bluuebunny/crossref_metadata_2025 huggingface dataset:
The impact of three-dimensional structure on the expression of PlA alloantigens on human integrin beta 3
Authors: Honda S , Honda Y , Bauer B , Ruan C , Kunicki TJ
Published: October 2019

We have compared the binding of affinity-purified anti-PlA1 IgG from seven nonrelated donors with chimeric integrin subunit beta 3 molecules expressed in the baculovirus-Spodoptera frugiperda insect cell system. Beta 3 chimeras were engineered to include segments of antigenic human beta 3 sequences spliced to intervening segments of nonantigenic Xenopus beta 3 sequence. Our results clearly show that antibodies from all seven donors will bind to nondenatured molecules containing the antigenic human beta 3 Cys26-Cys38 loop only when it is presented in a correct orientation that must be maintained by noncontiguous human sequences. Key downstream sequences are located within the region beta 3(288–490), flanking either side of the putative long-range disulfide at Cys435. Although our results confirm unambiguously that the Leu/Pro polymorphism at position 33 in human beta 3 is necessary for the expression of PlA epitopes, they also indicate that this polymorphic sequence alone is not sufficient. The requirement for additional human beta 3 sequence transcends the need to maintain a correct orientation within the Cys26-Cys38 loop itself, because the murine monoclonal antibody SZ21, which recognizes the sequence beta 3(28–35) contained within the Cys26-Cys38 loop, binds to all chimeras containing this loop, even if the same chimeras are not recognized by anti-PlA1. Our results indicate that additional noncontiguous residues encompassed by the sequence 288–490 either directly contribute to the composition of the PlA1 epitope or, more likely, maintain the Cys26-Cys38 loop in a proper orientation with respect to the remainder of the beta 3 molecule and thereby maintain proper antigenic presentation of the sequences in that loop.
Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture
Authors: Guerriero R , Testa U , Gabbianelli M , Mattia G , Montesoro E , Macioce G , Pace A , Ziegler B , Hassan HJ , Peschle C
Published: October 2019

Cellular and molecular analysis of megakaryocytopoiesis has been hampered thus far by the lack of pure and abundant megakaryocyte (MK) cell populations. In this study, hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood, were induced to megakaryocytic differentiation/maturation in serum-free liquid suspension culture treated with a growth factor cocktail (interleukin-3 [IL-3], c-kit ligand, and IL-6) and/or recombinant mpl ligand (mpIL). In particular, (1) the growth factor cocktail induced the growth of a 40% MK population, ie, 4 x 10(4) cells at day 0 generated 2 x 10(5) MK at terminal maturation; (2) further addition of mpIL increased the MK purity level to 80% with a final yield of 4 x 10(5) MKs; (3) treatment with mpIL alone resulted in a 97% to 99% MK population, with a mild increase of cell number (to 1.5 x 10(5) cells). In mpIL-supplemented culture, morphological evaluation indicated the presence of putative mononuclear MK precursors and then of mature polynucleated platelet- forming MKs, peaking at days 5 and 12, respectively. Membrane phenotype analysis showed a gradual decrease of CD34+ HPCs, coupled with an inverse increase of MK-specific antigens (eg, CD61/62/42b) starting before mature MK detection by morphology analysis. In situ hybridization showed the expression of MK-specific von Willebrand gene in both MK precursors and mature MKs. Furthermore, MKs synthesize and secrete low but significant amounts of both IL-6 and granulocyte- macrophage colony-stimulating factor. Comparative culture studies were performed on purified bone marrow CD34+/38hi or CD34+/38lo cells stimulated by mpIL alone. Both populations generated a highly enriched MK progeny (62% and 93% MKs at day 12 of culture, respectively) but showed either little or no proliferation. In conclusion, the purified peripheral blood HPC differentiation culture system allows for growth of a relatively large number of highly purified or “pure” megakaryocytic precursors and then mature MKs, thus providing an in vitro experimental tool to dissect the cellular and molecular basis of megakaryocytopoiesis.
Changing the differentiation program of hematopoietic cells: retinoic acid-induced shift of eosinophil-committed cells to neutrophils
Authors: Paul CC , Mahrer S , Tolbert M , Elbert BL , Wong I , Ackerman SJ , Baumann MA
Published: October 2019

