bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of functional CD40 antigen on Reed-Sternberg cells and Hodgkin's disease cell lines
Authors: Carbone A , Gloghini A , Gattei V , Aldinucci D , Degan M , De Paoli P , Zagonel V , Pinto A
Published: October 2019

CD40 is a member of the nerve growth factor receptor family, showing a significant homology to the Hodgkin's disease (HD)-associated antigen CD30 and is capable of transduce growth signals in a number of cell types. A series of 312 lymphoma samples, including 139 cases of HD, 32 cases of CD30+ anaplastic large cell (ALC) lymphomas, 141 cases of other non-Hodgkin's lymphomas (NHLs), and a panel of HD- or NHL-derived cell lines, were evaluated for CD40 expression by immunostaining of paraffin embedded sections, cell smears and flow cytometry. CD40 was strongly expressed with a highly distinct pattern of staining on Reed- Sternberg (RS) cells and variants in 100% (139/139) of HD cases, irrespective of their antigenic phenotype (T, B, non T-non B) and histologic subtype of HD. Conversely, CD40 was immunodetected on only one third (12/32; 37%) of ALC lymphoma cases and on 105 of 127 B-cell NHLs. The relative cell density of CD40 on HD cell lines (L-428, KM-H2, HDLM-2) as assessed by flow cytometry was significantly higher than on all other lymphoma cells analyzed. Engagement of CD40 by its soluble ligand (CD40L) enhanced both clonogenic capacity and colony cell survival of HD cell lines. Such effect was potentiated by interleukin-9 costimulation in KM-H2 cells. Finally, we have shown that in vitro rosetting of activated CD4+ T cells to HD cells (L-428) is mediated in part by the CD40/CD40L adhesion pathway. Our data indicate that CD40 is a useful antigen for immunodetection and identification of tumor cells in all subtypes of HD, and suggest that it may play a role in the regulation of RS cell expansion and the contact-dependent interactions of these cells with cytokine-producing T lymphocytes.
Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines
Authors: Kitayama H , Kanakura Y , Furitsu T , Tsujimura T , Oritani K , Ikeda H , Sugahara H , Mitsui H , Kanayama Y , Kitamura Y
Published: October 2019

The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val- 559-->Gly (G559) mutation in the juxtamembrane domain and Asp-814-->Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B-type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies.
Leukocyte depletion of random single-donor platelet transfusions does not prevent secondary human leukocyte antigen-alloimmunization and refractoriness: a randomized prospective study
Authors: Sintnicolaas K , van Marwijk Kooij M , van Prooijen HC , van Dijk BA , van Putten WL , Claas FH , Novotny VM , Brand A
Published: October 2019

We studied the value of leukocyte depletion of platelet transfusions for the prevention of secondary human leukocyte antigen (HLA)- alloimmunization in patients with a high-risk of prior immunization induced by pregnancies. Seventy-five female patients with hematologic malignancies (mostly acute leukemia) and a history of pregnancy were randomized to receive either standard random single-donor platelet transfusions (mean leukocytes, 430 x 10(6) per transfusion) or leukocyte-depleted random single-donor platelet transfusions. Leukocyte depletion to less than 5 x 10(6) leukocytes per platelet transfusion (mean leukocytes, 2 x 10(6) per transfusion) was achieved by filtration. Of the 62 evaluable patients, refractoriness to random donor platelets occurred in 41% (14 of 34) of the patients in the standard group and in 29% (8 of 28) of the patients in the filtered group (P = .52); anti-HLA antibodies developed in 43% (9 of 21) of individuals in the standard group and 44% (11 of 25) of cases in the filtered group. The time toward refractoriness and development of anti- HLA antibodies was similar for both groups. We conclude that leukocyte depletion of random single-donor platelet products to less than 5 x 10(6) per transfusion does not reduce the incidence of refractoriness to random donor platelet transfusion because of boostering of anti-HLA antibodies.
Clinical and molecular characterization of a rare syndrome of acute promyelocytic leukemia associated with translocation (11;17)
Authors: Licht JD , Chomienne C , Goy A , Chen A , Scott AA , Head DR , Michaux JL , Wu Y , DeBlasio A , Miller WH Jr
Published: October 2019

Analysis of a variant translocation t(11;17) in a case of acute promyelocytic leukemia (APL) led to discovery of a novel zinc finger gene, PLZF, fused to the retinoic acid receptor-alpha (RAR alpha) gene. We reviewed the clinical and molecular features of five additional patients with t(11;17)-associated APL. The clinical course of three patients was characterized by early death and three experienced disseminated intravascular coagulation. Morphologically all of the patients fell in a unusual morphologic spectrum of APL, with features intermediate between M2 and M3 AML. All six patients had PLZF-RAR alpha gene fusion as detected by reverse transcription/polymerase chain reaction assay, Southern blotting, or pulsed-field gel electrophoresis. Five of the six patients failed to achieve complete remission after initial chemotherapy or differentiation therapy with all-trans retinoic acid (ATRA). A sixth patient responded to initial chemotherapy, but on relapse failed to respond to ATRA. When tested in vitro, cultured cells from three of the patients failed to differentiate in response to ATRA. APL associated with t(11;17) and fusion of the PLZF and RAR alpha genes is a discrete clinico-pathologic syndrome with a distinctly worse prognosis than t(15;17) APL.
Molecular basis of the Kell (K1) phenotype
Authors: Lee S , Wu X , Reid M , Zelinski T , Redman C
Published: October 2019

