ALBERT

Description: Background: Exposure to red blood cell (RBC) alloantigens during pregnancy or transfusion can lead to the development of alloantibodies and result in transfusion-related complications, including hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. However, the factors that regulate RBC alloimmunization remain incompletely understood. Several studies suggest that alterations in factors that regulate RBC clearance may impact RBC uptake and antigen presentation, directly influencing the likelihood of RBC alloimmunization. To test this, we directly examined the potential role of CD47, a master regulator of RBC removal previously shown to be altered during RBC senescence and cold storage. To accomplish this, we crossed transgenic mice that express the model HOD antigen (a fusion protein consisting of hen egg lysozyme fused to ovalbumin and human Duffy b) with CD47 knock out (KO) mice to generate HOD RBC donors with wild type, heterozygous or homozygous KO levels of CD47 and used these donors to define the impact of CD47 on antibody formation following RBC transfusion. Methods: HOD transgenic mice expressing the HOD antigen exclusively on RBCs were crossed with CD47-/- mice to produce HOD CD47+/- or HOD CD47-/- mice. HOD and CD47 levels were assessed by flow cytometric analysis using anti-HOD and anti-CD47 antibodies. HOD CD47+/+, HOD CD47+/- or HOD CD47-/- RBCs were transfused into C57BL6 recipients, followed by serum collection on days 14 and 28 post transfusion and evaluation of anti-HOD antibodies by flow cytometry crossmatch. To determine the CD4 T cell response to transfusion, TCR transgenics specific to ovalbumin (OTII) were labeled with CFSE, followed by adoptive transfer, transfusion of HOD CD47+/+, HOD CD47+/- or HOD CD47-/- RBCs and evaluation of T cell proliferation, activation and cytokine secretion. Cellular removal of HOD RBCs was determined by flow cytometric examination of CFSE-labeled HOD RBCs. Finally, antigen levels on HOD RBCs was determined by staining cells with anti-HEL antibodies followed by flow cytometric examination. All three groups were subjected to one-way ANOVA analysis with a p value

Description: The promoter region of the Bβ fibrinogen gene containing the polymorphic site (G−455-A) shows an increase in fibrinogen levels for individuals containing an adenine rather than a guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were performed using specific DNA probes containing base sequences pertinent to the allelic site. Specific DNA binding proteins were detected and their binding characteristics were determined. Secondly, we placed DNA fragments containing different −455 nucleotide substitutions of the Bβ promoter upstream of a luciferase reporter gene and transfected them into HepG2 cells to determine their effect on transactivation. An adenine at position −455 resulted in greater luciferase activity than when a guanine was present. UV cross-linking bound protein to the DNA demonstrated a 47-kD protein binding preferentially to the site when a guanine rather than an adenine was present at −455. We hypothesize that a transactivation protein complex associates with the site, but its association is stronger when guanine is present, thereby slowing downstream Bβ gene transcription. These data provide the first molecular evidence that accounts for the increase in fibrinogen in individuals carrying this allele. © 1998 by The American Society of Hematology.

Description: Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Because unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that all unusually large factor V in platelets was associated with multimerin and it remained associated in 0.5 M salt. Multimerin immunodepletion of the normal pooled platelet lysate removed 100 ± 0% of multimerin and 47.0 ± 2.4% of total factor V antigen, whereas sham immunodepletion removed 12.0 ± 3.0 % of multimerin and 4.0 ± 4.0% of factor V antigen (means ± 1 S.D. for 3 experiments). Analyses of serial factor V immunopurified samples indicated that platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The suggestion that only a subpopulation of multimerin was covalently linked to factor V was consistent with the estimated 17 fold molar excess of multimerin subunits to factor V molecules in platelets. The disulfide-linked complexes of multimerin and factor V in platelets, that are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Multimerin could function to hold about half of the platelet pool of factor V in covalent and noncovalent linkages, until granule release occurs and thrombin cleavages liberate factor Va for prothrombinase assembly on the platelet surface, akin to the way supporting scaffolds hold pieces of plastic models in a unit until their removal for model assembly is desired.

