Effects of a 7-day military training exercise on inflammatory biomarkers, serum hepcidin, and iron status.

BACKGROUND: Hepcidin, a peptide that is released into the blood in response to inflammation, prevents cellular iron export and results in declines in iron status. Elevated serum and urinary levels of hepcidin have been observed in athletes following exercise, and declines in iron status have been reported following prolonged periods of training. The objective of this observational study was to characterize the effects of an occupational task, military training, on iron status, inflammation, and serum hepcidin. FINDINGS: Volunteers (n = 21 males) included Norwegian Soldiers participating in a 7-day winter training exercise that culminated in a 3-day, 54 km ski march. Fasted blood samples were collected at baseline, on day 4 (PRE, prior to the ski march), and again on day 7 (POST, following the ski march). Samples were analyzed for hemoglobin, serum ferritin, soluble transferrin receptor (sTfR), interleukin-6 (IL-6), and serum hepcidin. Military training affected inflammation and serum hepcidin levels, as IL-6 and hepcidin concentrations increased (P < 0.05) from the baseline to POST (mean ± SD, 9.1 ± 4.9 vs. 14.5 ± 8.4 pg/mL and 6.5 ± 3.5 vs. 10.2 ± 6.9 ng/mL, respectively). Iron status was not affected by the training exercise, as sTfR levels did not change over the course of the 7-day study. CONCLUSIONS: Military training resulted in significant elevations in IL-6 and serum hepcidin. Future studies should strive to identify the role of hepcidin in the adaptive response to exercise, as well as countermeasures for the prevention of chronic or repeated elevations in serum hepcidin due to exercise or sustained occupational tasks which may result in longer term decrements in iron status.

Legionella and non-Legionella bacteria in a biological treatment plant.

Legionella pneumophila were previously identified in the aeration ponds (up to 10(10) CFU/L) of a biological wastewater treatment plant at Borregaard Ind. Ltd., Sarpsborg, Norway, and in air samples (up to 3300 CFU/m(3)) collected above the aeration ponds. After 3 outbreaks of Legionnaires' disease reported in this area in 2005 and 2008, the aeration ponds of the plant were shut down by the Norwegian authorities in September 2008. The aim of the present work was to analyze the Legionella and non-Legionella bacterial communities in the aeration ponds before and during the shutdown process and to identify potential human pathogens. The non-Legionella bacterial community was investigated in selected samples during the shutdown process by 16S rDNA sequencing of clone libraries (400 clones) and growth analysis. The concentration of L. pneumophila and Pseudomonas spp. DNA were monitored by quantitative PCR. Results showed a decrease in the concentration of L. pneumophila and Pseudomonas spp. during the shutdown. This was accompanied by a significant change in the composition of the bacterial community in the aeration ponds. This study demonstrated that several advanced analytical methods are necessary to characterize the bacterial population in complex environments, such as the industrial aeration ponds.

One week of multifactorial high-stress military ranger training affects Gram-negative signalling.

BACKGROUND: Toll-like receptor 4 (TLR4), especially expressed on monocytes/macrophages, connects microbial and sterile innate immune activation. Lipopolysaccharide (LPS) from Gram-negative bacteria and several endogenous molecules, among others saturated fatty acids (SFAs), are able to induce signalling through this receptor. Downstream inflammatory cytokines orchestrate the immune response. Our aim was to investigate how long-lasting multifactorial stress affects Gram-negative signalling and search for possible correlations between cytokine production and TLR4 expression or SFA concentration. METHODS: Eight healthy males were studied during a 7-day ranger-training course with semi-continuous physical strain, together with energy and sleep restrictions. Blood drawn on days 0, 3, 5 and 7 was incubated ex vivo for 6 h with or without LPS 10 ng/mL, whereupon surface expression of TLR4 on CD14⁺ monocytes and supernatant concentrations of inflammatory cytokines (TNF-α, IL-1ß and IL-6) were measured. In addition, plasma free fatty acids were quantified. RESULTS: Monocyte TLR4 expression was elevated throughout the course (p < 0.05 vs. baseline). Corresponding results were found for SFAs. The concentration of TNF-α increased significantly on day 3 and thereafter normalized, and a similar pattern was seen for IL-1ß. No correlations were found between cytokine concentrations and monocyte TLR4 expression or plasma SFAs. CONCLUSION: Multifactorial stress significantly affected ex vivo production of TNF-α and monocyte surface expression of TLR4. In addition, mobilization of fat resulted in increased plasma concentrations of SFAs. No associations between inflammatory cytokines and monocyte TLR4 expression or SFAs were found.

