John P. Campbell

Acute exercise mobilises CD8+ T lymphocytes exhibiting an effector-memory phenotype.

An acute bout of exercise evokes mobilisation of lymphocytes into the bloodstream, which can be largely attributed to increases in CD8+ T lymphocytes (CD8TLs) and natural killer (NK) cells. Evidence further suggests that, even within these lymphocyte subsets, there is preferential mobilisation of cells that share certain functional and phenotypic characteristics, such as high cytotoxicity, low proliferative ability, and high tissue-migrating potential. These features are characteristic of effector-memory CD8TL subsets. The current study therefore investigated the effect of exercise on these newly-identified subsets. Thirteen healthy and physically active males (mean+/-SD: age 20.9+/-1.5 yr) attended three sessions: a control session (no exercise); cycling at 35% Watt(max) (low intensity exercise); and 85% Watt(max) (high intensity exercise). Each bout lasted 20 min. Blood samples were obtained before exercise, during the final min of exercise, and +15, and +60 min post-exercise. CD8TLs were classified into naïve, central memory (CM), effector-memory (EM), and CD45RA+ effector-memory (RAEM) using combinations of the cell surface markers CCR7, CD27, CD62L, CD57, and CD45RA. In parallel, the phenotypically distinct CD56(bright) regulatory and CD56(dim) cytotoxic NK subsets were quantified. The results show a strong differential mobilisation of CD8TL subsets (RAEM>EM>CM>naïve); during high intensity exercise the greatest increase was observed for RAEM CD8Tls (+450%) and the smallest for naïve cells (+84%). Similarly, CD56(dim) NK cells (+995%) were mobilised to a greater extent than CD56(bright) (+153%) NK cells. In conclusion, memory CD8TL that exhibit a high effector and tissue-migrating potential are preferentially mobilised during exercise. This finding unifies a range of independent observations regarding exercise-induced phenotypic and functional changes in circulating lymphocytes. The selective mobilisation of cytotoxic tissue-migrating subsets, both within the NK and CD8TL population, may enhance immune-surveillance during exercise.

Rudimentary signs of immunosenescence in Cytomegalovirus-seropositive healthy young adults

Ageing is associated with a decline in immune competence termed immunosenescence. In the elderly, this process results in an accumulation of differentiated ‘effector’ phenotype memory T cells, predominantly driven by Cytomegalovirus (CMV) infection. Here, we asked whether CMV also drives immunity towards a senescent profile in healthy young adults. One hundred and fifty-eight individuals (meanu2009±u2009SD; age 21u2009±u20093xa0years, body mass index 22.7u2009±u20092.7xa0kgxa0m2) were assessed for CMV serostatus, the numbers/proportions of CD4+ and CD8+ late differentiated/effector memory cells (i.e. CD27−CD28−/CD45RA+), plasma interleukin-6 (IL-6) and antibody responses to an in vivo antigen challenge (half-dose influenza vaccine). Thirty percent (48/158) of participants were CMV+. A higher lymphocyte and CD8+ count (both pu2009<u20090.01) and a lower CD4/CD8 ratio (pu2009<u20090.03) were observed in CMV+ people. Eight percent (4/58) of CMV+ individuals exhibited a CD4/CD8 ratio <1.0, whereas no CMV− donor showed an inverted ratio (pu2009<u20090.001). The numbers of CD4+ and CD8+CD27−CD28−/CD45RA+ cells wereu2009~u2009fourfold higher in CMV+ people (pu2009<u20090.001). Plasma IL-6 was higher in CMV+ donors (pu2009<u20090.05) and showed a positive association with the numbers of CD8+CD28− cells (pu2009<u20090.03). Finally, there was a significant negative correlation between vaccine-induced antibody responses to the A/Brisbane influenza strain and CMV-specific immunoglobulin G titres (pu2009<u20090.05). This reduced vaccination response was associated with greater numbers of total CD8+ and CD4+ and CD8+CD27−CD28−/CD45RA+ cells (pu2009<u20090.05). This study observed marked changes in the immune profile of young adults infected with CMV, suggesting that this virus may underlie rudimentary aspects of immunosenescence even in a chronologically young population.

Serum free immunoglobulin light chain evaluation as a marker of impact from intraclonal heterogeneity on myeloma outcome

Intraclonal heterogeneity was recently described in multiple myeloma (MM), but its full impact on disease progression and relapse has not been entirely explored. The immunoglobulin type produced by myeloma cells provides an excellent marker to follow changes in clonal substructure over time. We have prospectively evaluated serial paraprotein and serum free light chain (FLC) measurements and found that 258 of 520 and 54 of 520 patients who presented with a whole paraprotein relapsed with paraprotein only (PO) and FLC escape, respectively. The median overall survival of PO patients was longer, when compared with patients whose relapse manifested as an increase in FLC both alone and with a whole paraprotein, as a result of a significantly shorter survival from relapse of the latter groups. These observations fit a model in which 1 clone is able to produce a complete antibody, whereas the other secretes only FLC; the type of relapse represents the outgrowth of different clones, some of which are more resistant to therapy. To our knowledge, this is the largest series describing patients who have relapsed with FLC escape and highlights the importance of monitoring FLC when there is a suspicion of clinical relapse. This study was registered at www.isrctn.org as ISRCTN68454111.

Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine

Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1 mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5 mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated.

Exercise intensity does not influence the efficacy of eccentric exercise as a behavioural adjuvant to vaccination.

Acute exercise prior to vaccination can improve the antibody response to influenza vaccination. However, both the optimal exercise protocol and the mechanisms underpinning this adjuvant effect remain unclear. The aim of the current study was to determine whether exercise intensity influenced the efficacy of the intervention. One hundred and sixty healthy young adults were randomly assigned to a resting control group or one of three intervention groups, who exercised at an intensity of 60%, 85%, or 110% of their pre-determined concentric one repetition maxima. The exercise groups performed 50 repetitions of the eccentric portion of both bicep curl and lateral raise movements. All participants then immediately received a reduced dose (50% recommended dose) influenza vaccine. Antibody titres to the three viral strains contained in the vaccine were measured at baseline and at 28 days post-vaccination. Compared to the control group, exercise enhanced the antibody response to the least immunogenic of the three strains (B/Florida). In addition, the exercise groups showed an augmented response to the A/Uruguay strain compared to control; however, this effect was observed only in men. The intervention had no effect on the antibody responses to the most immunogenic strain, A/Brisbane. Finally, antibody responses were unrelated to the intensity of the exercise bout. In conclusion, our findings provide further evidence of exercise as an adjuvant to enhance vaccination responses. The results further show that responses to the low-immunogenic antigens are particularly responsive to augmentation by acute eccentric exercise.

The effects of vaccine timing on the efficacy of an acute eccentric exercise intervention on the immune response to an influenza vaccine in young adults

An acute bout of exercise prior to vaccination can improve the antibody and cell-mediated responses to influenza vaccination. The mechanisms underpinning this adjuvant effect remain unclear, and further investigation to determine the optimal exercise protocol is warranted. The aim of the current study was to determine whether exercise augmented the immune response to vaccination, and whether the timing of exercise relative to vaccination affected the efficacy of the intervention. One hundred and fifty-six (76 men) healthy participants were randomly assigned to a control group or one of three intervention groups who exercised immediately, 6h or 48 h prior to administration of a standard trivalent influenza vaccine. The exercise groups performed 50 repetitions of the eccentric portion of both the bicep curl and lateral raise movements at an intensity eliciting 85% of each participants pre-determined concentric one repetition maxima. Antigen-specific serum antibody titres were measured at baseline and 28 days post-vaccination as indicators of the humoral response. All three viral strains elicited strong antibody responses; however, eccentric exercise did not further augment any antibody responses compared to the control group. Cell-mediated immunity at 28 days post-vaccination was determined by measuring the IFN-gamma response to in vitro stimulation of the blood with whole vaccine. There were no differences in cell-mediated immunity among the groups. Although these null findings were unexpected, they are consistent with previous research showing that exercise-induced immunoenhancement was only observed when the control group had relatively poor responses. In conclusion, it is likely that the robust immune responses to the vaccine observed in this study may have limited any further immune enhancement by exercise.

Total lymphocyte CD8 expression is not a reliable marker of cytotoxic T-cell populations in human peripheral blood following an acute bout of high-intensity exercise

Cytotoxic T-lymphocytes co-express the T-cell receptor, CD3 and the MHC I restricted antigen CD8. Although total CD8 expression is often used to identify CD8(+) T-cells in blood, errors are associated with this method as some CD3 negative natural killer (NK)-cells are known to express CD8. As greater relative proportions of NK-cells are found in the blood compartment after exercise, these errors are likely to be amplified in post exercise blood samples. To test this, isolated blood lymphocytes obtained from aerobically trained male subjects before, immediately after and 1h after an exhaustive treadmill-running protocol were surface stained for CD3, CD4, CD8, CD16, and CD56 and analysed by multi-colour flow cytometry. It was found that 25.4+/-16.9% of all CD8(+) cells at rest were CD3 negative, CD8(dim+) and expressed the NK-cell markers CD16 and CD56. The magnitude of this error increased to 40.8+/-20.7% immediately after exercise due to an influx of CD8(dim+) NK-cells. Although all CD8(bright+) cells expressed CD3, gating around the CD8(bright+) cells only identified 79.2+/-8.7% of the total CD3(+)/CD8(+) T-cell population; however, the magnitude of this error did not change after exercise despite the altered proportions of CD8(bright+) and CD8(dim+) cells. In conclusion, total lymphocyte expression of CD8 should not be used as a single antigenic marker to identify CD8(+) T-cells after an acute bout of exercise. Although there are errors associated with using CD8(bright+) as a single antigenic marker to identify CD3(+) T-cells, these are not amplified in response to exercise.

