Fig 1.
Hpase expression in rejecting murine cardiac allografts versus syngeneic cardiac grafts.
Hpase expression is increased in graft-infiltrating lymphocytes (solid arrows) and endothelial cells (hollow arrows) during rejection of allogeneic grafts compared with syngeneic grafts. A, B) Hpase staining of syngeneic and allogeneic cardiac transplants on POD 7. Hematoxylin counterstained, 40x mag. C, D) HS (Red) present in the graft extracellular matrix is degraded in areas of CD3+ T cell (Green) infiltration during graft rejection compared with control heart. Dapi (Blue) nuclear stain, 40x mag.
Fig 2.
Increased number of Hpase-expressing graft-infiltrating lymphocytes (GILs) in allogeneic versus syngeneic cardiac grafts.
Hpase-expressing GILs were counted per high powered field (200X). Mean ± SEM shown in red; comparisons made by unpaired t-test.
Fig 3.
Serum HS levels are elevated in recipients of allogeneic prior to graft rejection.
Serum HS levels are elevated following allogeneic (N = 3), but not syngeneic (N = 3) cardiac transplants in mice. (*p<0.05).
Fig 4.
Activated T cell expression of Hpase.
Hpase expression is increased in CD4 and CD8 T cells activated by ConA and increases with time. Western blot analysis for Hpase of lysates from purified human CD4+ and CD8+ T cells treated with the pan-T cell activator, ConA, for 0, 48 and 96 hours. Blots were re-probed for GAPDH as a loading control.
Fig 5.
Cell surface expression of Hpase on activated T cells by flow cytometry. Cell surface expression of Hpase by activated T cells increases with time. A) FACS analysis of human CD4+ and CD8+ T cells treated with ConA for 0, 48 and 96 hrs for expression of Hpase. B) Mean fluorescent intensity (MFI) of FACS analyses performed in triplicate. (*P<0.05, Unpaired Student’s t-test).
Fig 6.
Plasma HS levels in kidney transplant recipients with biopsy-proven acute cellular rejection (red) versus those with no rejection on graft biopsy (blue).
Plasma HS levels are elevated in patients with biopsy-proven rejection (red, closed symbols) and not in patients without rejection on biopsy (blue, open symbols). All biopsies were performed for cause (elevated sCr).
Table 1.
Comparison of human plasma HS levels (average and maximum) for the four experimental groups.
Kruskal-Wallis test was used to determine if differences existed among groups.
Table 2.
P-values obtained from pairwise comparisons of plasma HS levels (average and maximum) for four human cohorts using Bonferroni adjustment for multiple testing.
Fig 7.
Correlation between plasma HS and serum creatinine.
(A) Average plasma HS versus serum creatinine, (B) Maximum plasma HS versus serum creatinine.
Table 3.
Comparison of serum Cr (average and maximum) within 14 days from graft biopsy in “Rejection” and “No rejection” cohorts.
Fig 8.
Serum HS is not elevated by BK viral infection (A), and is minimally elevated by CMV infection (B).
Fig 9.
Activated T cells express heparanase (Hpase), facilitating penetration of the glycocalyx and migration into tissues.
Heparan sulfate (HS) is released in the process, becoming detectable in serum assays.