Takashi Kumae

Impact of a competitive marathon race on systemic cytokine and neutrophil responses

PURPOSE To investigate whether cytokines and neutrophils mediate exercise-related pathogenesis, we examined their responses and possible association after exhaustive exercise. METHODS Plasma and urine samples were obtained from 10 male runners before and after a 42.195-km marathon race. Major cytokines and neutrophil activation markers [myeloperoxidase (MPO) and lactoferrin (LTF)] were measured by enzyme-linked immunosorbent assays. Functional modulation of standard neutrophils and monocytes by plasma was determined on their luminol-dependent chemiluminescence responses. RESULTS The race induced peripheral neutrophilia accompanied by an increase in band neutrophils and monocytosis. Plasma MPO and LTF concentrations increased significantly by 1.8 and 1.4 times after the race. There was a greater increase in urine concentrations of MPO and LTF, 12.3 and 3.5 times after exercise, respectively, suggesting that neutrophil activation occurred and that renal clearance exceeded the increase in plasma concentrations. Plasma interleukin (IL)-6, IL-8, IL-10, granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), and monocyte chemotactic protein 1 (MCP-1) increased significantly after the race, and urine IL-1beta, IL-6, G-CSF, M-CSF, and MCP-1 increased significantly. The plasma IL-6 responses correlated with the increases of band neutrophil count (r = 0.860, P < 0.01), suggesting IL-6-mediated bone marrow release of neutrophils. Furthermore, the increases in urine MPO concentration were correlated with increases in urine IL-6 (r = 0.868, P < 0.01) and G-CSF (r = 0.875, P < 0.01), suggesting that these cytokines promoted neutrophil activation. However, preincubation of neutrophils and monocytes with postexercise plasma could not cause priming responses, possibly because of the exercise-induced enhancement of plasma antioxidant activity. CONCLUSION Although many cytokines recruiting and priming neutrophils and monocytes were secreted and functional after exhaustive exercise, overwhelming antioxidant and antiinflammatory defenses were induced, preventing exercise-induced oxidative stress.

Measurement of chemiluminescence from neutrophils in a 96-well microplate using Lumi Box U-800 II.

We have developed a microplate photon counting system based on a cooled charge-coupled device (Lumi Box U-800 II) jointly with Maikurotekku Nition Company (Chiba, Japan). The system makes it possible to quantify chemiluminescence (CL) in a 96-well microplate automatically and simultaneously in a single experiment. We studied the measurement conditions for a luminol-dependent CL assay from neutrophils stimulated with opsonized zymosan (OZ) using this system. Parameters examined included the effect of OZ dose per well, mixing speed, mixing time and detection time on CL responses. The results indicated that this system allows the measurement of CL from phagocytes on a large number of samples using small amounts of sample and regents.

Effects of cefbuperazone on the chemiluminescence of human neutrophils.

To investigate the in vivo effect of antimicrobial agent on neutrophil chemiluminescence and serum opsonic activity, cefbuperazone sodium (CBPZ) was administered to healthy male volunteers via an intravenous drip. A peripheral blood sample (10 ml) was collected 7 times: prior to injection, then 1, 3, 6, 9, 12 and 24 h after injection. Neutrophil chemiluminescence and serum opsonic activity was measured by the simultaneous multiple measurement system based on luminol-dependent chemiluminescence and indicated by peak height and peak time. Neutrophils were activated at hour 1 from the point of view of peak height and statistical significance was observed. The fluctuation of serum opsonic activity was small and statistical significance was not observed. CBPZ has an in vivo effect on neutrophils which was not supposed from in vitro study.

Effects of 6 months of endurance training on neutrophil functions to produce reactive oxygen species and mental states in male long-distance runners.