K1 (K, Kell) is a strong immunogen; its antibodies can cause severe reactions if incompatible blood is transfused and may cause hemolytic disease of the newborn in sensitized mothers. K1 is a member of the Kell blood group system, which is complex, containing over 20 different antigens. Some of the antigens are organized in allelic pairs of high and low prevalence whereas others are independently expressed. K1, which is present in 9% of the population, is antithetical to the high- prevalence K2 (k) antigen. We have determined the molecular basis of the K1/K2 polymorphism by sequencing the 19 exons of the Kell gene (KEL) of a K1/K1 person. Polymerase chain reaction was performed on genomic DNA isolated from peripheral blood and the amplified products were either directly sequenced or subcloned and sequenced. Comparisons of K1/K1 and K2/K2 DNA showed a C to T base substitution in exon 6 that predicts a threonine to methionine change at amino acid residue 193. This amino acid substitution occurs at a consensus N-glycosylation site (Asn. X. Thr) and probably prevents N-glycosylation, leading to a change in phenotype. The C to T substitution creates a Bsm I restriction enzyme site, which was tested in 42 different samples to confirm that this base change identifies the K1/K1 genotype. This test differentiates genotypes, K1/K1, K2/K2, and the K1/K2 heterozygote and should prove useful in the prenatal diagnosis of K1-related hemolytic disease of the newborn.
The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro
Authors: Gronthos S , Simmons PJ
Published: October 2019

Factors that regulate the growth and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant growth factors for their ability to initiate and support clonogenic growth of fibroblast colony-forming cells (CFU- F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic- activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO- 1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU- F to exogenously added factors. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic growth of CFU- F in this culture system was absolutely dependent on an exogenous source of growth factor. Platelet-derived growth factor-BB (PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability to support colony growth. Colony formation was dose-dependent, with half- maximal colony numbers at approximately 0.2 ng/mL for either factor and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either factor alone or with the size of colonies elicited in control cultures by 20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF- 1 and NGF did not support colony growth. This report demonstrates the development of a simple, reproducible, and stringent culture system for the growth and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of growth factors in the regulation of stromal cell proliferation, differentiation, and development.
Identification of hematopoietic stem cell subsets on the basis of their primitiveness using antibody ER-MP12
Authors: van der Loo JC , Slieker WA , Kieboom D , Ploemacher RE
Published: October 2019

Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a physical separation of primitive long-term repopulating stem cells from more mature multilineage progenitors. When used in two-color immunofluorescence with ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow (BM) cells could be identified. These subsets were isolated using magnetic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colony-forming units in culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for day-12 spleen colony-forming units (CFU-S-12), and for cells with long-term repopulating ability using a recently developed alpha-thalassemic chimeric mouse model. Cells with long-term repopulation ability (LTRA) and day-12 spleen colony-forming ability appeared to be exclusively present in the two subpopulations that expressed the ER-MP12 cell surface antigen at either an intermediate or high level, but lacked the expression of Ly- 6C. The ER-MP12med20- subpopulation (comprising 30% of the BM cells, including all lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28 CAFC (CAFC-28), 75% of the CFU-S-12, and very low numbers of CFU-C. In contrast, the ER-MP12hi20- population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primitive CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 through day 14), of which 70% were ER-MP20- and 10% to 20% ER-MP20med/hi. In addition, erythroblasts, granulocytes, lymphocytes, and monocytes could almost be fully separated on the basis of ER-MP12 and ER-MP20 antigen expression. Functionally, the presence of ER-MP12 in a long-term BM culture did not affect hematopoiesis, as was measured in the CAFC assay. Our data demonstrate that the ER-MP12 antigen is intermediately expressed on the long-term repopulating hematopoietic stem cell. Its level of expression increases on maturation towards CFU-C, to disappear from mature hematopoietic cells, except from B and T lymphocytes.
The glycoprotein Ib-IX complex-specific monoclonal antibody SZ1 binds to a conformation-sensitive epitope on glycoprotein IX: implications for the target antigen of quinine/quinidine-dependent autoantibodies
Authors: Lopez JA , Li CQ , Weisman S , Chambers M
Published: October 2019