Description: Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a–null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a−/− myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a−/− HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a−/− mice. miR-146a−/− HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Description: Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p〈 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p

Description: No defects related to deficiency of the Wiskott-Aldrich Syndrome protein (WASp) have been described in osteoclasts. Here we show that there are significant morphologic and functional abnormalities. WASp-null cells spread over a much larger surface area and are highly polykaryotic. In their migratory phase, normal cells assemble clusters of podosomes behind their leading edges, whereas during the bone resorptive phase multiple podosomes are densely aggregated in well-defined actin rings forming the sealing zone. In comparison, WASp-null osteoclasts in either phase are markedly depleted of podosomes. On bone surfaces, this results in a failure to form actin rings at sealing zones. Complementation of WASp-null osteoclasts with an enhanced green fluorescent protein (eGFP)-WASp fusion protein restores normal cytoarchitecture. These structural disturbances translate into abnormal patterns of bone resorption both in vitro on bone slices and in vivo. Although physiologic steady-state levels of bone resorption are maintained, a major impairment is observed when WASp-null animals are exposed to a resorptive challenge. Our results provide clear evidence that WASp is a critical component of podosomes in osteoclasts and indicate a nonredundant role for WASp in the dynamic organization of these actin structures during bone resorption. (Blood. 2004;103:3552-3561)

Description: Iron accumulation and overload in beta thalassaemia patients are associated with significant morbidity and mortality. Iron chelators are used to manage iron accumulation but side effects and compliance issues restrict the use of available chelators. Deferitrin (Genzyme Corporation) is an orally available iron chelator intended for iron overload. Method: Patients were dosed in 4 cohorts, receiving 5, 10, 15 and 25 mg/kg/day of deferitrin. Deferitrin dosing in cohorts 1–3 was once daily for 12 weeks. Cohort 4 received deferitrin twice daily (BD) for 48 weeks (12.5mg/kg BD, 25 mg/kg/day). Pharmacokinetics (PK) were assessed in a subset of up to 5 patients in each cohort, pre-dose and 1, 2, 4 and 8 hours post dose. All patients had trough levels assessed at weeks 1, 6 and 12 (all Cohorts) and additionally at weeks 24, 36 and 48 for Cohort 4. PK parameters were determined by model independent (non-compartmental) analyses. Safety was assessed by collection of adverse events and laboratory assessments with renal parameters measured weekly due to observations of renal toxicity in preclinical testing. Efficacy (change in liver iron concentration (LIC)) was assessed by SQUID (superconducting quantum interference device) in Turin, Italy, between screening and end of study. Iron excretion and intake were estimated by calculation:Iron excretion due to deferitrin = Iron Intake (mg/kg/day) - TBI (mg/kg/day)Iron Intake (mg/kg/day) = [total mL pRBC (exclude last BT×) × 1.08] / [Weight (kg) × Days (Between 1st & last BT×)]TBI (mg/kg/day) = Change in LIC (mg Fe/g dry weight) × [10.6 (Angelucci Factor) / D (Days on deferitrin)] Key: pRBC = packed red blood cells, BT× = blood transfusion, TBI=Total Body Iron. Results: PK: PK for deferitrin dosed once daily was linear and dose proportional. The serum half-life was 1.3–1.8 hrs, clearance was 226–340 mL/min and mean residence time was 2.8–3.4 hrs for once daily dosing. PK data from BD dosing is not yet available. Safety: Deferitrin dosed once daily was generally well tolerated (Cohorts 1–3). Slight rises in transaminases were seen at 10 and 15 mg/kg/day. A large proportion of enrolled patients were hepatitis C positive (73%). When dosed BD (12.5 mg/kg BD in Cohort 4), 3 patients developed renal toxicity after 4–5 weeks of treatment. Two patients experienced increased proteinuria (max 3.73 g/L & 3.29 g/L) and one patient suffered acute renal failure (peak serum creatinine 4.1 mg/dL, lowest GFR 27 mmol/L). All patients recovered normal renal function after stopping treatment. No patients were re-challenged with deferitrin. Dosing was terminated in all patients because of safety concerns. Efficacy: Mean iron excretion in mg/kg/day (S.D) for Cohort 1 was 0.22 (0.22), Cohort 2 was 0.45 (0.14) and Cohort 3 was 0.33 (0.12). The reasons for the lack of dose proportionality in iron excretion are unclear. Efficacy could not be assessed in Cohort 4 due to early termination of the study. Conclusions: Deferitrin dosed once daily was generally well tolerated and associated with a mean iron excretion of 0.34 mg/kg/day. Deferitrin dosed BD (12.5mg/kg BD) was associated with unacceptable renal toxicity and led to study termination. Deferitrin does not appear to have an acceptable therapeutic margin to allow sufficient iron excretion for long-term administration.

