The King is Dead, Long Live the King: Entering A New Era of Stem Cell Research and Clinical Development

With the approval of the first therapeutic cancer vaccines for veterinarian and human use, the field reached a significant milestone after a considerable interval of tumultuous research and development marked by numerous ups and downs. As the mechanism of action and clinical benefit afforded by this class of agents are starkly different from that of conventional or small targeted therapies for cancer, there are still numerous hurdles that need to be overcome to fully unleash their potential. These challenges and efforts are illustrated in a book just published on this subject, a non-exhaustive yet representative synopsis of the latest advances in cancer vaccine technologies in various stages of development. Major lessons resulting from clinical testing of cancer vaccines and other immune interventions, are being integrated in novel, cutting edge platform technologies that blur the distinction between passive and active immunotherapies as well as carry the promise of fundamentally changing and improving the management of patients with cancer.     

Patient-specific transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-associated mortality. The inadvertent transfusion of neutrophil antibodies can cause pulmonary transfusion reactions and TRALI. However, not all patients transfused with neutrophil antibodies experience transfusion reactions. A 22-year-old man with severe aplastic anaemia (SAA) experienced TRALI after a platelet transfusion. The donor was found to be alloimmunized to human neutrophil antigen (HNA)-3a, an antigen expressed by neutrophils from approximately 90% of Caucasians. Eleven other platelet components from this donor were transfused prior to this event and two caused reactions: one chills and one TRALI. Both episodes of TRALI occurred in the same male patient with SAA. The fact that one patient experienced TRALI following both exposures to anti-HNA-3a from the same donor whereas nine other recipients did not adds evidence to the observation that patient factors make a significant contribution to neutrophil antibody-mediated transfusion reactions.     

Neutrophil and platelet antibodies in autoimmune lymphoproliferative syndrome

BACKGROUND AND OBJECTIVES: Autoimmune lymphoproliferative syndrome (ALPS), is an inherited disorder characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly, accumulation of T-cell receptor (TCR)-alphabeta+ CD4- CD8- T cells (double-negative T cells) and autoimmunity. We investigated the incidence and nature of neutrophil and platelet antibodies in patients with ALPS. MATERIALS AND METHODS: Sera from 26 patients with ALPS were tested for neutrophil antibodies by granulocyte immunofluorescence, granulocyte agglutination and monoclonal antibody immobilization assays of granulocyte antigens, and for platelet antibodies using a solid-phase antibody-detection system. RESULTS: Neutrophil antibodies were detected in 46% of patients with ALPS. Antibody specificity could be defined in eight of the 12 patients with neutrophil antibodies. Among these eight patients, four had antibodies directed against more than one antigen. Overall, 14 antibodies directed to specific antigens were identified: three were directed to the HNA-1a antigen of FcgammaRIIIb; two to the HNA-1b antigen of Fcgamma-RIIIb; two to epitopes common to all FcgammaRIIIb molecules; four to the HNA-2a antigen of the NB1 glycoprotein; and three to neutrophil beta2 integrins. Platelet antibodies were detected in 35% of patients with ALPS. No antibody specificities were identified among the platelet antibodies. There was no association between the detection of neutrophil antibodies and a history of clinical neutropenia, or between the detection of platelet antibodies and a history of clinical thromobocytopenia. CONCLUSIONS: Neutrophil and platelet antibodies are important markers of ALPS, but do not always cause clinical cytopenias. The specificities of neutrophil antibody were similar to those found in children with autoimmune neutropenia but without ALPS.     

Delayed donor red cell chimerism and pure red cell aplasia following major ABO-incompatible nonmyeloablative hematopoietic stem cell transplantation

Delayed donor red cell engraftment and pure red cell aplasia (PRCA) are well-recognized complications of major ABO-incompatible hematopoietic stem cell transplantation (SCT) performed by means of myeloablative conditioning. To evaluate these events following reduced-intensity nonmyeloablative SCT (NST), consecutive series of patients with major ABO incompatibility undergoing either NST (fludarabine/cyclophosphamide conditioning) or myeloablative SCT (cyclophosphamide/high-dose total body irradiation) were compared. Donor red blood cell (RBC) chimerism (initial detection of donor RBCs in peripheral blood) was markedly delayed following NST versus myeloablative SCT (median, 114 versus 40 days; P <.0001) and strongly correlated with decreasing host antidonor isohemagglutinin levels. Antidonor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median, 83 versus 44 days; P =.03). Donor RBC chimerism was delayed more than 100 days in 9 of 14 (64%) and PRCA occurred in 4 of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared with cases without, PRCA (30 versus 98 days; P =.008). Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased antidonor isohemagglutinin levels and resolution of PRCA following NST. These data indicate that significantly delayed donor erythropoiesis is (1) common following major ABO-incompatible NST and (2) associated with prolonged persistence of host antidonor isohemagglutinins. The clinical manifestations of these events are affected by the degree and duration of residual host hematopoiesis.     

Characterization of multiple quinine-dependent antibodies in a patient with episodic hemolytic uremic syndrome and immune agranulocytosis.