The monoclonal antibody SZ1 is of interest for two reasons: it was used to define complex formation between glycoprotein (GP) Ib and GP IX, and its epitope is likely to be identical to that recognized by most quinine- and quinidine-dependent autoantibodies that cause thrombocytopenia. To determine the location of the epitope for SZ1 within the GP Ib-IX complex (which consists of three subunits: GP Ib alpha, GP Ib beta, and GP IX), we tested the ability of the antibody to bind transfected cells that expressed different combinations of complex subunits, and compared this binding to the binding of antibodies of known specificity. SZ1 bound to cells that expressed the entire GP Ib- IX complex in the same pattern as did AN51 (an antibody specific for GP Ib alpha). However, unlike AN51, SZ1 did not bind alpha beta cells (ie, cells that express GP Ib alpha and GP Ib beta, but not GP IX), but did bind to beta IX and alpha IX cells. We then compared the binding patterns of SZ1 and FMC25, an antibody specific for GP IX. Both bound virtually identically to cell lines that expressed every combination of two of the three GP Ib-IX complex subunits. However, the epitopes of the two antibodies were not identical, because fixation with 4% paraformaldehyde of cells that expressed GP IX destroyed the SZ1 epitope while maintaining the FMC25 epitope. Because of the ability of SZ1 to block the binding of many quinine- and quinidine-dependent antibodies, these data strongly suggest that GP IX is the component of the GP Ib-IX complex recognized by those antibodies.
Six point mutations that cause factor XI deficiency
Authors: Pugh RE , McVey JH , Tuddenham EG , Hancock JF
Published: October 2019

We have identified six novel types of mutation that cause factor XI deficiency, an inherited bleeding disorder. Two are point mutations that interfere with the normal splicing of exons in the mRNA and four are point mutations that result in amino acid substitutions. One of these amino acid substitutions (Asp 16-->His) is near the amino terminal end of the protein. The other three amino acid substitutions (Leu 302-->Pro, Thr 304-->Ile, and Glu 323-->Lys) are in the fourth apple domain, a region that mediates dimerization of identical subunits of factor XI. All four amino acid substitutions cause a reduction in the amount of factor XI secreted from cells grown in vitro.
Enhanced production of B lymphocytes after castration
Authors: Wilson CA , Mrose SA , Thomas DW
Published: October 2019

Castration has long been recognized to stimulate thymic growth and augment cellular immunity. We sought to determine whether castration affects B lymphopoiesis by analyzing the phenotype of bone marrow and spleen cells from animals postcastration. In this report, we show that the bone marrow cells from castrated male mice show a sustained, twofold to threefold increase in numbers of B220+/IgM- cells and of newly formed B220+/IgM+ B cells. Most of the expanded B220+/IgM- cell population consisted of small, HSAhi, CD43- cells characteristic of pre- B cells. The castrated animals also showed increased numbers of splenic B cells, primarily consisting of small IgM+, IgDlo, B220lo, HSAhi cells. Taken together, these results show that castration causes dramatic, long-lived enhancement of B lymphopoiesis in bone marrow and increased numbers of mature B cells in the periphery.
Identification of soluble APO-1 in supernatants of human B- and T-cell lines and increased serum levels in B- and T-cell leukemias
Authors: Knipping E , Debatin KM , Stricker K , Heilig B , Eder A , Krammer PH
Published: October 2019

The cell-surface protein APO-1 is a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily. APO-1 mediates apoptosis in susceptible cells upon stimulation with the monoclonal antibody anti-APO-1 or upon binding of its natural ligand. Soluble receptors had previously been identified for most members of the NGF/TNF receptor superfamily. Recently, a soluble form of APO-1 (sAPO- 1) was described. We established a sandwich enzyme-linked immunosorbent assay to detect sAPO-1 in culture supernatants of human cell lines and in human sera. sAPO-1 was found in culture supernatants of different human B- and T-cell lines. Molecular weights of sAPO-1 and membrane APO- 1 were similar. In addition, in comparison to healthy donors, sera from patients with different high- and low-grade malignant B- and T-cell leukemias and lymphomas contained increased levels of sAPO-1. These findings may have implications for the growth of leukemias and the diagnostic monitoring of individual patients.
Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor [see comments]
Authors: Bensinger WI , Weaver CH , Appelbaum FR , Rowley S , Demirer T , Sanders J , Storb R , Buckner CD
Published: October 2019

Peripheral blood stem cells (PBSCs) are widely used in autologous transplantation because of ease of collection and rapid hematopoietic reconstitution. However, PBSCs have rarely been used for allogeneic transplantation because of concerns about donor toxicities from cytokine administration and the theoretical increased risk of graft- versus-host-disease (GVHD) from the large number of T cells infused. Eight patients with advanced malignancies received allogeneic PBSC transplants from genotypically HLA-identical sibling donors. All donors received 5 days of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 16 micrograms/kg/day) subcutaneously and were leukapheresed for 2 days. After treatment of the patient with total body irradiation and cyclophosphamide (n = 7) or etoposide, thiotepa, and cyclophosphamide (n = 1), PBSCs were infused immediately after collection and without modification. All patients received cyclosporine and either methotrexate (n = 6) or prednisone (n = 2) for GVHD prophylaxis, rhG-CSF was well tolerated with mild bone pain requiring acetaminophen occurring in two donors. All patients engrafted and in seven hematopoietic recovery was rapid, with 500 neutrophils/microL achieved by day 18 and 20,000 platelets/microL by day 12. Complete donor engraftment was documented by Y chromosome analysis in all four sex-mismatched donor-recipient pairs tested and by DNA analysis in two sex-matched pairs. One patient died on day 18 of veno-occlusive disease of the liver with engraftment but before chromosome analysis could be performed (results are pending in 1 patient). A second patient died of fungal infection 78 days after transplant. Grade 2 acute GVHD occurred in two patients and grade 3 GVHD occurred in one patient. One patient is 301 days from transplant in remission with chronic GVHD; the remaining five patients are alive and disease free 67 to 112 days after transplantation. Preliminary results indicate that allogeneic PBSCs mobilized by rhG-CSF can provide rapid hematologic recovery without an appreciably greater incidence of acute GVHD than would be expected with marrow. Further follow-up is required to determine the incidence of chronic GVHD and any potential beneficial effects on relapse after transplant.
Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts [see comments]
Authors: Korbling M , Przepiorka D , Huh YO , Engel H , van Besien K , Giralt S , Andersson B , Kleine HD , Seong D , Deisseroth AB
Published: October 2019