To clarify changes of neutrophil functions, mental conditions and relationships among them, 19 male elite long-distance runners participated in this study for 6 months. Examinations, with informed consent, were carried out once a month. According to the results of physical characteristics, it was thought that training intensity was reduced after the main race, Hakone-Ekiden. Neutrophil functions were estimated by indices of reactive oxygen species production, determined by luminol- and lucigenin-dependent chemiluminescence (LmCL and LgCL, respectively) and cytochrome c reduction methods. The peak times (PT) in LmCL and LgCL (LgPT) were most prolonged in January and December, respectively. The peak heights (PH) in LmCL (LmPH) were enhanced in February. Decreased levels of negative categories in the profile of mood state (POMS) questionnaire and the total mood state (TMS) of POMS were observed in February without significance. Correlation analysis using measured values revealed significant negative correlation between LmPH and negative categories in POMS; however, these correlations were possibly a mere appearance, caused by personal differences. After eliminating personal differences, LgPT correlated positively to depression (p< 0.05), anger (p< 0.05), fatigue (p < 0.01) and TMS (p< 0.05). These results suggest that the mean time from the recognition of foreign matter to the maximum production of superoxide from neutrophils is prolonged in the mentally suppressed conditions found under continuous physical training.

A study for prevention of chronic fatigue. part 2. effects of strenuous physical exercise performed in a training camp on serum enzyme activity levels and subjective fatigue.

The principal objective of this paper is to develop a simple and rapid method of estimating levels of fatigue so that chronic fatigue can be prevented. Long-distance runners belonging to a successful corporate team (Group A; 25 males) and representative runners at the prefectural level (Group B; 14 males) participated in this study. We examined the effects of strenuous physical exercise on serum enzyme activity and the fatigue level felt by the runners (subjective fatigue).The following parameters were measured on two consecutive mornings during a training period: physical characteristics, serum-biochemistry using the dry-chemistry method, and subjective fatigue determined using the questionnaire regarding subjective symptoms authorized by the Japan Association of Industrial Health and the Profile of Mood State (POMS). Group A was divided into A-Senior (17 males; highest performance level) and A-Freshman (8 males) subgroups according to the length of employment within the corporation (one year or more and less than one year, respectively).The levels of serum aspartate aminotransferase (AST) and creatine kinase (CK) were significantly lower in the A-Senior group than the other groups and this group displayed the “iceberg” POMS profile at both examinations. Some significant correlations between the elements of POMS and serum enzyme activity levels were observed among all three groups during both examinations. The decline of serum CK levels tended to accompany a decrease in “Fatigue” according to POMS among 9 of 17 members of the A-Senior group. This tendency between the A-Senior and the A-Freshman groups statistically differed according to theX - square analysis.Our results suggest that the effects of physical stress on serum enzyme activity levels and subjective fatigue are affected by performance levels. Physical fatigue seemed to be reflected by serum CK levels. Monitoring subjective fatigue while measuring serum enzyme activity levels using the dry-chemistry method immediately provides clinical value to players and coaches on site, and should therefore help to prevent a shift from “overreaching” to “overtraining”.

Effects of summer training camp on serum opsonic activity and plasma cytokine levels in female long-distance runners

In a summer training camp, strenuous physical exercises are carried out to improve endurance ability. The effects of the training stress on the immune system have been studied extensively. However, less attention has been paid to non-specific immunological changes. Serum opsonic activity (SOA) is a more direct and suitable indicator of non-specific humoral immunity. In this study, we used the luminal-dependent chemiluminescence (LmCL) to measure reactive oxygen species generation from pooled human neutrophils as an indicator of SOA. We also measured plasma myeloperoxidase (MPO) and plasma cytokine levels. Twenty-two female college runners took part in this study after giving their written informed consent. There was no significant difference in the physical characteristics and serum enzyme levels. However, significant differences were observed in the changes of blood property and plasma cytokine levels. According to MPO levels, neutrophils in vivo may be deactivated during the camp. Positive correlations between fluctuations of pro- and anti-inflammatory cytokine levels were observed in this study. According to the results of peak time and peak height of LmCL, SOA seems to increase at first qualitatively and then quantitatively during the summer camp. The increased SOA level may compensate for the decrease in neutrophil activity.

Development of a new simple estimating method for protein, fat, and carbohydrate in cooked foods.

