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N. Luebbering et al.
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Figure 2. The formation and clearance of actin-vitamin D binding protein (VDBP) complexes after hematopoietic stem cell transplant (HSCT) and the impact of VDBP genotype on modulation of VDBP levels after HSCT. (A) Western blot probed for VDBP showing serum collected pre-transplant, and at days 0, 7, and 14 after transplant from two HSCT recipients. A lower molecular weight band corresponding to VDBP (unbound to filamentous actin) is seen in all samples including pre-trans- plant serum (baseline [BL]). However, in post-transplantation serum a larger slower migrating band was also observed corresponding to VDBP bound to actin collected on days 0, 7 and 14, demonstrating the presence of circulating actin-VDBP complex in samples from one patient, unique patient number (UPN) 198. Similar increase in levels of circulating actin-VDBP complex is not seen in patient UPN 436, illustrating inter-individual variability. BL: sample collected before the start of transplant. (B) Western blot probed for VDBP showing clearance of increased actin-VDBP complexes from the circulation, with complexes largely cleared by days 21-28 after HSCT in two different transplant recipients (UPN 324 and UPN 336). (C) VDBP levels according to genotype, measured at BL, days 0, 7, 14, 30 and 100 after HSCT, showing decreased levels at day 14 in all genotypes.
Statistical analysis
Categorical and continuous demographics are described by fre- quency (percent) and median (range), respectively. Time to event data, non-relapse mortality (NRM), GvHD and TA-TMA are described at 1 year and with cumulative incidence curves using the Kaplan-Meier method incorporating death as a competing risk. In both the day 30 and 100 analysis patients with an event preceding the date of measurement were excluded. This was done to ensure that the measured VDBP preceded the respective NRM, GvHD or TA-TMA event. Gray’s method for competing risks was used to test for differences in time to event between groups. Glycan data are summarized by mean and levels were compared between day 0 and day 100 using a paired t-test.
Results
Circulating filamentous actin is associated with clinical outcomes of hematopoietic stem cell transplant
Our overall goal was to understand the mechanism of endothelial injury after HSCT. We hypothesized that F-actin is released into the circulation during lysis of hematopoietic cells caused by chemotherapy and radia-
tion given prior to HSCT. F-actin is known to be toxic to endothelial cells and is generally not present in the circu- lation. First we looked for detectable F-actin in the circula- tion at three timepoints after HSCT (baseline, days 0, 7, 14) in an unselected cohort of 96 consecutive transplant recipients who consented to participate in our institution- al HSCT sample repository and scored any finding of F- actin as positive. Cases with no F-actin measured at any of the three timepoints were scored as negative. Fifty of 96 (52%) cases analyzed had detectable F-actin. We then examined the impact of F-actin presence in the first 14 days of transplant on outcomes of transplant. We found higher risk of TA-TMA in those with F-actin in the circu- lation (61% vs. 37% at 1 year, P=0.03, Figure 1A). Adjustment of the data for age and HLA-match yielded a P-value of 0.06. Moreover, NRM was increased in those with F-actin in the circulation (29% vs. 13%, P=0.028, Figure 1B). Adjustment of the NRM data for age and human leukocyte antigen (HLA)-match yielded a P-value of 0.07. In an analysis of factors that might modify likeli- hood of increased F-actin we found that 41% of recipients of myeloablative preparative regimen had detectable F-actin compared with 64% of recipients of reduced inten- sity conditioning (P=0.04).
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haematologica | 2021; 106(5)