La importancia de la motivación como aporte del docente para superar el

Many studies have utilised a range of physical stimuli in order to study the acute phase response (Arai, Yorifuji et al. 1990; Gleeson, Almey et al. 1995; Shek, Sabiston et al. 1995; Camus, Poortmans et al. 1997; Rohde, MacLean et al. 1997; Ostrowski, Hermann et al. 1998; Ostrowski, Rohde et al. 1998; Ostrowski, Rohde et al. 1999; Moldoveanu, Shephard et al. 2000; Smith, Anwar et al. 2000; Takarada, Nakamura et al. 2000). Whilst the markers measured often differed between studies, most reported a physiological change similar to that following sepsis or trauma. However a number of problems are encountered when utilising such stressors to dissect the genetic factors that are important in modulating such responses. These include differences in the racial background, age, sex and environmental factors both before and after the exercise that will impact upon the quality and quantity of response generated. For these reasons there are several advantages in studying a sample of military recruits that require further discussion.

4.4.1 Advantages and Disadvantages O f Using Military Recruits To Study The Acute Inflammatory Response

All subjects enrolled in the current study were physically healthy and of closely related ages. Recruits spend their entire 10-week training period living in the same barracks, eating the same food and undergoing identical training. They are thus exposed to very similar environmental conditions, thereby reducing possible external confounders. Only Caucasians were selected for the final cohort of the present study in order to reduce the potential of racial bias. Additionally, at the outset of the study, it was decided only to approach male recruits, as there were too few female soldiers to make recruitment worthwhile, without introducing potential sex-bias into the results. However, the large numbers of recruits present at their induction afforded an excellent opportunity to prospectively screen potential volunteers on the basis of a specific genotype (IL6 - 174G>C and p-fibrinogen -455G>A). Without this prospective selection the study size would have needed to be very much larger to include a significant number of subjects carrying the least common genotypes (such as p-fibrinogen -455AA).

Despite these obvious advantages, so too are there a number of disadvantages of the present model of acute phase response, one of which relates to the study design. Due to the enormity of the task set at the study outset, of collecting serial blood samples over a twelve-month period of time from more than 50 military platoons (each over 4 separate sample time points) it was not possible to collect daily post-FME samples from each recruit. Furthermore, it was not felt to be ethically permissible to subject the study volunteers to five consecutive daily blood tests after the completion of the final exercise whilst they were still receiving physical training. Nonetheless, despite the more conservative blood-sampling protocol, many of the recruits still required careful explanation before they agreed to enter the study.

At the time of the study inception, it was anticipated that volunteer compliance would fall if any more than three serial post-FME samples would be requested (even if taken on alternate days). Indeed one of the greatest practical difficulties was not only in tracing the recruits at the 48-hour and 96-hour times post FME, whilst they were still undergoing the final stages of basic training, but also in obtaining all the follow-up blood samples. Thus the final number of samples available for assaying at the 96-hour

At the outset it was decided to take blood samples 2-hours after FME, rather than immediately following the end of FME. This timing was determined by logistic necessity, since the final exercises take place outside ATR Bassingboum requiring a 45- minute drive back to the barracks following their conclusion. Nonetheless in the light of the published data (Ostrowski, Rohde et al. 1998; Ostrowski, Rohde et al. 1999); Ostrowski, Hermann et al. 1998) it was still felt that even 2-hours beyond the end of FME, (and the predicted IL6 peak), IL6 levels would still be elevated several fold above pre-exercise levels.

An alternative approach would have been to duplicate the sampling protocol of the original Bassingboum study (Montgomery, Clarkson et al. 1996) where subjects each gave only two blood samples, one pre-training and one post-FME. In this manner smaller sub-groups of individuals would provide the second sample at different time intervals. Whilst there would have been no longitudinal data on any one individual’s response to exercise, and thus a reduction in the number of samples from subjects with the least frequent genotypes, the overall response to exercise would have been profiled more fully. Such an approach might have helped explain the timing of the secondary rise in fibrinogen levels 96-hours post-FME. This was attributed to the response following a 3-km mn that all recmits undertook following during the first two days after their returning from the FME, since fibrinogen levels are known to increase within a few hours of such exertion (Weight, Alexander et al. 1991).

