The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

Resistance to arsenic and/or all-trans retinoic acid (ATRA) is a challenging problem in the clinical management of acute promyelocytic leukemia (APL). Acquired genetic mutations in the PML moiety of the PML-RARA fusion gene are found in some patients with relapsed/refractory APL. Whether all of the identified point mutations play a role and have a similar function in the mechanisms of arsenic resistance remains unknown. Here we performed in vitro functional analyses and a retrospective analysis of APL patients to investigate the effect of PML-RARA mutations in mediating resistance to arsenic trioxide. Among the 5-point mutations in the PML part of PML-RARA identified in patients with relapsed APL, we found that A216V, S214L, and A216T mutations could attenuate the negative regulation of arsenic on PML-RARA, resulting in the retention of oncoproteins. In contrast, L217F and S220G mutations functioned weakly in this context. Furthermore, we demonstrated that either increasing the concentration of arsenic trioxide or combining it with ATRA could overcome the mutation-triggered arsenic resistance in vitro. In addition to presenting more evidence to reinforce the correlation of genetic mutations in PML-RARA with arsenic efficacy, we provide novel insight into the functional difference of acquired mutations of PML-RARA both in vitro and in the clinical setting. Our findings may help predict the prognosis and select more effective strategies during APL therapy.

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML.

Kaposi sarcoma-associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL) with a poor prognosis in immunocompromised patients. However, it still lacks effective treatment which urgently requires the identification of novel therapeutic targets for PEL. Here, we report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated by KSHV in vitro and in vivo. The selective c-MET inhibitor, PF-2341066, can induce PEL apoptosis through cell cycle arrest and DNA damage, and suppress tumor progression in a xenograft murine model. By using microarray analysis, we identify many novel genes that are potentially controlled by HGF/c-MET within PEL cells. One of the downstream candidates, ribonucleoside-diphosphate reductase subunit M2 (RRM2), also displays the promising therapeutic value for PEL treatment. Our findings provide the framework for development of HGF/c-MET-focused therapy and implementation of clinical trials for PEL patients.

Chronic graft-versus-host disease (GVHD) is a pleotropic syndrome that lacks validated methods of measuring response in clinical trials, although several end points have been proposed. To investigate the prognostic significance of these proposed end points, such as the 2005 National Institutes of Health (NIH) response measures, 2014 NIH response measures, clinician-reported response, and patient-reported response, we tested their ability to predict subsequent overall survival (OS), nonrelapse mortality (NRM), and failure-free survival (FFS). Patients (n = 575) were enrolled on a prospective chronic GVHD observational trial. At 6 months, clinician-reported response (P = .004) and 2014 NIH-calculated response (P = .001) correlated with subsequent FFS, and clinician-reported response predicted OS (P = .007). Multivariate models were used to identify changes in organ involvement, laboratory values, and patient-reported outcomes that were associated with long-term outcomes. At 6 months, a change in the 2005 NIH 0 to 3 clinician-reported skin score and 0 to 10 patient-reported itching score predicted subsequent FFS. Change in the Lee skin symptom score and Functional Assessment of Cancer Therapy-Bone Marrow Transplant score predicted subsequent OS. Change in the Lee skin symptom score predicted subsequent NRM. This study provides evidence that clinician-reported response and patient-reported outcomes are predictive of long-term survival. The trial was registered at www.clinicaltrials.gov as #NCT00637689.

There are >24 million registered adult donors, and the numbers of unrelated donor transplantations are increasing. The optimal strategy for prioritizing among comparably HLA-matched potential donors has not been established. Therefore, the objective of the current analyses was to study the association between donor characteristics (age, sex, parity, cytomegalovirus serostatus, HLA match, and blood group ABO match) and survival after transplantation for hematologic malignancy. The association of donor characteristics with transplantation outcomes was examined using either logistic or Cox regression models, adjusting for patient disease and transplantation characteristics associated with outcomes in 2 independent datasets: 1988 to 2006 (N = 6349; training cohort) and 2007 to 2011 (N = 4690; validation cohort). All donor-recipient pairs had allele-level HLA typing at HLA-A, -B, -C, and -DRB1, which is the current standard for selecting donors. Adjusting for patient disease and transplantation characteristics, survival was better after transplantation of grafts from young donors (aged 18-32 years) who were HLA matched to recipients (P < .001). These findings were validated for transplantations that occurred between 2007 and 2011. For every 10-year increment in donor age, there is a 5.5% increase in the hazard ratio for overall mortality. Increasing HLA disparity was also associated with worsening survival. Donor age and donor-recipient HLA match are important when selecting adult unrelated donors. Other donor characteristics such as sex, parity, and cytomegalovirus serostatus were not associated with survival. The effect of ABO matching on survival is modest and must be studied further before definitive recommendations can be offered.