Moderate temperature alterations affect Gram-negative immune signalling in ex vivo whole blood.

BACKGROUND: Alterations in body temperature may influence immune system function and consequently affect the risk of infection and inflammatory diseases. Lipopolysaccharide (LPS) from gram-negative bacteria induces production of inflammatory cytokines after ligand binding to Toll-like receptor 4 (TLR4) on immune cells (especially monocytes/ macrophages). Our aim was to explore how clinically relevant hypo- and hyperthermia affect this signalling in an ex vivo whole blood model, and investigate if the cytokine response was correlated with monocyte TLR4 expression level. METHODS: Blood from 11 healthy volunteers was incubated with LPS 10 ng/ml for 6 h at 33, 37 or 40°C. The concentrations of selected pro-inflammatory (tumour necrosis factor-α (TNF-α) and interleukin (IL)-1ß) and anti-inflammatory (IL-10) cytokines were measured in plasma, and the surface expression of TLR4 was quantified on CD14 + monocytes. RESULTS: Monocyte TLR4 expression and plasma IL-1ß were inversely related to temperature. The TNF-α production was unaffected by hypothermia but increased significantly during hyperthermia, whereas plasma IL-10 was significantly reduced during both hypo- and hyperthermic incubation. No correlation was found between TLR4 expression and cytokine concentrations. During hypothermia, the TNF-α/IL-10 and IL-1ß/IL-10 ratios increased seven and nine times, respectively. Hyperthermia increased the TNF-α/IL-10 ratio, but to a lesser extent (doubling), whereas the IL-1ß/IL-10 ratio remained unchanged. CONCLUSION: Hypothermia significantly changed the cytokine ratios in the pro-inflammatory direction. In comparison, the effect of hyperthermia was sparse, with a modest increase in the TNF-α/IL-10 ratio only. No association was found between LPS-stimulated cytokine production and TLR4 expression on CD14 + monocytes.

Alternative routes for dissemination of Legionella pneumophila causing three outbreaks in Norway.

Three outbreaks of Legionnaires' disease were reported in the Fredrikstad/Sarpsborg community, Norway, in 2005 and 2008 caused by the L. pneumophila ST15 and ST462 strains determined by sequence based typing. In this retrospective study, we suggest that the aeration ponds, a part of the biological treatment plant at Borregaard Ind. Ltd., are the main amplifiers and primary disseminators of the outbreak L. pneumophila strains. This result is supported by the finding that the ST15 and ST462 strains were not able to survive in air scrubber liquid media more than two days of incubation at the scrubber's operating conditions during the 2005 and 2008 outbreaks. In 2008, >10¹° CFU/L of L. pneumophila ST462 were detected in the aeration ponds. ST15 and ST462 were also detected in the river Glomma in 2005 and 2008, respectively, downstream of the wastewater outlet from the treatment plant (105CFU/L). These findings strongly suggest that the presence of L. pneumophila in the river is due to the release of wastewater from the industrial aeration ponds, demonstrating that the river Glomma may be an additional disseminator of L. pneumophila during the outbreaks. This work emphasizes the need for preventive actions against the release of wastewater containing human pathogens to the environment.

N-Acetylcysteine administered as part of the immediate post-traumatic resuscitation regimen does not significantly influence initiation of inflammatory responses or subsequent endotoxin hyporesponsiveness.

Polytrauma and resuscitative efforts induce extensive alterations in the host's internal environment and cellular responses that may be a serious threat to these patients. Administration of exogenous thiols has been recommended to modulate the post-traumatic inflammatory responses. In this study, we have investigated the effect of N-acetylcysteine (NAC) on the early markers of leukocyte activation and subsequent endotoxin hyporesponsiveness. Twenty-eight pigs were exposed to a standardized gunshot injury. First aid treatment and initial life saving surgery was started without delay. One group (n = 14) was randomised to receive NAC 200 mg kg(-1) over 20 min, the remaining group was given the same volume of vehicle. Blood samples drawn at time points 0 and 75 min were also studied in vitro and stimulated with LPS or LPS plus NAC. Selected physiologic variables and degree of organ injury were equal in both groups. TNF-alpha, IL-1beta, and reactive oxygen species (ROS) tended to be lower in the NAC-group (NS). In vitro, NAC significantly reduced the release of the same cytokines after the LPS challenge in blood drawn before injury. NAC did not influence post-traumatic endotoxin tolerance. Adding NAC to the immediate resuscitation fluid did not influence the early post-traumatic organ injury, and initiation of inflammatory responses significantly, or endotoxin tolerance. In vitro, NAC significantly reduced proinflammatory cytokine release, but only in normal blood. The clinical value of this treatment regimen is probably restricted, both due to the unfavourable post-traumatic internal environment and imposed dosing limitations.