One final issue to consider was the possibility of sample degradation between the time of centrifugation and freezing. This might have been particularly relevant to the IL6 measures, as this cytokine is easily degraded (in contrast to CRP and fibrinogen that are much more stable)(Flower, Ahuja et al. 2000). In order to discount this occurrence, prior to the commencement of the study, a small number of samples were drawn pre- and post-FME and split into two aliquots that were each handled differently. One set of serum samples was frozen immediately after centrifugation (representing the optimum conditions), and the other kept at 4®C until transportation back to the laboratory for freezing (reflecting the typical handling protocol). Following IL6 results were similar between the two pairs of samples (Table 3).

4.4.2 M ilitary Exercise Induces A n A cute Phase Response

As has been described in Chapter 1, during exercise the body generates an acute phase response. This is associated with increased circulating levels of cytokines (for example, IL l, TN Fa and IL6) together with raised concentrations of acute phase proteins such as CRP and fibrinogen. In contrast to the extreme inflammatory responses seen post- CABG (Chapter 3), the inflammatory response precipitated by the current FME can only be described as low-grade. Despite peak increases in IL6, fibrinogen and CRP of 99%, 16% and 112 % respectively, levels of all three markers remained within the normal clinical reference ranges. Peak 1L6 levels 2-hours post-FME correlated most strongly with the 48-hour fibrinogen and CRP values whilst the 48-hour 1L6 values correlated most strongly with fibrinogen and CRP at the 96-hour time point (Table 6). However the presence of a significant positive correlation between increases in fibrinogen and CRP at all three time points post-FME, confirms the internal consistency of the present study as a true model of the acute inflammatory response. These observations are analogous to those seen post-CABG, although the lack of daily post- FME samples prevents a fuller profile of the post-exercise changes in fibrinogen and CRP. Nonetheless these data are as expected based on in vitro studies that show 1L6 increases hepatic acute phase protein synthesis (Castell, Gomez-Lechon et al. 1989; Gauldie, Northemann et al. 1990; Kushner 1993).

4.4.3 Exercise Models O f Inflammatory Response

A number of earlier studies have been published describing the effect of exercise on acute inflammation, reporting a wide range of responses that vary with the nature of stressor used. Having reviewed the literature, those studies involving runners most closely resembled the present study of military exercise (Taylor, Rogers et al. 1987; Drenth, Krebbers et al. 1998);(Strachan, Noakes et al. 1984; Drenth, Van Uum et al. 1995; Weinstock, Konig et al. 1997; Ostrowski, Hermann et al. 1998; Ostrowski, Rohde et al. 1998; Ostrowski, Rohde et al. 1999); (Fallon 2001).

In a report by Drenth following a 5 km run, serum 1L6 was below the level of detection, with CRP levels rising at 24 hours by 2.5 fold to a mean of only 500 pg/ml (Drenth,

(Taylor, Rogers et al. 1987), CRP increased 300% 24-hours after the race with values again not significantly beyond those found of the healthy physiological range.

In two studies of marathon runners by (Ostrowski, Rohde et al. 1998; Ostrowski, Rohde et al. 1999)((Ostrowski, Rohde et al. 1998; Ostrowski, Rohde et al. 1999), IL6 increased from mean baseline values of 1.5 pg/ml and was shown to peak at the end of the run (reaching values of 80 and 94 pg/ml respectively). IL6 levels fell 2-hours into recovery although mean values remained significantly elevated (at 20 pg/ml and 22 pg/ml). In a study of subjects who ran on a treadmill for 2.5 hours, there was a 25-fold increase in EL6 from 1.5 to 35pg/ml, with comparable 2-hour post exercise IL6 levels (20pg/ml) to those seen in the marathon groups (Ostrowski, Hermann et al. 1998), however it was noted that IL6 levels already increased four fold after 30 minutes of running. Finally, a recent report by Fallon from a study of ultra marathon running, in whom competitors ran an excess of 100 km per day on six consecutive days (Fallon 2001), peak CRP increases of almost 2000% over baseline (1.9mg/l) were reported, with mean peak CRP values up to 38 mg/1.

Thus despite the low post-exercise IL6, fibrinogen and CRP values recorded for the current cohort of subjects, these data are broadly comparable to those previously reported in several of the earlier series, studies such as those by Fallon and Taylor (Taylor, Rogers et al. 1987; Fallon 2001).

