Leukocyte infiltration into inflamed tissues is considered to involve sequential steps of rolling over the endothelium, adhesion, and transmigration. In this model, the leukocyte adhesion molecule L-selectin and its ligands expressed on inflamed endothelial cells are involved in leukocyte rolling. We show that upon experimental and human renal ischemia/reperfusion, associated with severe endothelial damage, microvascular basement membrane (BM) heparan sulfate proteoglycans (HSPGs) are modified to bind L-selectin and monocyte chemoattractant protein-1. In an in vitro rolling and adhesion assay, L-selectin-binding HSPGs in artificial BM induced monocytic cell adhesion under reduced flow. We examined the in vivo relevance of BM HSPGs in renal ischemia/reperfusion using mice mutated for BM HSPGs perlecan (Hspg2(Delta3/Delta3)), collagen type XVIII (Col18a1(-/-)), or both (cross-bred Hspg2(Delta3/Delta3)xCol18a1(-/-)) and found that early monocyte/macrophage influx was impaired in Hspg2(Delta3/Delta3)xCol18a1(-/-) mice. Finally, we confirmed our observations in human renal allograft biopsies, showing that loss of endothelial expression of the extracellular endosulfatase HSulf-1 may be a likely mechanism underlying the induction of L-selectin- and monocyte chemoattractant protein-1-binding HSPGs associated with peritubular capillaries in human renal allograft rejection. Our results provide evidence for the concept that not only endothelial but also (microvascular) BM HSPGs can influence inflammatory responses.

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myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosisregulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p ‫؍‬ 0.0067 and p ‫؍‬ 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p ‫؍‬ 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Despite good remission rates observed in acute myeloid leukemia (AML) patients, the 5-year event-free survival rates reach only 35-40% in adults and 60 -70% in children (1, 2). Apoptosis is one of the crucial mechanisms influencing survival of AML cells, and its deregulation can possibly lead to chemotherapy resistance and eventually relapse (3-5). The ability of cells to undergo apoptosis is largely defined by the relative expression of anti-(i.e. BCL-2, BCL-X long isoform -BCL-XL, or MCL-1) and proapoptotic (i.e. BAX, BH3 interacting domain death agonist -BID, caspases) proteins. Several studies have shown that the levels of BCL-2 and BCL-2/BAX ratio are a determinant of apoptosis-resistance in AML blasts and are associated with survival in AML patients (3,6). We have previously demonstrated that the expression of several apoptosis-related proteins, such as BCL-2, BCL-XL, MCL-1, From the ‡Dept.

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.

Summary:Stem cell doses necessary for engraftment after myeloablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34 + cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze-thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen-thawed grafts, threshold numbers for adequate haematological recovery of 2.8-3.0 ؋ 10 6 CD34 + cells/kg body weight determined for fresh grafts, now decreased to 1.2-1.3 ؋ 10 6 CD34 + cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34 + cells were sufficient for recovery (0.3-0.4 ؋ 10 6 CD34 + cells/kg). In contrast to freeze-thawing of leucapheresis material, a high viability of CD34 + cells was preserved during storage for 3 days at 4؇C, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen-thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34 + threshold doses for haematological recovery.