Vaccination response following aerobic exercise: Can a brisk walk enhance antibody response to pneumococcal and influenza vaccinations?

High intensity acute exercise at the time of vaccination has been shown to enhance the subsequent antibody response. This study examines whether an acute moderate intensity aerobic intervention prior to vaccination can enhance antibody response to pneumonia and half dose influenza vaccination. Sixty young (age (SD)=22.0 (6.1) years) and 60 older (age (SD)=57.5 (6.5) years) adults attended the laboratory on two separate occasions. At the first session, baseline antibody titres were determined, before participants completed either a brisk walk around campus at >55% of their age-predicted heart rate maximum, or a resting control condition, for 45 min. After the intervention, all participants received a full-dose pneumococcal vaccination and a half-dose influenza vaccination. Four weeks later, participants returned for a follow up blood sample. Multivariate ANOVA revealed an increase in total antibody titres against the influenza vaccine (F((12,106))=25.76, p<.001, η(2)=.75) and both the IgM (F((12,106))=17.10, p<.001, η(2)=.66) and IgG (F((12,106))=25.76, p<.001, η(2)=.75) antibody titres against the pneumococcal vaccine. However, there were no significant Time×Group interactions (ps all >.15), indicating that a 45 min brisk walk prior to vaccination did not affect antibody response to either the influenza or pneumonia vaccine. The results suggest that higher intensity exercise is necessary to augment antibody response to vaccination.

Acute exercise enhancement of pneumococcal vaccination response: a randomised controlled trial of weaker and stronger immune response

Acute exercise at the time of vaccination can enhance subsequent immune responses. However, the potential benefit of this effect will be its efficacy in boosting poor responses, and thus protection in at-risk populations. The current study tested the effect of exercise on the response to either a full- or half-dose Pneumococcal (Pn) vaccination to elicit stronger and weaker responses. Subjects were 133 young healthy adults, randomised to one of four groups: exercise or control task, receiving a full- or half-dose Pn vaccination. Prior to vaccination, exercise groups completed a 15 min arm and shoulder exercise task, control groups rested quietly. Antibody levels to 11 Pn strains were evaluated at baseline and 1-month. Across all participants, exercise groups showed significantly greater increase in antibody levels than control groups. When doses were compared, it emerged that those who exercised had significantly larger responses than those who rested in the half-dose group, but in the full-dose groups responses were similar. This data indicates the effectiveness of exercise as a vaccine adjuvant, particularly in weaker responses. Thus, given the potential public health benefits of no-cost behavioural intervention to enhance response to vaccination, testing in at-risk populations should be pursued.

The antioxidant enzyme peroxiredoxin-2 is depleted in lymphocytes seven days after ultra-endurance exercise

Abstract Purpose. Peroxiredoxin-2 (PRDX-2) is an antioxidant and chaperone-like protein critical for cell function. This study examined whether the levels of lymphocyte PRDX-2 are altered over 1 month following ultra-endurance exercise. Methods. Nine middle-aged men undertook a single-stage, multi-day 233 km (145 mile) ultra-endurance running race. Blood was collected immediately before (Pre), upon completion/retirement (Post), and following the race at Day 1, Day 7 and Day 28. Lymphocyte lysates were examined for PRDX-2 by reducing and non-reducing SDS-PAGE with western blotting. In a sub-group of men who completed the race (n = 4), PRDX-2 oligomeric state (indicative of redox status) was investigated. Results. Ultra-endurance exercise caused significant changes in lymphocyte PRDX-2 (F(4,32) 3.409, p = 0.020, η2 = 0.299): 7 days after the race, PRDX-2 levels in lymphocytes had fallen to 30% of pre-race values (p = 0.013) and returned to near-normal levels at Day 28. Non-reducing gels demonstrated that dimeric PRDX-2 (intracellular reduced PRDX-2 monomers) was increased in three of four race completers immediately post-race, indicative of an ‘antioxidant response’. Moreover, monomeric PRDX-2 was also increased immediately post-race in two of four race-completing subjects, indicative of oxidative damage, which was not detectable by Day 7. Conclusions. Lymphocyte PRDX-2 was decreased below normal levels 7 days after ultra-endurance exercise. Excessive accumulation of reactive oxygen species induced by ultra-endurance exercise may underlie depletion of lymphocyte PRDX-2 by triggering its turnover after oxidation. Low levels of lymphocyte PRDX-2 could influence cell function and might, in part, explain reports of dysregulated immunity following ultra-endurance exercise.