A 23-year-old woman experienced six distinct episodes of severe combined neutropenia and thrombocytopenia. At least one of the episodes was accompanied by hemodialysis-requiring acute renal failure and fragmentation hemolysis (hemolytic uremic syndrome [HUS]). In retrospect, all episodes were probably associated with the ingestion of quinine. Quinine-dependent antibodies to platelets, neutrophils, T lymphocytes, and red blood cells (RBCs) were detected in the patient's serum. By a monoclonal antibody antigen capture assay, the patient's serum contained IgG antibodies, which in the presence, but not absence, of quinine reacted with platelet glycoprotein (GP) complexes Ib/IX and IIb/IIIa, but not Ia/IIa. By immunoprecipitation assay, the serum, after addition of quinine, reacted strongly with an 85-Kd neutrophil membrane protein and weakly with 130- and 60-Kd moieties. Serum adsorbed with RBCs in the presence of quinine continued to react with platelets and neutrophils, and serum that was absorbed with platelets continued to react with neutrophils and RBCs, indicating that the antigenic targets were different on platelets, neutrophils, and RBCs. Since platelets and endothelial cells share some antigens, we tested patient serum for antibodies to human umbilical vein endothelial cells (HUVEC); no quinine-dependent antibodies to HUVEC were detected. However, her quinine-dependent antibodies not only bound to platelets and neutrophils, but also activated neutrophils. Thus, the patient's serum with quinine aggregated neutrophils, but neither agent alone caused activation. Moreover, the patient's serum with quinine (but not without) augmented the adherence of neutrophils to HUVEC. Treatment of the patient's serum with staphylococcal protein A removed the quinine neutrophil aggregation cofactor, suggesting it was due to IgG. In both neutrophil aggregation and adherence assays, decomplementation of the patient's serum markedly blunted its effect. Furthermore, the patient's serum failed to aggregate formalin-inactivated neutrophils, suggesting neutrophil activation, probably by activated complement, was necessary for aggregation and adhesivity to endothelium. We conclude that our patient's neutropenia, thrombocytopenia, lymphopenia, and anemia were due to quinine-dependent antibodies, and that these antibodies recognized epitopes that were different in the three target cell populations. We further suggest that HUS was likely secondary to the activation and adhesion of neutrophils to endothelium.     

Factors affecting the formation of white particulate matter in red blood cell components

BACKGROUND: Recently white particulate matter (WPM) in red blood cell (RBC) components has received increased attention. The nature and causes of WPM formation were investigated. STUDY DESIGN AND METHODS: Whole-blood units were collected from 18 healthy subjects with three different types of collection sets. Six units were collected into each type. Units were divided into four equal parts and stored for 4 hours: two parts at room temperature and two at 4 degrees C. RBCs were prepared from each quarter-unit: two by heavy centrifugation (5000 x g) and two by light centrifugation (2000 x g). Whole blood was inspected for WPM over 4 hours and RBCs over 1 hour. RESULTS: No WPM was detected in whole blood, but WPM was detected in at least one RBC component from 9 of the 18 donations. The 36 components prepared by heavy centrifugation were more likely to contain WPM than the 36 prepared by light centrifugation (50% vs. 19%; p < 0.02). The incidence of WPM was similar among RBCs stored at room temperature and 4 degrees C. Donors of RBCs with WPM had higher total cholesterol levels than donors of components without WPM (191 +/- 20 mg/dL vs. 163 +/- 32 mg/dL; p < 0.04), but there was no difference in triglyceride levels between the two groups. CONCLUSIONS: WPM is an expected consequence of standard RBC manufacturing methods, but it is more frequent in RBCs prepared by heavy centrifugation and from donors with higher cholesterol levels.     

CD177 polymorphisms: correlation between high-frequency single nucleotide polymorphisms and neutrophil surface protein expression

BACKGROUND: Human neutrophil antigen-2a (NB1) is located on NB1 glycoprotein, which is expressed on subpopulations of neutrophils. PRV-1 is a gene that is over-expressed in neutrophils from patients with polycythemia rubra vera. The gene encoding NB1 differs from PRV-1 at four nucleotides. The purpose of this study was to determine if PRV-1 and NB1 are alleles of the same gene or two separate genes; and, moreover, if they are alleles, to explore potential correlations to NB1 glycoprotein expression. STUDY DESIGN AND METHODS: Primer pairs were used to amplify WBC DNA in the regions surrounding the four NB1 polymorphic sites. The four resulting amplicons were sequenced. The size of the neutrophil population in each donor that stained brightly with CD177 antibody was assessed by flow cytometry. RESULTS: If PRV-1 and NB1 are separate genes, then all people should be heterozygous for the PRV-1/NB1 polymorphisms. Because 11 of 23 donors were homozygous for PRV-1 polymorphisms at all four sites, PRV-1 and NB1 are alleles of the same gene, CD177. When the sequenced exons were compared with PRV-1, 13 single nucleotide polymorphisms (SNPs) that result in amino acid changes were found. The G42C NB1 polymorphism, found in 10 donors, was the most common SNP. The size of the CD177 bright-staining neutrophil population was greater in 42C/C donors than in 42G/G donors (66 +/- 20% vs. 41 +/- 21%, p = 0.004). CONCLUSIONS: PRV-1 and NB1 are alleles of the polymorphic gene CD177. The most common SNP in bp 42 predicted an amino acid change that may have an effect on protein expression.