A randomized controlled trial evaluating recovery and survival of 6% dimethyl sulfoxide-frozen autologous platelets in healthy volunteers.

BACKGROUND: Availability of platelets (PLTs) is severely limited by shelf life in some settings. Our objective was to determine and compare to Food and Drug Administration (FDA) criteria the PLT recovery and survival of autologous PLTs cryopreserved at -65°C or less in 6% dimethyl sulfoxide (DMSO) reconstituted with a no-wash method (cryopreserved PLTs [CPPs]) compared to autologous fresh PLTs. STUDY DESIGN AND METHODS: This was a randomized, Phase I study analyzing PLT viability and in vitro function in consenting healthy subjects. Apheresis PLTs (APs) were collected in plasma. APs were suspended in 6% DMSO, concentrated, and placed at not more than -65°C for 7 to 13 days. Frozen CPPs were thawed at 37°C and resuspended into 25 mL of 0.9% NaCl. Control PLTs (fresh autologous) and CPPs were labeled with (111) In or (51) Cr, and recovery and survival after reinfusion were determined using standard methods. A panel of in vitro assays was completed on APs and CPPs. RESULTS: After frozen storage, CPPs retained 82% of AP yield and showed increased PLT associated P-selectin and reduced responses to agonists. CPP 24-hour recovery (41.6 ± 9.7%) was lower than for fresh PLTs (68.4 ± 8.2%; p < 0.0001) and did not meet the current FDA criterion. CPPs had diminished survival compared to fresh PLTs (7.0 ± 2.1 days vs. 8.4 ± 1.2 days, respectively; p = 0.018), but did meet and exceed the FDA criterion for survival. CONCLUSION: While 24-hour recovery does not meet FDA criteria for liquid-stored PLTs, the CPP survival of circulating PLTs was surprisingly high and exceeded the FDA criteria. These data support proceeding with additional studies to evaluate the clinical effectiveness of CPPs.

Vav3 collaborates with p190-BCR-ABL in lymphoid progenitor leukemogenesis, proliferation, and survival.

Despite the introduction of tyrosine kinase inhibitor therapy, the prognosis for p190-BCR-ABL(+) acute lymphoblastic leukemia remains poor. In the present study, we present the cellular and molecular roles of the Rho GTPase guanine nucleotide exchange factor Vav in lymphoid leukemogenesis and explore the roles of Vav proteins in BCR-ABL-dependent signaling. We show that genetic deficiency of the guanine nucleotide exchange factor Vav3 delays leukemogenesis by p190-BCR-ABL and phenocopies the effect of Rac2 deficiency, a downstream effector of Vav3. Compensatory up-regulation of expression and activation of Vav3 in Vav1/Vav2-deficient B-cell progenitors increases the transformation ability of p190-BCR-ABL. Vav3 deficiency induces apoptosis of murine and human leukemic lymphoid progenitors, decreases the activation of Rho GTPase family members and p21-activated kinase, and is associated with increased Bad phosphorylation and up-regulation of Bax, Bak, and Bik. Finally, Vav3 activation only partly depends on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to inhibit CrkL activation and impair leukemogenesis in vitro and in vivo. We conclude that Vav3 represents a novel specific molecular leukemic effector for multitarget therapy in p190-BCR-ABL-expressing acute lymphoblastic leukemia.

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells.

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21(cip1.) and p16(INK4a), and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors.

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Bmi1 reprograms CML B-lymphoid progenitors to become B-ALL-initiating cells.

The characterization and targeting of Philadelphia chromosome positive (Ph(+)) acute lymphoblastic leukemia (ALL)-initiating cells remains unresolved. Expression of the polycomb protein Bmi1 is up-regulated in patients with advanced stages of chronic myelogenous leukemia (CML). We report that Bmi1 transforms and reprograms CML B-lymphoid progenitors into stem cell leukemia (Scl) promoter-driven, self-renewing, leukemia-initiating cells to result in B-lymphoid leukemia (B-ALL) in vivo. In vitro, highly proliferating and serially replatable myeloid and lymphoid colony-forming cultures could be established from BCR-ABL and Bmi1 coexpressing progenitors. However, unlike in vivo expanded CML B-lymphoid progenitors, hematopoietic stem cells, or multipotent progenitors, coexpressing BCR-ABL and Bmi1 did not initiate or propagate leukemia in a limiting dilution assay. Inducible genetic attenuation of BCR-ABL reversed Bmi1-driven B-ALL development, which was accompanied by induction of apoptosis of leukemic B-lymphoid progenitors and by long-term animal survival, suggesting that BCR-ABL is required to maintain B-ALL and that BCR-ABL and Bmi1 cooperate toward blast transformation in vivo. Our data indicate that BCR-ABL targeting itself is required to eradicate Ph(+)/Bmi1(+) B-ALL-initiating cells and confirm their addiction to BCR-ABL signaling.

Atypical protein kinase C (aPKCzeta and aPKClambda) is dispensable for mammalian hematopoietic stem cell activity and blood formation.

The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6, and atypical protein kinase C (aPKCζ and aPKCλ), which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However, the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that, in contrast to accepted paradigms, polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice, having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ, respectively, have normal hematopoiesis, including normal HSC self-renewal, engraftment, differentiation, and interaction with the bone marrow microenvironment. Furthermore, inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization, self-renewal, engraftment, or lineage repopulation. In addition, aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together, the expression of aPKCζ, aPKCλ, or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis, contrary to the hypothesis of a unique, evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination.

In vivo viability of stored red blood cells derived from riboflavin plus ultraviolet light-treated whole blood.

BACKGROUND: A novel system using ultraviolet (UV) light and riboflavin (Mirasol System, CaridianBCT Biotechnologies) to fragment nucleic acids has been developed to treat whole blood (WB), aiming at the reduction of potential pathogen load and white blood cell inactivation. We evaluated stored red blood cell (RBC) metabolic status and viability, in vitro and in vivo, of riboflavin/UV light-treated WB (IMPROVE study). STUDY DESIGN AND METHODS: The study compared recovery and survival of RBCs obtained from nonleukoreduced WB treated using three different UV light energies (22, 33, or 44 J/mL(RBC)). After treatment, WB from 12 subjects was separated into components and tested at the beginning and end of component storage. After 42 days of storage, an aliquot of RBCs was radiolabeled and autologously reinfused into subjects for analysis of 24-hour recovery and survival of RBCs. RESULTS: Eleven subjects completed the in vivo study. No device-related adverse events were observed. By Day 42 of storage, a significant change in the concentrations of sodium and potassium was observed. Five subjects had a 24-hour RBC recovery of 75% or more with no significant differences among the energy groups. RBC t(1/2) was 24 ± 9 days for the combined three groups. Significant correlations between 24-hour RBC recovery and survival, hemolysis, adenosine triphosphate (ATP), and CO(2) levels were observed. CONCLUSIONS: This study shows that key RBC quality variables, hemolysis, and ATP concentration may be predictive of their 24-hour recovery and t(1/2) survival. These variables will now be used to assess modifications to the system including storage duration, storage temperature, and appropriate energy dose for treatment.