Peripheral blood stem cells (PBSCs) have been used rarely for allogeneic transplantation because of concerns regarding graft failure and graft-versus-host disease (GVHD). We evaluated the results of allogeneic PBSC transplantation (allo-PBSCT) in 9 patients with refractory leukemia or lymphoma receiving myeloablative therapy followed by allo-PBSCT from an HLA-identical sibling donor. Three patients had relapsed 11 to 21 months after allogeneic bone marrow transplantation (allo-BMT) and underwent allo-PBSCT using the same donor. Six patients received PBSCs as their initial allogeneic transplant. Filgrastim-mobilized PBSCs were collected from the donors in 3 to 4 aphereses and cryopreserved. The apheresis collections contained a median nucleated cell count of 16.5 x 10(8)/kg (range, 10.8 to 28.7 x 10(8), 10.7 x 10(6) CD34+ cells/kg (range, 7.5 to 22.5 x 10(6)), and 300.0 x 10(6) CD3+ cells/kg (range, 127.8 to 1,523.2 x 10(6)). The median recovery of CD34+ progenitor cells after freezing, thawing, and washing was 106.4% (range, 36.7% to 132.0%). All patients received filgrastim posttransplant through engraftment, and cyclosporine and methylprednisolone were used for GVHD prophylaxis. Neutrophil recovery to greater than 0.5 x 10(9)/L and greater than 1.0 x 10(9)/L occurred at a median of 9 (range, 8 to 10) and 9 days (range, 8 to 11) posttransplant, respectively, which was similar to historical controls after allo-BMT and granulocyte colony-stimulating factor therapy. Platelets recovered to greater than 20 x 10(9)/L and greater than 50 x 10(9)/L at a median of 12 (range, 8 to 25) and 15 days (range, 11 to 59), respectively, which was significantly more rapid than for the controls (P < .01). Donor cell engraftment was documented by cytogenetics, fluorescence in situ hybridization, and/or restriction fragment length polymorphisms with longest follow-up of 283 + days. Three patients developed grade 2 acute GVHD involving only the skin. Three of five evaluable patients show limited chronic GVHD. Cryopreserved, filgrastim-stimulated allogeneic PBSCs may be a suitable alternative to allogeneic marrow for transplantation with the advantage of more rapid platelet recovery. Acute GVHD was minimal despite the infusion of 1 log more CD3 cells than with marrow allografts. Further studies are required to assess long-term risks of chronic GVHD.
Pediatric myelodysplasia: a study of 68 children and a new prognostic scoring system
Authors: Passmore SJ , Hann IM , Stiller CA , Ramani P , Swansbury GJ , Gibbons B , Reeves BR , Chessells JM
Published: October 2019

Clinical, morphologic, and cytogenetic features were examined in a group of 68 children with myelodysplasia (MDS) referred to a single institution between 1971–1991. The morphologic French-American-British (FAB) system of classification proved of limited value in this group of patients because 50% of the cases were categorized as chronic myelomonocytic leukemia and three patients with eosinophilia and MDS were unclassifiable. Cytogenetic analysis was performed in 63 cases and clonal abnormalities were detected in 55%; the most common chromosome involved was number 7. Modification of the FAB system to incorporate additional diagnostic features such as pretreatment fetal hemoglobin (Hb F) and cytogenetics allowed incorporation of the categories of juvenile chronic myeloid leukemia (JCML) and infantile monosomy 7 syndrome (IMo7). The resulting groups of patients had highly significant differences in survival (P = .00009). The overall 5-year survival for the patients was 31.9% (95% CI 21.7 to 44.1) and factors influencing prognosis included: modified FAB type, platelet count, Hb F level, and cytogenetic complexity. We developed a scoring system (“FPC”) where each of the following findings at diagnosis scored one point: HbF greater than 10%, platelets < or = 40 x 10(9)/L, and complex karyotypic changes (two or more clonal structural/numerical abnormalities), which produced groups with highly significant differences, patients with a score of 0 having a 5-year survival of 61.6% (CI 33% to 84%), whereas those with a score of two or three all died within 4 years of diagnosis. The revised classification and scoring system may prove helpful in making treatment choices in pediatric MDS and now needs to be tested prospectively in large scale population-based studies.
The oxidation of exogenously added organic anions by platelets facilitates maintenance of pH during their storage for transfusion at 22 degrees C
Authors: Murphy S
Published: October 2019

