55 and 35 °C sludge. In the acetate conversion study, residual acetate (79 mg l⁻¹) at 70 °C was three to five times higher than that at 55 and 35 °C. Further, the methane produced as percentage of the acetate consumed at 70 °C (89%) was lower than that at 55 (95%) and 35 °C (97%). At 70 °C, 10% of the ISMA remained after 15 days of starvation as compared to 26% (55 °C) and 92% (35 °C) after 30 days of starvation. Thus, the kinetics of the 70 °C granular sludge seem to differ from those at 55 and 35 °C. Received: 1 February 1999 / Accepted: 20 March 1999     

Thermal maxima for juvenile shortnose sturgeon acclimated to different temperatures

Many populations of shortnose sturgeon, Acipenser brevirostrum, in the southeastern United States continue to suffer from poor juvenile recruitment. High summer water temperatures, which may be exacerbated by anthropogenic activities, are thought to affect recruitment by limiting available summer habitat. However, information regarding temperature thresholds of shortnose sturgeon is limited. In this study, the thermal maximum method and a heating rate of 0.1°C min⁻¹ was used to determine critical and lethal thermal maxima for young-of-the-year (YOY) shortnose sturgeon acclimated to temperatures of 19.5 and 24.1°C. Fish used in the experiment were 0.6 to 35.0 g in weight and 64 to 140 days post hatch (dph) in age. Critical thermal maxima were 33.7°C (±0.3) and 35.1°C (±0.2) for fish acclimated to 19.5 and 24.1°C, respectively. Critical thermal maxima significantly increased with an increase in acclimation temperature (p < 0.0001). Lethal thermal maxima were 34.8°C (±0.1) and 36.1°C (±0.1) for fish acclimated to 19.5 and 24.1°C, respectively. Lethal thermal maxima were significantly affected by acclimation temperature, the log₁₀ (fish weight), and the interaction between log₁₀(fish weight) and acclimation temperature (p < 0.0001). Thermal maxima were used to estimate upper limits of safe temperature, thermal preferences, and optimal growth temperatures of YOY shortnose sturgeon. Upper limits of safe temperature were similar to previous temperature tolerance information and indicate that summer temperatures in southeastern rivers may be lethal to YOY shortnose sturgeon if suitable thermal refuge cannot be found.     

Increased circulating heat shock protein 70 levels in pregnant asthmatics

Asthma is one of the most common diseases complicating pregnancy and represents a risk factor for several maternal and perinatal complications. The natural history of asthma is known to change in pregnancy, but very few data are available in the terms of pathomechanism of this change during gestation. Circulating heat shock protein 70 (Hsp70) levels are decreased in healthy pregnancy, which might reflect physiological immunotolerance. The aim of our study was to determine the serum levels of Hsp70 in asthmatic women during gestation. Forty pregnant women with bronchial asthma and 40 healthy pregnant women matched for maternal and gestational age were involved in this case-control study. Serum Hsp70 levels were measured using the ELISA Kit of R&D Systems. Spirometry and oxygen saturation measurements were performed in asthmatic patients. In asthmatic pregnant women, an increase of serum Hsp70 levels was observed compared to healthy pregnant women (median (25–75 percentile): 0.44 ng/ml (0.36–0.53) versus 0.21 ng/ml (0–0.27), p < 0.001). Fetal birth weight of asthmatic mothers was significantly smaller than of healthy controls, but in the normal range (3,230 g (2,690–3,550) versus 3,550 g (3,450–3,775), p < 0.05). A statistically significant negative correlation between maternal age and serum Hsp70 concentrations (Spearman R = −0.48, p = 0.0018) and a significant positive correlation between gestational age and serum Hsp70 levels (Spearman R = 0.83, p < 0.001) were detected in healthy pregnant women. In conclusion, this study proves an elevation of circulating Hsp70 levels during asthmatic pregnancy compared to healthy pregnant women. However, further studies are warranted to determine the role of circulating Hsp70 in the pathogenesis of maternal and perinatal complications of asthma in pregnancy.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

Influence of the Selected Antioxidants on the Stability of the Celsior Solution Used for Perfusion and Organ Preservation Purposes

The purpose of the following research was to improve the original Celsior solution in order to obtain a higher degree of stability and effectiveness. The solution was modified by the addition of selected antioxidants such as vitamin C, cysteine, and fumaric acid in the following concentrations: 0.1, 0.3, and 0.5 mmol/l. The solution’s stability was estimated using an accelerated stability test based on changes in histidine concentrations in the solution using Pauly’s method for determining concentrations. Elevated temperatures, the factor accelerating substances’ decomposition reaction rate, were used in the tests. The research was conducted at four temperatures at intervals of 10°C: 60 ± 0.2°C, 70 ± 0.2°C, 80 ± 0.2°C, and 90 ± 0.2°C. It was stated that the studied substances’ decomposition occurred in accordance with the equation for first-order reactions. The function of the logarithmic concentration (log%C) over time was revealed to be rectilinear. This dependence was used to determine the kinetics of decomposition reaction rate parameters (the rate constant of decomposition k, activation energy E _a, and frequency factor A). On the basis of these parameters, the stability of the modified solution was estimated at +5°C. The results obtained show that the proposed antioxidants have a significant effect on lengthening the Celsior solution’s stability. The best results were reached when combining two antioxidants: vitamin C and cysteine in 0.5 mmol/l concentrations. As a result, the Celsior solution’s stability was lengthened from 22 to 299 days, which is 13.5 times. Vitamin C at a concentration of 0.5 mmol/l increased the solution’s stability by 5.2 times (t ₉₀ = 115 days), cysteine at a concentration of 0.5 mmol/l caused a 4.4 times stability increase (t ₉₀ = 96 days), and fumaric acid at a concentration of 0.5 mmol/l extended the stability by 2.1 times (t ₉₀ = 48 days) in relation to the original solution.     

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta)

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34 °C and 36 °C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36 °C, 3 h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events.