Evaluations of daily nutrient intakes with practical accuracy contribute not only to public but also to personal health. To obtain accurate estimations of nutrient intake, chemical analyses of a duplicate sample of all foods consumed are recommended. But these analytical methods are expensive, time consuming, and not practically applicable for field surveys dealing with numerous food samples. To solve this problem, a new rapid and simple method of estimating nutrients is developed here. Elemental compositions of cooked foods were examined using a high speed and high performance carbon, hydrogen, and nitrogen autoanalyzer, and the results showed good reproducibility. A significant correlation between Kjeldahl’s and the autoanalyzer methods was observed in the nitrogen measurement (n=20; r =0.999; p< 0.001), and very good agreement was observed between the methods. Therefore, the nitrogen amount obtained by the autoanalyzer was used for the estimation of the protein proportion in the cooked foods. The fat and carbohydrate proportions estimated by the new method correlated with the values obtained by the chemical method (p< 0.001 each). There were also good agreements of fat and carbohydrate proportions between the chemical and the new estimation methods. According to these results, the new, rapid and simple estimation method established in this study should be applicable to nutritional research.

A study for prevention of chronic fatigue Part 1. Effects of endurance running during one month on blood properties and subjective fatigue.

The principal objective of this paper is basic research on the development of methods for the prevention of chronic fatigue. Overtraining was paid special attention to as one of the types of chronic fatigue condition. Six healthy male participants in a distance-running competition were recruited and used as subjects for this study with informed consent for all examinations. Subjects, that is, participants in the competition, tried to achieve their objective maximum running distance designated before the competition during one month, while in the same period performing their routine work duties. It was reported that the competition caused participants to exhibit an overtrained condition. In this paper, effects of endurance running during one month on blood properties and the fatigue level felt by the subject (subjective fatigue) were studied.Examinations carried out in this research were as follows; 1) physical examination was conducted 3 times during the study; 1 week before the competition, immediately before and 3 weeks after the end of the competition, 2) blood sampling was conducted 4 times; prior to the competition, 1 week after the beginning of and immediately before the end of the competition, and about 3 weeks after the competition, 3) subjective fatigue levels were monitored 8 times; 1 week before and one time in every week during the competition, and 1 week and again 3 weeks after the competition.Immediately before the end of the competition, when subjects might be expected to exhibit the overtrained condition, minor influences on fatigue levels were found. However, effects of the endurance run seemed to remain more than 3 weeks after the competition as indicated by changes of the population of white blood cells and blood hormone levels.The above mentioned results suggest that it is important to determine signs of overtraining at an early stage. To determine overtraining at an early stage, norepinephrine, testosterone and the ratio of testosterone to cortisol may be the most useful indices. These, as well as the element of “drowsiness and dullness” in the questionnaire on subjective symptoms of fatigue authorized by the Japan Association of Industrial Health and the elements of “anger”, “fatigue”, and “confusion” in the Profile of Mood State (POMS) questionnaire are thought to be the most important subjective indices.

In vivo and in vitro effects of sizofiran on the human neutrophils and the serum opsonic activity

In this paper, we examine the effects of SPG, which is a well known BRM, both in vivo and in vitro on the neutrophilic ROS production and the serum opsonic activity by the chemiluminescence technique using luminol as a probe.To investigate the in vivo effects, SPG was administered to 12 healthy male volunteers and two phases of enhancement of the neutrophilic ROS production and the serum opsonic activity were observed. In vitro, the addition of SPG showed a dose-dependent suppression. To investigate the mechanisms in these contradictory effects of SPG, supernatants of a lymphocytes culture medium in the presence of SPG with or without mitogen were added to the neutrophils. The addition of supernatants at a lower concentration of SPG (0.01 mg/ml) with mitogens showed significant preventive effects on the neutrophilic ROS production for the duration of incubation.This suggests that cytokines derived from lymphocytes may contribute to the in vivo effects of SPG. SPG can play an important role in the host’s defense against microbe infections by enhancing it’s effect on non-specific immunity when administered in vivo