Correction of the PNH defect by GPI protein transfer: still an open question.

Sloand et al1 report that in human red blood cells (RBCs) the paroxysmal nocturnal hemoglobinuria (PNH) defect can be corrected by transfer of glycosylphosphatidylinositol (GPI)-anchored proteins to GPI-deficient cells. It has been demonstrated before by a number of authors that cell-to-cell transfer of GPI-anchored proteins can occur, and I have no doubt that a similar event also takes place between erythrocytes and microvesicles enriched in GPI-anchored proteins. However, in my opinion, it can be questioned whether the experiments performed by Sloand et al1 warrant the conclusions that were drawn. In Figure 1B the authors show an immunoblot using a monoclonal antibody (mAb) against CD55. This antibody reacts with more than a dozen bands (see lanes 1, 2), making it surprising that the authors are able to identify CD55 among many other bands of similar intensities in lane 3 of the same figure. The antibody apparently reacts with such a large number of proteins that the immunoblot shown is by no means a proof for the presence of CD55 in the sample—a result that the authors rely on in a number of subsequent experiments in the paper. Such a broad reactivity of a monoclonal antibody would prompt me to question its specificity or check the methodology for immunoblotting. Similarly, in Figure 1C the authors show a blot with an mAb against CD59 that reacts with 2 bands of similar intensities. Although in this case the identification of CD59 may not give rise to much criticism, one should be aware that the same antibody is used throughout the paper in flow cytometry experiments, and it is not clear with what it may react in these experiments. The situation is even more worrisome in the case of the mAb against CD55, which is also used in their flow cytometric analyses. Even more importantly, the authors claim that in order for a transfer of GPI-anchored proteins to RBCs to occur, the GPI anchors must be intact. This conclusion is based solely on their observation that treatment of the material containing the GPI-anchored proteins with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished transfer. Interestingly, however, it is well known from the literature that the GPI anchors of human RBC proteins are substituted with a fatty acyl chain on the inositol,2-5 which renders the structures insensitive to cleavage by PI-PLC.2,5 A careful study of the literature would have revealed this fact. Thus, their observation that transfer of proteins to RBCs was decreased after PI-PLC treatment cannot be explained by GPI-anchor hydrolysis, and I wonder what might have caused the altered findings. Inhibition of HIV-1 replication by alloantigen-stimulated supernatants derived from PBMC cultures of healthy blood bank donors and HIV-1– infected individuals