The ubiquitin ligase Triad1 inhibits myelopoiesis through UbcH7 and Ubc13 interacting domains.

Ubiquitination plays a major role in many aspects of hematopoiesis. Alterations in ubiquitination have been implicated in hematological cancer. The ubiquitin ligase Triad1 controls the proliferation of myeloid cells. Here, we show that two RING (really interesting new gene) domains in Triad1 differentially bind ubiquitin-conjugating enzymes, UbcH7 and Ubc13. UbcH7 and Ubc13 are known to catalyze the formation of different poly-ubiquitin chains. These chains mark proteins for proteasomal degradation or serve crucial non-proteolytic functions, respectively. In line with the dual Ubc interactions, we observed that Triad1 catalyzes the formation of both types of ubiquitin chains. The biological relevance of this finding was studied by testing Triad1 mutants in myeloid clonogenic assays. Full-length Triad1 and three mutants lacking conserved domains inhibited myeloid colony formation by over 50%. Strikingly, deletion of either RING finger completely abrogated the inhibitory effect of Triad1 in clonogenic growth. We conclude that Triad1 exhibits dual ubiquitin ligase activity and that both of its RING domains are crucial to inhibit myeloid cell proliferation. The differential interaction of the RINGs with Ubcs strongly suggests that the ubiquitination mediated through UbcH7 as well as Ubc13 plays a major role in myelopoiesis.

Feasibility of a new in vitro approach to evaluate cellular damage following co-infusion of red blood cell concentrates and intravenous drug solutions.

INTRODUCTION: Transfusion guidelines may result in unwanted delay in infusion schemes, as simultaneous infusion of blood components and drug solutions is universally prohibited. The aim of this study was to measure possible damage to red cells by drug solutions, as manifested by haemolysis, using a dynamic model that resembles the clinical setting. METHODS: Stored filtered and irradiated RBC concentrates and drug solutions were co-infused in an in vitro dynamic model. Also, incubation in a static model was performed. The haemolytic potency of the drug solutions was measured by determining free haemoglobin (fHb) levels. RESULTS AND DISCUSSION: Neither in the dynamic tests nor in the static tests did fHb levels exceed the maximally acceptable standard for filtered RBC concentrates according to Dutch specification guidelines. In the static test model, fHb levels were slightly elevated compared with those of control samples, as well as those in the dynamic test model. CONCLUSION: A novel in vitro dynamic infusion system appears to represent a useful technique to calculate possible damage to RBCs resulting from co-infused drug solutions. Co-infusion of the drug solutions tested with filtered and irradiated RBC concentrates did not produce fHb levels above the levels accepted by the Dutch national guidelines. Apart from haemolysis, other parameters reflecting RBC damage should be investigated in future studies.

Interferon-gamma-based mixed lymphocyte culture as a selection tool for allogeneic bone marrow donors other than identical siblings.

Selection procedures in bone marrow transplantation (BMT) would benefit from the development of easy-to-perform cellular assays with high discriminative power. We tested a cytokine-based mixed lymphocyte culture (MLC) and compared its outcome to the routinely used MLC, helper T-lymphocyte precursor (HTLp)-f and cytotoxic T-lymphocyte precursor (CTLp)-f assays. Interferon (IFN)gamma was selected as a marker cytokine for (deleterious) T-helper 1 like responses and 36 (potential) BMT donor-recipient pairs were analysed. The IFNgamma-MLC appeared sensitive to HLA class II (subtype) differences, but not to isolated class I differences, or to mismatches other than HLA (identical siblings). The test enabled a distinction between combinations with positive MLC (proliferation) and HTLp-f, exemplified by the fact that although high IFNgamma levels were observed in the class II mismatched group, certain DRB3, DQB1-subtype and DRB1-subtype mismatches did not give rise to IFNgamma production. This might be of relevance for the detection of so-called permissible mismatches. With regard to prediction of acute graft-versus-host disease (aGVHD) in unrelated BMT, the data indicated that high levels of IFNgamma coincided with severe aGVHD, whereas low levels were largely associated with grades 0-I. However, in the case of isolated class I mismatches the test had no predictive value. The cell-saving IFNgamma-MLC provides an alternative for the assays currently in use, but should be employed along with an assay that is sensitive to class I differences to correct for false negatives. Consequently, a combination of IFNgamma-MLC and CTLp-f assays seems most promising for donor selection, other than identical siblings.