4.4.4 Differences Between Past And Present Studies O f Military Exercise

Whilst there are no previous data describing the IL6 or CRP response following military exercise, differences exist between these data describing changes in fibrinogen post- FME and those from an earlier exercise study (Montgomery, Clarkson et al. 1996). Despite similar baseline fibrinogen values, the magnitude of fibrinogen increase in the current study cohort (representing increases of 15.7%, 3.4% and 7.6% 2-, 48- and 96 hours post-FME) is lower than in the first study, where peak fibrinogen increased by a mean of 37% 48-hours after exercise. These findings reflect changes in the content of the current two-day final exercise, which is now less intense than when first studied some 5 years ago. Recruits in the present cohort still complete several long endurance runs during FME, however there is no longer the same emphasis on exercises combining heavy lifting with endurance running as in previous years. This type of very

severe exertion at the end of the 48-hour FME might account for the elevated fibrinogen levels that peaked two days after exercise in the first study. In contrast, the present data show the highest fibrinogen levels to be on return from exercise.

The current study improves and expands upon the original observation in several ways. All subjects gave several samples for fibrinogen assay collected over several days, whilst in the original study, paired samples were obtained (one pre-exercise and one post-FME) with no individual giving more than one post-exercise sample. Thus the present sampling protocol will more accurately reflect an individual’s change in fibrinogen levels with exercise. Secondly, the current study was designed prospectively to select subjects on the basis of their genotype. In this way it was possible to enrich the final cohort to contain 19 subjects of the rare -455A A genotype, compared to just three in the original study. Finally, not only did the present study look at the association between the IL6 -174G>C and -572G>C polymorphisms and the fibrinogen response to exercise but data were also available describing the changes in IL6 and CRP post-FME also enabling the effects of IL6 and CRP genotype to be studied.

4.4.5 The Association Between 1L6 Genotype And Post-Exercise IL6 Response.

Despite the low-grade inflammatory response following FME, with peak increase of only 99% (compared to the 50-fold increases post-CABG, described in Chapter 3) these data support the presence of a significant association between IL6 -572G>C genotype and peak post-exercise IL6. However in contrast to the association of genotypes - 572GC/CC and -174CC with higher post CABG EL6, the genotype -572GC was associated with lower post-FME IL6 levels. Similarly, IL6 levels were higher in subjects carrying one or more -174G-allele, although at no time point was this difference statistically significant.

How then can these apparently contradictory data be reconciled with the results of the CABG study? There are a number of possible explanations, although it is likely that a combination of factors is involved. Firstly, as stated above the overall magnitude of inflammatory response generated by the present military exercise model is clearly much less severe than that generated by long-distance running and a fraction of the response to CABG. Thus any conclusions about the effect on genotype exercise-induced APR

to other scenarios. Secondly, it is possible that in the face of a more profound inflammatory stimulus (which, for the sake of discussion, might have generated CRP values in the range of 10-15 mg/1 or IL6 values of 5-10 pg/ml), a different effect of genotype might have been revealed. Finally, there might be no true association between genotype and exercise-induced inflammatory response at all, although the strength of the association appears relatively robust (there is an approximately 1 in 40 probability of the difference in 2-hour post-FME IL6 between IL6 -572G>C genotypes having occurred by chance alone). Whilst these data conflict with those describing the IL6 response post-CABG, they remain internally consistent with the observed association between genotype -572GC and lower post-FME fibrinogen (see section 4.4.7). However there is one important caveat. The overall magnitude of APR induced by the present exercise-model of inflammation is unlikely to represent the maximal inflammatory response to exercise, especially when these results are compared to data from studies such as those by (Ostrowski, Rohde et al. 1998; Ostrowski, Rohde et al. 1999). Thus any association between IL6 genotype and EL6 response should again be kept in the correct context.

It is possible that in the face of a more rigorous physical challenge, that would generate “inflammatory” levels of IL6, CRP and fibrinogen, that a different effect of genotype would be revealed. To prospectively test such a hypothesis would require a rigid and more readily controlled exercise protocol. It might be possible to recruit healthy subjects selected by genotype, to undergo a formal 30-minute treadmill run with serial sampling pre-and post-exercise. For instance, performing such a study with 25 subjects in each genotype group (-572GG and -572GC) would have 80% power to detect a difference between peak IL6 values of 2.0 and 2.5 pg/ml for the two groups, (with total SD + 0.7) representing a difference that was statistically significant at p=0.05.