Previous studies of platelet metabolism during the storage of platelet concentrates (PCs) at 22 degrees C for transfusion have shown high rates of both oxygen consumption and aerobic glycolysis with little oxidation of pyruvate produced by glycolysis. During storage in plasma, free fatty acids are major oxidative fuels. Glutamine is also present as a potential fuel but relatively little is metabolized beyond glutamate. In synthetic media, acetate is oxidized and provides a poorly understood buffering function. In the current work, acetate and pyruvate (6 to 25 mmol/L, sometimes with their 14-C-labeled counterparts) were added to PCs stored in plasma. They were both vigorously metabolized, predominantly to CO2 (0.51 +/- 0.08 and 0.31 +/- 0.06 mmol/d/10(12) platelets for acetate and pyruvate, respectively). The metabolism of these exogenous substrates was associated with significantly increased oxygen consumption and decreased glucose consumption, fatty acid oxidation, and bicarbonate utilization for buffering. In a more limited number of studies, similar findings were observed with addition of beta-hydroxy-butyrate. Superior maintenance of pH in the presence of these additives could be attributed to the fact that the metabolism of an organic anion requires that a proton be brought with the anion into the metabolic pathway, thus providing an alkalinizing effect. Pyruvate (but not acetate) also stimulated the metabolism of glutamate. Studies with 14-C-labeled glucose suggested significant activity of the hexose-monophosphate shunt during PC storage and confirmed that little, if any, pyruvate derived from glucose was fully oxidized. Taken together, the results provide a relatively complete picture of the pathways of energy metabolism used by platelets during PC storage and suggest a strategy by which organic anions such as acetate can be used to improve the results of such storage.
Blood transfusions and immunophenotypic alterations of lymphocyte subsets in sickle cell anemia. The Transfusion Safety Study Group
Authors: Wong WY , Powars DR , Operskalski EA , Hassett J , Parker JW , Sarnaik S , Pegelow CH , Hilgartner MW , Johnson CS , Zhou Y
Published: October 2019

Transfusions purportedly induce dysfunction of cell-mediated immunity in sickle cell anemia (SCA). We studied hematologic and lymphocytic indices in 173 human immunodeficiency virus (HIV)-negative subjects with SCA and 131 black controls. Children aged 1 to 7 years with SCA had leukocyte counts and percentages of granulocytes, monocytes, natural killer cells, and T-cell markers (CD2+CD11b+, CD4+CD26+, CD4+CD29+) that were significantly higher than those for control children. Percent total lymphocytes was decreased for this age group, but the total number of lymphocytes and T and B cell counts were similar to controls. Platelets were not increased. Adolescents (aged 8 to 17 years) and adults (aged > or = 18 years) with SCA had increased total leukocytes and monocytes and lymphocytes counts that remained level instead of decreasing, as did comparably aged controls. Lymphocyte subsets typically increased in count, but their percentage remained similar to children. The exception was CD56+ cell counts, which were increased in adolescents and adults. No lymphocytic subset change suggested impaired cellular immunity, and none could be related to transfusion. Prophylactically transfused patients had higher granulocyte counts, but these may arise from the complications of SCA itself.
Identification of signaling motifs within human Fc gamma RIIa and Fc gamma RIIb isoforms
Authors: Van den Herik-Oudijk IE , Capel PJ , van der Bruggen T , Van de Winkel JG
Published: October 2019

To assess the functional capacity of the heterogeneous Fc gamma RII (CD32) family and to identify critical regions for functioning, we generated a panel of B-cell transfectants. The Fc gamma R-negative B-cell line IIA1.6 was transfected with wild-type or mutant human Fc gamma RIIa and IIb molecules. Solely Fc gamma RIIa-expressing IIA1.6 cells were capable of phagocytosing opsonized Staphylococcus aureus bacteria, and cross-linking of Fc gamma RIIa triggered a rapid induction of tyrosine phosphorylation after 20 seconds. Analysis of Fc gamma RIIa mutants identified the immunoreceptor tyrosine-based activation motif (ITAM; previously described as ARH-1 motif) within the IIa cytoplasmic tail to be critical for B-cell activation. In contrast, Fc gamma RIIb isoforms triggered tyrosine phosphorylation on cross-linking with much slower kinetics (> 3 minutes) than Fc gamma RIIa. Furthermore, solely Fc gamma RIIb molecules proved capable of downregulating [Ca2+]i and interleukin-2 production on co-cross-linking with sIgG in IIA1.6. The Fc gamma RIIb-mediated functions were absent in Fc gamma RIIb mutants in which the tyrosine or leucine within the YSLL motif in a conserved 13-aa region (now known as immunoreceptor tyrosine-based inhibitor motif [ITIM]) were changed into phenylalanines. In conclusion, these data show the presence of functionally critical motifs within Fc gamma RII cytoplasmic tails. Fc gamma RIIa contains an ITAM involved in B-cell activatory functions, whereas the downregulatory activity of Fc gamma RIIb isoforms is linked to an ITIM.
Glycophorin SAT of the human erythrocyte membrane is specified by a hybrid gene reciprocal to glycophorin Dantu gene
Authors: Huang CH , Reid ME , Okubo Y , Daniels GL , Blumenfeld OO
Published: October 2019