Analysis of the genetic distribution among members of Clostridium botulinum group I using a novel multilocus sequence typing (MLST) assay.

Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partial sequencing of the adk, atpH, gyrB, proC, rpoD and spo0A genes from 51 various C. botulinum/sporogenes isolates was performed, resulting in 37 different sequence types (STs). Analysis of the sequence data revealed a genetic distribution in five larger clusters with a loose correlation to the BoNT serotypes. The developed MLST assay had a slightly lower resolution ability when compared to the MLVA (multilocus variable number of tandem repeat analysis), but the two methods resulted in similar subclusters of the strains possessing the BoNT serotypes A, B and F. The current work presents the development of a novel MLST assay useful for genotyping C. botulinum related to basic phylogenetic research and trace-back analysis in microbial forensic studies.

Effects of winter military training on energy balance, whole-body protein balance, muscle damage, soreness, and physical performance.

Physiological consequences of winter military operations are not well described. This study examined Norwegian soldiers (n = 21 males) participating in a physically demanding winter training program to evaluate whether short-term military training alters energy and whole-body protein balance, muscle damage, soreness, and performance. Energy expenditure (D2(18)O) and intake were measured daily, and postabsorptive whole-body protein turnover ([(15)N]-glycine), muscle damage, soreness, and performance (vertical jump) were assessed at baseline, following a 4-day, military task training phase (MTT) and after a 3-day, 54-km ski march (SKI). Energy intake (kcal·day(-1)) increased (P < 0.01) from (mean ± SD (95% confidence interval)) 3098 ± 236 (2985, 3212) during MTT to 3461 ± 586 (3178, 3743) during SKI, while protein (g·kg(-1)·day(-1)) intake remained constant (MTT, 1.59 ± 0.33 (1.51, 1.66); and SKI, 1.71 ± 0.55 (1.58, 1.85)). Energy expenditure increased (P < 0.05) during SKI (6851 ± 562 (6580, 7122)) compared with MTT (5480 ± 389 (5293, 5668)) and exceeded energy intake. Protein flux, synthesis, and breakdown were all increased (P < 0.05) 24%, 18%, and 27%, respectively, during SKI compared with baseline and MTT. Whole-body protein balance was lower (P < 0.05) during SKI (-1.41 ± 1.11 (-1.98, -0.84) g·kg(-1)·10 h) than MTT and baseline. Muscle damage and soreness increased and performance decreased progressively (P < 0.05). The physiological consequences observed during short-term winter military training provide the basis for future studies to evaluate nutritional strategies that attenuate protein loss and sustain performance during severe energy deficits.

Seven days' around the clock exhaustive physical exertion combined with energy depletion and sleep deprivation primes circulating leukocytes.

Both exhaustive physical exertion and starvation have been reported to induce depression of immune function. The aim of the present study was to investigate the inflammatory environment and state of activation and mediator-producing potential of circulating leukocytes during prolonged physical activity with concomitant energy and sleep deprivation. Eight well-trained males were studied during 7 days of semi-continuous physical activity. Sleep was restricted to about 1 h/24 h, energy intake to 1.5- 3.0 MJ/24 h. Blood was drawn at 07.00 A.M.: on days 0, 2, 4, and 7. Plasma levels of inflammation markers were measured. The response of circulating leukocytes to lipopolysaccharide (LPS; 1 microg mL(-1)), and the effect of added hydrocortisone (10 and 100 nmol L(-1)), were measured in the supernatant after 3 h of incubation in an ex vivo whole blood model. Activation of leukocytes steadily increased as measured by plasma matrix metalloproteinase-9, tumour necrosis factor-alpha, interleukin-1beta, and interleukin-6. Inhibitors of systemic inflammation were either unaltered (tissue inhibitor of matrix metalloproteinase-1) or elevated (plasma interleukin-1 receptor antagonist). Cortisol levels increased on days 2 and 4, but thereafter reverted to baseline values. The leukocytes responded to LPS activation with increasing release of inflammatory cytokines throughout the study period. The anti-inflammatory potency of hydrocortisone decreased. Prolonged multifactorial stress thus activated circulating immune cells and primed them for an increased response to a subsequent microbial challenge.