Description: Monoclonal chronic lymphocytic leukemia (CLL)–phenotype cells are detectable in 3.5% of otherwise healthy persons using flow cytometric analysis of CD5/CD20/CD79b expression on CD19-gated B cells. To determine whether detection of such CLL-phenotype cells is indicative of an inherited predisposition, we examined 59 healthy, first-degree relatives of patients from 21 families with CLL. CLL-phenotype cells were detected in 8 of 59 (13.5%) relatives, representing a highly significant increase in risk (P = .00002). CLL-phenotype cell levels were stable with time and had the characteristics of indolent CLL. Indolent and aggressive clinical forms were found in family members, suggesting that initiation and proliferation involves distinct factors. The detection of CLL-phenotype cells provides a surrogate marker of carrier status, potentially facilitating gene identification through mapping in families and direct analysis of isolated CLL-phenotype cells.

Description: The National Marrow Donor Program maintains a registry of volunteer donors for patients in need of a hematopoietic stem cell transplantation. Strategies for selecting a partially HLA-mismatched donor vary when a full match cannot be identified. Some transplantation centers limit the selection of mismatched donors to those sharing mismatched antigens within HLA-A and HLA-B cross-reactive groups (CREGs). To assess whether an HLA mismatch within a CREG group (“minor”) may result in better outcome than a mismatch outside CREG groups (“major”), we analyzed validated outcomes data from 2709 bone marrow and peripheral blood stem cell transplantations. Three-hundred and ninety-six pairs (15%) were HLA-DRB1 allele matched but had an antigen-level mismatch at HLA-A or HLA-B. Univariate and multivariate analyses of engraftment, graft-versus-host disease, and survival showed that outcome is not significantly different between minor and major mismatches (P = .47, from the log-rank test for Kaplan-Meier survival). However, HLA-A, HLA-B, and HLA-DRB1 allele–matched cases had significantly better outcome than mismatched cases (P 〈 .001). For patients without an HLA match, the selection of a CREG-compatible donor as tested does not improve outcome.

Description: Antitumor effects of metformin are widely known. Unfortunately their use in the treatment of acute lymphoblastic leukemia is limited. Study design The study consisted of two stages Phase 1: Cell assay adding metformin (40mM) assessing the viability and cell cycle in the cell line molt 4 Phase 2: Pilot study adding Metformin (850mg three time day) during the pre-treatment with steroids and the induction remission phase versus a control group (2:1 randomization) Statistical analysis Chi-square analysis was used to corroborate the hypothesis. The odds ratio was calculated for both, the absence of Good steroid response and refractory leukemia. Cox regression analysis and Kaplan-Meier curves were used for the survival analysis Results Celular assay. Metformin inhibited cell viability at 120hours reducing the percentage of cells in phase S. Clinical assay. 151 patients were studied, 44 (29.1%) on Metformin arm, of these 59.1% ( n=26) archived a GSR compared with the control group (26.2%, n=28). A greater number of complete remission were presented in Metformin arm (81.8% versus 57.9%) shown to be a protective factor for GSR and complete remissions (odds ratio ; 0.2454 y 0.3062 respectively). In the Cox regression analysis, Metformin significantly impact in the global survival (p=0,012,95% IC) versus the other variables studied. Conclusions Metformin is useful both in vivo and in vitro treatment of adult acute lymphoblastic leukemia. Disclosures: No relevant conflicts of interest to declare.