Previous studies of two unrelated Japanese families showed that two isoforms of glycophorin were associated with the expression of SAT antigen on the erythrocyte membrane. Here we report the molecular basis for one form of this MNSs-related surface marker that displayed an altered immunoblotting pattern. Evidence is presented that glycophorin SAT (GPSAT) is encoded by a hybrid gene resulting from unequal homologous recombination between GPA and GPB genes. Analysis of SAT genomic DNAs by Southern blots showed gross alterations in the glycophorin gene cluster. Those restriction fragments characteristic of the GPA 3′ and GPB 5′ ends were absent from the SAT homozygote and showed reduced intensity in SAT heterozygotes. Reticulocyte RNA polymerase chain reaction showed the presence in the SAT homozygote of GPSAT and GPE transcripts but no GPA and GPB transcripts. Direct sequencing of the amplified SAT cDNA showed that its sequence from exon I to exon IV was identical with the N allele of GPA, whereas its 3′ portion, including exon V and exon VI, was derived from the GPB gene. The GPSAT protein in its mature form should contain 104 amino acid residues and bear a novel sequence, Ser-Glu-Pro-Ala-Pro-Val, encoded by the junction of GPA exon IV and GPB exon V. This sequence interfaces the extracellular and transmembrane domains and could be the epitope site of the SAT antigen. The formation of such a hybrid junction not only explains why SAT homozygous erythrocytes lack S, s, and U antigens but also shows a reciprocal arrangement with respect to the B-A hybrid GPDantu gene.
Four new mutations in the NADH-cytochrome b5 reductase gene from patients with recessive congenital methemoglobinemia type II
Authors: Vieira LM , Kaplan JC , Kahn A , Leroux A
Published: October 2019

Recessive congenital methemoglobinemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases. In the type I form, cyanosis is the only symptom, and the soluble enzyme is defective in red blood cells. In the type II form, cyanosis is associated with severe mental retardation and neurologic impairment; the enzymatic defect is systemic, involving both soluble and membrane-bound isoforms. We characterized mutations responsible for cytb5r deficiency in three unrelated patients with severe RCM type II. The first patient presented a homozygous exon 5 skipping. The only mutation detected was a homozygous G to C transversion at position +8, downstream from the 5′ splice site of exon 5. We suggest that this unusual mutation might be responsible for the abnormal splicing of the primary transcripts, resulting in frameshift with premature STOP codon. The second mutation found corresponds to a homozygous C to T transition changing the Arg-218 codon to a premature STOP codon in exon 8. The third case was a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation with replacement of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele carried a 3-bp deletion (TGA) of nucleotides 815 to 817, modifying two contiguous codons in exon 9 of the cDNA with loss of Met-272. These results confirm the genetic polymorphism of cytb5r gene mutations identified in RCM type II, as observed for the mutations described in the RCM type I, and shed light on the molecular bases of the two different diseases associated with cytb5r deficiency.
Thrombopoietic effects and toxicity of interleukin-6 in patients with ovarian cancer before and after chemotherapy: a multicentric placebo- controlled, randomized phase Ib study
Authors: D'Hondt V , Humblet Y , Guillaume T , Baatout S , Chatelain C , Berliere M , Longueville J , Feyens AM , de Greve J , Van Oosterom A
Published: October 2019

Recombinant human interleukin-6 (IL-6) has previously been shown to increase platelet counts in normal and sublethally irradiated mice, dogs, and primates. To assess its tolerance and efficacy in clinical use, we performed a randomized phase Ib study in patients with ovarian carcinoma. IL-6 was administered during an initial 7-day cycle before any chemotherapy. Beginning 7 days later, six cycles of chemotherapy containing carboplatin were administered every 3 weeks. During chemotherapy cycles 2 to 6, IL-6 was administered from day 4 through day 17 at escalating dose levels from 0.5 to 10 micrograms/kg/d. At each level, three patients received IL-6 and one patient received a placebo. During the prechemotherapy cycle of IL-6, a dose-dependent increase in platelet count was observed from day 12 to 15 and was maximal on day 15 (r = .77; P < .01). The median ploidy of bone marrow megakaryocytes shifted from 16 N to 32 N after 7 days of the initial prechemotherapy IL-6 administration. Dose-dependent increases in C-reactive protein (CRP) and fibrinogen levels were observed on day 8 (P < .0001 for both). A significant decrease in hemoglobin level occured rapidly after initiation of IL-6 therapy and was maximal on day 8 (P < .001). When given after chemotherapy, IL-6 accelerated platelet recovery after chemotherapy cycles 2 to 6. Postponements of scheduled chemotherapy due to thrombocytopenia were less frequent in patients treated with IL-6. No difference in either neutrophils or peripheral blood progenitor assays was observed during or after IL-6 treatment. Toxicity of IL-6 appeared mild and was not dose-limiting up to 10 micrograms/kg/d. Systemic symptoms such as fever, headache, and myalgia were the main side effects and were easily relieved by acetaminophen administration. No biologic toxicity was observed. The data indicate that IL-6 is a well-tolerated cytokine and capable of accelerating platelet recovery in patients receiving chemotherapy.
Unrelated bone marrow donor transplants for children with leukemia or myelodysplasia
Authors: Casper J , Camitta B , Truitt R , Baxter-Lowe LA , Bunin N , Lawton C , Murray K , Hunter J , Pietryga D , Garbrecht F
Published: October 2019

