Self-reported QOL increases following IVIG infusion
Self-reported quality of life was assessed on the day of infusion — the presumed QOL nadir — and at day 7, when the positive effect of IVIG was likely to have onset and unlikely to have diminished (Fig 1). Changes over this interval were modeled with random intercept by subject to control for inter-subject variation. Under this model, the overall VAS score increased (improved) at day 7 (p=0.0003, Table 2, Fig 2a). In the modified SF-12 questionnaire, fatigue (question 5) and general assessment of health for the prior week (question 2) decreased (improved) over this same period (p=0.03, p=0.03, Table 2, Fig 2c & 2d). We then tested for correlation between the VAS and individual items from the modified SF-12. Changes in VAS score were significantly correlated with modified SF-12 question 2 (health for the prior week) on day 7 (Kruskal Wallis, p=0.001, Fig 2b) but did not correlate with question 5 (fatigue) either at day 7 or as the difference in fatigue scores between infusion day and day 7 (p=0.13, p=0.32, Fig S2). These findings support the concept of IVIG “wear-off”. VAS scores were consistently lower on the day of infusion compared to 7 days later. Question 2 of the modified SF-12 confirms this observation, as on day 7, subjects reported their health was worse 7 days prior to questionnaire administration. In addition, fatigue over the preceding week decreased from infusion day to day 7; however, fatigue scores did not correlate with the VAS score or day 7 report of prior weeks. Therefore, the questionnaires captured two independent quality of life measures – general health and fatigue – that changed from infusion day to day 7.
Table 2
Significant Relationships Between QOL Measures on Infusion Day and Day 7.
Measure
|
Coefficients
|
p-values
|
CI_2.5
|
CI_97.5
|
Weekly Health (1-5 scale, 1 is high)
|
-0.5088
|
0.0295
|
-0.9552
|
-0.0624
|
Suffer from fatigue?
|
-0.4840
|
0.0320
|
-0.9154
|
-0.0527
|
VAS
|
4.6440
|
0.0003
|
2.1439
|
7.1441
|
Treg numbers increase following IVIG infusion
Treg numbers were measured at 3 time points during the cycle: before infusion, one hour after infusion completion, and 7 days after the infusion. There were no significant changes in numbers between the pre-infusion and post-infusion blood draw; however, levels increased at the visit 7 days following the infusion (p=0.01, Fig 3 and Fig S3A). We tested whether Treg levels on infusion day, day 7, or change from pre-infusion to day 7 correlated with changes in VAS score or fatigue score over the same time interval. There was no correlation between Treg levels and changes in VAS score or changes in fatigue score (Fig S3B & S3C), suggesting either the absence of a relationship between increases in Treg numbers and the assessed QOL measures, or insufficient numbers of measurements to identify such a relationship.
Changes in serum cytokine and chemokine levels during the infusion cycle
Multiple changes in cytokine and chemokine levels occurred between the pre- and 1h post-infusion blood draws. CCL2, CCL3, CCL4, TNF-a, granzyme B, IL-10, IL-1RA, IFN-g, IL-8, and CCL20 increased while IL-25 decreased (Fig S8). After stratification of naïve vs. IVIG-experienced subjects, (Fig 4) additional significant increases in IL-6 and GM-CSF and significant decreases in EGF and CD40L were seen in IVIG-naïve subjects. In IVIG-experienced subjects, CCL3, CCL4, and granzyme B were the only significant cytokine increases, with increases in CCL2, TNF-a, IL-10, IL-1RA, IFN-g, IL-8, and CCL20 no longer attaining significance. Interestingly, the decrease in IL-25 level identified in the non-stratified subject sample was entirely attributable to the non-naïve group and was not detected in IVIG-naïve subjects (Fig 4). Thus, IVIG infusion in IVIG-naïve subjects was rapidly followed by an increase in a subset of inflammatory cytokines in the blood. In IVIG-experienced subjects, a partially overlapping set of changes was identified with similar but lower magnitude changes in CCL3, CCL4, and granzyme B, and a distinct decrease in IL-25 that was not detected in the naïve subjects.
To determine whether these changes represented large changes in few subjects or whether these changes were widespread, a direction of change was assigned for each interval and the proportions of direction of change were examined (Fig 5). CCL3, CCL4, granzyme B, TNF-a, and IFN-g increased for the majority of pre-to-post infusion intervals, whereas IL-10, CCL2, IL-6, GM-CSF, IL-8, and IL-1RA were increased in only the minority. Among the cytokines found to decrease significantly, CD40 ligand, IL-25, and EGF decreased for the majority of pre-to-post infusion intervals (Fig 5, Fig S4, Fig S5, Fig S6). These findings suggest that increases in the levels of CCL3, CCL4, granzyme B, TNF-a, and IFN-g are typical changes in response to IVIG infusion, as are decreases in CD40 ligand, IL-25, and EGF.
Comparing pre-infusion to day 7, no changes in cytokine levels reached statistical significance of adjusted p-value < 0.05; however, IL-25 trended towards a decrease during the interval (p = 0.051). As IL-25 was decreased immediately after infusion, we further determined that the log-fold change in IL-25 between the pre-infusion and post-infusion time point was not different than the pre-infusion and day 7 time points (paired T-test, p=0.46, Fig S7). Therefore, in the IVIG-experienced group, IL-25 levels decrease immediately following IVIG infusion and frequently remain low at least 7 days post-infusion. IL-25 decreases occurred in 55% of pre-to-post intervals and 58% of pre-to-day-7 intervals and increases occurred in 30% of both intervals. Thus, IVIG infusions are frequently associated with immediate and prolonged decreases in IL-25 in a subset of subjects.
Correlation analysis links clusters of cytokines
To better understand the relationship between cytokines, we calculated correlation coefficients for changes in cytokine/chemokine levels before and immediately after infusion. In IVIG-experienced subjects, CCL2, CCL3, CCL4, TNFa, IL-8, IL-1RA, granzyme B, and IL10 were highly correlated (Fig 6). This suggests that these cytokines were secreted by the same cell or at least were induced in response to the same stimulus. IL-25 was not correlated with this group of cytokines, indicating that decreases in IL-25 occurred independently of increases in the identified inflammatory cytokines and chemokines.
Relationships between cytokine changes and QOL measures
Changes in cytokine levels were tested for relationships with either the change in VAS or fatigue score from infusion day to day 7. IVIG-naïve subjects were not evaluated independently as the number of measurements was inadequate to power such an analysis. No linear relationship was seen between change in VAS or change in fatigue score and any of the cytokines or chemokines that were significantly changed in the study. Since the largest changes in cytokine levels were detected in the small number (3) of IVIG-naïve subjects, this result was not surprising but warrants further analysis in a larger cohort.