Conversely, whilst the post-FME response might only be low-grade, it might in fact be a better reflection of the sub-clinical changes in acute phase markers that occur throughout a person’s life, rather than the extreme APR following CABG. In particular, the levels of peak IL6 and fibrinogen generated after exercise are similar to those seen in smokers. This is of extreme importance since smoking is associated with an approximate two-fold increase in the risk of cardiovascular death. It is tempting to speculate that small differences in inflammatory markers may lead to a long-term

increase in cumulative inflammatory burden that in turn might increase cardiovascular risk in a similar fashion.

4A .6 The Effect O f Fibrinogen Genotype On Fibrinogen Post-Fxercise

The present study data confirm the previous report of an association between fibrinogen -455G>A genotype and the fibrinogen response to exercise (Montgomery, Clarkson et al. 1996). Carriers of one or more -455A-allele had significantly higher fibrinogen levels after exercise than -455GG homozygotes. However aside from the differences in study design (that have already been discussed) there were also slightly different observations between these two studies.

The first study by Montgomery et al showed a “dose-effect” of fibrinogen -455G>A genotype on fibrinogen levels, determined by the number of -455A-alleles, with the highest fibrinogen levels for genotype -455AA and intermediate values for genotype - 455GA. In contrast, the current data show very similar fibrinogen values in subjects of genotype -455A A and -455GA. Although there is no clear explanation for this difference the effect of genotype might be explained by a “threshold effect”, where a very low-level inflammatory stimulus is insufficient to drive fibrinogen production. In such a scenario, subjects of genotype -455AA fail to generate higher fibrinogen than individuals of genotype -455GA, but in the face of a more severe stimulus the -455A- allele homozygotes are able to generate the highest fibrinogen. Since the magnitude of inflammatory stimulus in the present study was lower than in the original report by Montgomery et al, based on the lower fibrinogen levels, this might account for the difference.

In contrast to other studies that suggest functionality of the -854G>A polymorphism (Behague, Poirier et al. 1996; v an 't Hooft, von Bahr et al. 1999), these data do not support the presence of a significant association between the -854A-allele and higher resting fibrinogen levels, nor do they identify any further effect of this polymorphism following exercise. This may reflect a genuine lack of in vivo functionality. Alternatively, the degree of gene-environment interaction may be too weak to detect in a sample of the present size, or may have only been detectable in a situation where the fibrinogen response was greater than that actually observed with the present model.

4.4 .7 The E ffect O flL 6 Genotype On Fibrinogen Post-Exercise

The association of the IL6 -174G>C and -572G>C promoter polymorphisms, with the fibrinogen response following FME has not been previously described. At baseline there appears to be a codominant effect of the -174G>C polymorphism, such that fibrinogen levels are highest for genotype -174GG and lowest for genotype -174CC. This observation is broadly in keeping with the expected effect, based on the findings of Fishman (Fishman, Faulds et al. 1998). Thus in the subjects of genotype -174GG, who have the highest IL6 levels, one would predict there to be the highest fibrinogen and CRP) following an inflammatory stimulus. Although IL6 levels were higher in -174G- allele carriers at no point was this difference statistically significant. However, this contradicts the predictions one might make based on IL6 data from the CABG model (Chapter 3), where IL6 genotype -174CC and -572GC/CC were associated with the highest IL6 levels. Following FME there was no significant association between IL6 -

174 genotype and fibrinogen levels.

There was no difference in baseline fibrinogen levels between -572C-allele carriers and subjects of genotype -572GG. Carriers of the -572C-allele did have lower mean fibrinogen levels following FME, a difference that was significant 2-hours post FME, ANOVA p=0.026 (ANCOVA, p<0.03). This observation is particularly intriguing, since

in vitro data suggest the -572C-allele to have greater promoter strength than the C-allele (Le Anh Luong, personal communication) which would suggest that IL6, CRP and fibrinogen values should be higher in -572C-allele carriers. This association is again opposite to the expected result based on the finding of higher IL6 levels following