Allogeneic bone marrow transplantation is the treatment of choice for many childhood leukemias. The donor of choice-an HLA matched sibling-is only available about 30% of the time. Unrelated donors are an alternative choice. In this report, we describe the results of unrelated donor bone marrow transplants (BMT) in 50 children with leukemia (25 acute lymphoblastic leukemia [ALL], 3 acute myeloid leukemia [AML], 3 juvenile chronic myelogenous leukemia [JCML], 10 chronic myeloid leukemia [CML]) or myelodysplastic syndrome (MDS; 9). The median age of the 31 male and 19 female patients was 9 years (range 2 to 18). Only 13 patients were serologically matched at HLA-A, B, DR, and DQ with their donors; 6 of these were reactive in mixed lymphocyte culture. The other 37 patients were mismatched for one (36 patients) or more (1 patient) HLA antigens. Pretransplant conditioning included cytosine arabinoside, cyclophosphamide, fractionated total body irradiation (TBI) (with lung, liver, and more recently, kidney shielding), and methylprednisolone. High-risk patients also received busulfan. Graft-versus-host disease (GVHD) prophylaxis consisted of T- cell depletion with IgM monoclonal antibody T10B9 plus complement and posttransplant cyclosporine-A. Forty-nine patients (98%) engrafted. Median times to greater than 500 polymorphonuclear leukocytes (PMN)/microL and greater than 25,000 platelets/microL were 18 and 20 days, respectively. Acute GVHD > or = grade II occurred in 16 patients (33%); 13 (81%) of these died. Chronic GVHD developed in 30 of 40 patients at risk, but was extensive in only 5. Event-free survival (EFS) for all patients was 44% +/- 7% (median follow-up was 49 months), and overall survival was 50 +/- 7%. Patients with low-risk disease (ALL or AML in first or second remission and CML in chronic phase) had a better EFS than children with high-risk disease (60% v 34%, P = .07). There was no significant difference in EFS between patients who were serologically matched with their donors (46%) and those who were partially mismatched (43%) (P = .97). These data compare favorably with published reports for children transplanted with HLA-matched sibling donors and should encourage earlier consideration of unrelated donor BMT in children with leukemia or myelodysplasia.
Sphingosine-1-phosphate: a platelet-activating sphingolipid released from agonist-stimulated human platelets
Authors: Yatomi Y , Ruan F , Hakomori S , Igarashi Y
Published: October 2019

Sphingosine-1-phosphate (Sph-1-P) is the initial product of catabolism of sphingosine by sphingosine kinase and is cleaved by Sph-1-P lyase to a fatty aldehyde and ethanolamine phosphate. This phosphorylated sphingoid base is not only an intermediary catabolite, but also a bioactive lipid with important functions, including stimulation of cell proliferation in Swiss 3T3 fibroblasts and inhibition of tumor cell motility. In the present study, we examined functional roles of Sph-1-P in human platelets. Sph-1-P induced platelet shape change and aggregation reactions, although it failed to elicit secretion. Sphingosine, ceramide, sphingomyelin, and N,N-dimethylsphingosine did not mimic the positive effects of Sph-1-P on platelets. Subthreshold concentrations of Sph-1-P and weak platelet agonists such as adenosine diphosphate (ADP) and epinephrine synergistically elicited aggregation, which may be important for efficient amplification of platelet activation. Sph-1-P induced intracellular Ca2+ mobilization and the dose-response for Ca2+ release correlated closely with the concentration required for induction of shape change. On addition of [3H]sphingosine to intact platelets, the label was rapidly converted to Sph-1-P, and subsequently to ceramide and sphingomyelin. Interestingly, the Sph-1-P formed was specifically released into medium on stimulation of platelets with physiologic agonists. The amount of Sph-1-P in platelets, as measured by its conversion into radiolabeled N-acetyl-Sph- 1-P, was 1.4 nmol/10(9) cells and was about four times higher than the mass of Sph present. When compared by mole percent Sph-1- P/phospholipid, the value for platelets is over 10 times higher than that for neutrophils. Our results suggest that Sph-1-P, rapidly converted from sphingosine, abundantly stored in platelets, and released on the cell activation, may play a physiologic role in thrombosis, hemostasis, and the natural wound-healing processes.
Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and Shc in human blood platelets
Authors: Miyakawa Y , Oda A , Druker BJ , Kato T , Miyazaki H , Handa M , Ikeda Y
Published: October 2019