Cytokine storm syndromes, such as familial hemophagocytic lymphohistiocytosis (FHL), are lethal disorders caused by uncontrolled, systemic immune activation. In the murine model of FHL, in which perforin-deficient (Prf1(-/-)) mice are infected with lymphocytic choriomeningitis virus (LCMV), disease is driven by overabundant interferon (IFN)γ-producing LCMV-specific CD8(+) T cells thought to arise from excessive antigen stimulation through the T-cell receptor. However, this paradigm is insufficient to explain several fundamental aspects of FHL, namely, the inability of many pathogenic antigens to induce hyperinflammation, and the previously identified role of MyD88 in the disease. We now show a novel role for the MyD88-dependent interleukin-33 (IL-33) receptor, ST2, in FHL. Expression of IL-33 and ST2 is upregulated in LCMV-infected Prf1(-/-) mice. Blockade of ST2 markedly improves survival of LCMV-infected Prf1(-/-) mice and reduces the severity of multiple disease parameters, including serum levels of IFNγ. This decrease in IFNγ corresponds to a reduction in both the frequency of IFNγ(+) LCMV-specific CD8(+) and CD4(+) T cells and the magnitude of IFNγ expression in these cells. These findings demonstrate that disruption of ST2 signaling in the murine model of FHL reduces T cell-mediated production of IFNγ and suggest a revised paradigm in which danger signals such as IL-33 are crucial amplifiers of immune dysregulation in FHL. Furthermore, this study provides evidence to support blockade of ST2 as a novel therapeutic strategy for FHL.

Immunoassays are essential in the workup of patients with suspected heparin-induced thrombocytopenia. However, the diagnostic accuracy is uncertain with regard to different classes of assays, antibody specificities, thresholds, test variations, and manufacturers. We aimed to assess diagnostic accuracy measures of available immunoassays and to explore sources of heterogeneity. We performed comprehensive literature searches and applied strict inclusion criteria. Finally, 49 publications comprising 128 test evaluations in 15 199 patients were included in the analysis. Methodological quality according to the revised tool for quality assessment of diagnostic accuracy studies was moderate. Diagnostic accuracy measures were calculated with the unified model (comprising a bivariate random-effects model and a hierarchical summary receiver operating characteristics model). Important differences were observed between classes of immunoassays, type of antibody specificity, thresholds, application of confirmation step, and manufacturers. Combination of high sensitivity (>95%) and high specificity (>90%) was found in 5 tests only: polyspecific enzyme-linked immunosorbent assay (ELISA) with intermediate threshold (Genetic Testing Institute, Asserachrom), particle gel immunoassay, lateral flow immunoassay, polyspecific chemiluminescent immunoassay (CLIA) with a high threshold, and immunoglobulin G (IgG)-specific CLIA with low threshold. Borderline results (sensitivity, 99.6%; specificity, 89.9%) were observed for IgG-specific Genetic Testing Institute-ELISA with low threshold. Diagnostic accuracy appears to be inadequate in tests with high thresholds (ELISA; IgG-specific CLIA), combination of IgG specificity and intermediate thresholds (ELISA, CLIA), high-dose heparin confirmation step (ELISA), and particle immunofiltration assay. When making treatment decisions, clinicians should be a aware of diagnostic characteristics of the tests used and it is recommended they estimate posttest probabilities according to likelihood ratios as well as pretest probabilities using clinical scoring tools.

Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)γRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.