A cDNA for the thrombopoietin has been cloned by several groups. The recombinant thrombopoietin has been reported to stimulate the megakaryocytopoiesis and thrombopoiesis. Little is known regarding the molecular basis of its effects. To elucidate the molecular mechanism involved in signal transduction, we have investigated the effects of thrombopoietin on platelet tyrosine phosphorylation. We report here that thrombopoietin induced time- and dose-dependent tyrosine phosphorylation of several proteins including Janus kinase 2 (Jak2) and a 52-kD protein, Shc, in human blood platelets. Both Jak2 and Shc were tyrosine phosphorylated within 15 seconds after stimulation. The tyrosine phosphorylation of Jak2 was accompanied by increased kinase activity, whereas Shc tyrosine phosphorylation induced its association with a 25-kD protein, Grb2. Thus, our data suggest that Jak2, Shc, and Grb2 may be involved in signal transduction after ligand binding to c- mpl in human platelets.
Th1 and Th2 T-helper cells exert opposite regulatory effects on procoagulant activity and tissue factor production by human monocytes
Authors: Del Prete G , De Carli M , Lammel RM , D'Elios MM , Daniel KC , Giusti B , Abbate R , Romagnani S
Published: October 2019

The role of T-cell subsets in the induction of tissue factor (TF) production by human monocytes in vitro was investigated. Mitogen stimulation enabled both unfractionated T cells and their CD4+ or CD8+ subsets to promote procoagulant activity (PCA). After mitogen or antigen activation, all seven T-cell clones with Th1 cytokine profile, but none of seven Th2 clones, induced TF production and PCA. T-cell blasts from four Th1 activated clones were fixed with paraformaldehyde and added to monocytes in the presence of medium alone or their supernatants. Addition of either fixed Th1 cells or their supernatants induced low TF production (0.2 to 0.6 ng/mL), whereas addition of both resulted in much higher TF synthesis (1.8 to 3.4 ng/mL). Among Th1-type cytokines, only interferon-gamma (IFN-gamma) induced minimal TF production (0.1 to 0.4 ng/mL). No TF synthesis was induced by activated and fixed Th2 cells and/or their supernatants, whereas combined addition of fixed Th2 cells and Th1 supernatants or IFN-gamma induced noticeable TF production. The addition of either anti-IFN-gamma antibody or Th2 supernatants to monocytes stimulated with activated and fixed Th1 cells plus their supernatant resulted in a dose-dependent inhibition of TF synthesis, which was partially restored by neutralization of interleukin-4 (IL-4) or IL-10. Addition of recombinant IL-4, IL-13, or IL-10, but not IL-5, inhibited the Th1- induced TF production by monocytes. Data indicate that both CD8+ and CD4+ Th1, but not Th2, T cells can help TF production and PCA. Both cell-to-cell contact with activated T cells and Th1-type cytokines, in particular IFN-gamma, are required for optimal TF synthesis, whereas Th2-derived cytokines (IL-4, IL-13, and IL-10) are inhibitory. This may be of potential interest for future therapeutic strategies.
Increased levels of oxidized glutathione in CD4+ lymphocytes associated with disturbed intracellular redox balance in human immunodeficiency virus type 1 infection
Authors: Aukrust P , Svardal AM , Muller F , Lunden B , Berge RK , Ueland PM , Froland SS
Published: October 2019

We investigated the intracellular glutathione redox status in isolated lymphocyte subpopulations and monocytes in patients with human immunodeficiency virus type 1 (HIV-1) infection and in healthy controls. CD4+ lymphocytes from HIV-1-infected patients were primarily characterized by a substantial increase in oxidized glutathione levels and a considerable decrease in the ratio of reduced to total glutathione, in most cases below 0.5 in patients with symptomatic HIV-1 infection, rather than decreased levels of reduced glutathione. The increase in oxidized glutathione was strongly correlated with low numbers of CD4+ lymphocytes in peripheral blood and impaired stimulated interleukin-2 production and proliferation in peripheral blood mononuclear cells, which is compatible with an immunopathogenic role for these redox disturbances. The HIV-1-infected patients with the most advanced clinical and immunologic disease were also characterized by an increase in levels of reduced glutathione in monocytes, suggesting that the glutathione redox cycle may be differentially regulated in CD4+ lymphocytes and monocytes. We could not confirm previous reports suggesting cysteine deficiency as a major cause of disturbed glutathione homeostasis during HIV-1 infection. The demonstrated glutathione abnormalities were correlated with raised serum levels of tumor necrosis factor alpha. These findings suggest that a therapeutical approach, which can restore the glutathione redox dysbalance in CD4+ lymphocytes and decrease the inflammatory stress, may be worthwhile exploring in HIV-1 infection.
RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB- MYH11 transcripts
Authors: van der Reijden BA , Lombardo M , Dauwerse HG , Giles RH , Muhlematter D , Bellomo MJ , Wessels HW , Beverstock GC , van Ommen GJ , Hagemeijer A
Published: October 2019

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT- PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.