Mean platelet volume (MPV), a measure of platelet size, is a potential biological marker of platelet function. To date, a comprehensive analysis including known genetic and nongenetic factors that determine MPV is still lacking. MPV has been evaluated in 15 010 individuals from the population-based Gutenberg Health Study. Genetic information was available for 4175 individuals. Our results showed that age (β, 0.0346; 95% confidence interval [CI], 0.0255 to 0.0436), cardiovascular risk factors (CVRFs) such as smoking (β, 0.178; 95% CI, 0.128 to 0.229), hypertension (β, 0.05; 95% CI, 0.00289 to .0981), and high glucose level (β, 0.00179; 95% CI, 0.0006 to 0.00299) were linked with higher MPV in males only. Intake of oral contraceptives (β, 0.150; 95% CI, 0.0649 to 0.236) and menstruation (β, 0.123; 95% CI, 0.0231 to 0.224) were strongly associated with higher MPV in females. Seven single nucleotide polymorphisms (SNPs) for females and 4 SNPs for males were associated with higher MPV. The full model, including age, CVRFs, laboratory parameters, medications, and genetic variation, explained 20.4% of the MPV variance in females and 18.6% in males. The curves of cumulative mortality, stratified for sex, showed worse survival for males only with MPV > 9.96 fL vs MPV ≤ 9.96 fL (P < .0001). This study provides evidence for heterogeneity in the profile of determinants for MPV between sexes. The observed interactions between genetic variability, CVRFs, and MPV and its association with the development of cardiovascular disease or thrombotic risk need to be further investigated.

The prognosis of multiple myeloma is mainly dependent upon chromosomal changes. The 2 major abnormalities driving poor outcome are del(17p) and t(4;14). However, the outcome of these high-risk patients is not absolutely uniform, with some patients presenting long survival. We hypothesized that these better outcomes might be related to concomitant "good-risk" chromosomal changes exploring hyperdiploidy. We analyzed a large series of 965 myeloma patients, including 168 patients with t(4;14) and 126 patients with del(17p), using high-throughput single-nucleotide polymorphism arrays after plasma cell sorting. As expected, trisomic chromosomes were highly associated. Using the LASSO model, we found that only chromosome 3, when trisomic, was associated with a longer progression-free survival and that 3 trisomies modulated overall survival (OS) in myeloma patients: trisomies 3 and 5 significantly improved OS, whereas trisomy 21 worsened OS. In patients with t(4;14), trisomies 3 and/or 5 seemed to overcome the poor prognosis. For the first time, using a specific modeling approach, we show that not all trisomies display the same prognostic impact. This finding could be important for routine assessment of prognosis in myeloma, and some high-risk patients with a traditional evaluation could in fact be standard-risk patients.

Fc receptor for IgG IIA (FcγRIIA)-mediated platelet activation is essential in heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. There is considerable interindividual variation in platelet FcγRIIA activation, the reasons for which remain unclear. We hypothesized that genetic variations between FcγRIIA hyper- and hyporesponders regulate FcγRIIA-mediated platelet reactivity and influence HIT susceptibility. Using unbiased genome-wide expression profiling, we observed that human hyporesponders to FcγRIIA activation showed higher platelet T-cell ubiquitin ligand-2 (TULA-2) mRNA expression than hyperresponders. Silent interfering RNA-mediated knockdown of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase following FcγRIIA activation in HEL cells. Significantly, we found miR-148a-3p targeted and inhibited both human and mouse TULA-2 mRNA. Inhibition of miR-148a in FcγRIIA transgenic mice upregulated the TULA-2 level and reduced FcγRIIA- and glycoprotein VI-mediated platelet αIIbβ3 activation and calcium mobilization. Anti-miR-148a also reduced thrombus formation following intravascular platelet activation via FcγRIIA. These results show that TULA-2 is a target of miR-148a-3p, and TULA-2 serves as a negative regulator of FcγRIIA-mediated platelet activation. This is also the first study to show the effects of in vivo miRNA inhibition on platelet reactivity. Our work suggests that modulating miR-148a expression is a potential therapeutic approach for thrombosis.

In this issue of Blood, Price et al report the results of a phase 3 prospective randomized trial comparing the clinical outcome of infected, severely neutropenic patients after receiving or not receiving granulocyte transfusions.

In this issue of Blood, Zhu et al and Bougie et al provide the first evidence for a drug to alter the conformation of a drug-dependent antiplatelet antibody, thereby adding a new dimension to the mechanism of drug-induced immune thrombocytopenia (DITP).,