The mechanisms by which hematopoietic progenitor cells become lineage- committed remain poorly understood. A cloned subline of the AML14 cell line (AML14.3D10) that spontaneously differentiates to eosinophilic myelocytes in the absence of cytokine stimulation was obtained by limiting dilution. This subline exhibits augmented expression of interleukin-5 (IL-5) receptor alpha subunit mRNA and synthesizes all major eosinophil granule proteins. Exposure of this cell line to all- trans retinoic acid (ATRA) causes loss of eosinophilic granules and fast green staining within 48 hours, without cell death. In addition, mRNA for the IL-5 receptor alpha subunit becomes undetectable by 48 hours and the cells lose responsiveness to IL-5. Major basic protein, measured as a marker of eosinophilic granule content, decreases from more than 16 pg/cell to undetectable levels by 5 days after ATRA. Concomitant with the loss of major basic protein and fast green staining, surface expression of CD16 becomes detectable and is maximum by 10 days after ATRA. mRNA for the granulocyte colony-stimulating factor (G-CSF) receptor becomes detectable by day 5, and the cells become responsive to G-CSF. At this time, the cells appear morphologically as mature neutrophils and can reduce nitroblue tetrazolium. With continued culture, the neutrophilic cells die and the culture becomes repopulated with eosinophilic myelocytes. These findings show that it is possible to change the differentiation program of hematopoietic cells even after they show evidence of advanced lineage commitment. The AML14.3D10 subclone of AML14 will be a valuable model for study of the transcriptional regulation of the eosinophil and neutrophil differentiation programs and lineage-specific gene expression.
The Wiskott-Aldrich syndrome and X-linked congenital thrombocytopenia are caused by mutations of the same gene
Authors: Zhu Q , Zhang M , Blaese RM , Derry JM , Junker A , Francke U , Chen SH , Ochs HD
Published: October 2019

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, small platelets, eczema, recurrent infections, and immunodeficiency. Besides the classic WAS phenotype, there is a group of patients with congenital X-linked thrombocytopenia (XLT) who have small platelets but only transient eczema, if any, and minimal immune deficiency. Because the gene responsible for WAS has been sequenced, it was possible to correlate the WAS phenotypes with WAS gene mutations. Using a fingerprinting screening technique, we determined the approximate location of the mutation in 13 unrelated WAS patients with mild to severe clinical symptoms. Direct sequence analysis of cDNA and genomic DNA obtained from patient-derived cell lines showed 12 unique mutations distributed throughout the WAS gene, including insertions, deletions, and point mutations resulting in amino acid substitutions, termination, exon skipping, or splicing defects. Of 4 unrelated patients with the XLT phenotype, 3 had missense mutations affecting exon 2 and 1 had a splice-site mutation affecting exon 9. Patients with classic WAS had more complex mutations, resulting in termination codons, frameshift, and early termination. These findings provide direct evidence that XLT and WAS are caused by mutations of the same gene and suggest that severe clinical phenotypes are associated with complex mutations.
Role of collagen-adherent platelets in mediating fibrin formation in flowing whole blood
Authors: Kirchhofer D , Tschopp TB , Steiner B , Baumgartner HR
Published: October 2019

Activated platelets provide assembly sites for coagulation enzyme complexes and in this way can mediate coagulation during hemostasis and thrombosis. In this study, we examined the procoagulant activity of platelets adhering directly to fibrillar collagen, a main thrombogenic constituent of subendothelium. For this purpose, we used a human ex- vivo thrombosis model in which collagen-coated coverslips were exposed to flowing nonanticoagulated blood (shear rate, 65/s) for 5.5 minutes, which led to the deposition of adherent platelets, platelet thrombi, and fibrin. To examine the procoagulant activity of adherent platelets only, a selective antagonist of the platelet GPIIb-IIIa complex, Ro 44- 9883, was infused via a mixing device, resulting in a complete abrogation of platelet thrombus formation but leaving the collagen- adherent platelet layer intact. This platelet layer generated increased postchamber fibrinopeptide A (FPA) levels (203 +/- 33 ng/mL) as compared with control experiments without infusion of inhibitor (95 +/- 13 ng/mL). Concomitantly, fibrin deposition measured by morphometric analysis of cross-sections was also increased, as was the platelet adhesion to collagen. An immunochemical staining of fibrin fibers further showed that the adherent platelets formed the nuclei for fibrin fiber formation. This increase in fibrin deposition was mediated by the intrinsic factor X (F.X) activation complex on adherent single platelets, because almost complete inhibition of FPA generation (9 ng/mL) and fibrin deposition (0.4% +/- 0.2% coverage) was achieved upon coinfusion of the GP IIb-IIIa antagonist and active site-inhibited F.IXa. The large platelet thrombi that were deposited in control experiments contained no significant amounts of immunodetectable fibrin except at the thrombus base, where adherent platelets anchored the thrombi to the collagen surface. These results suggest that the collagen-adherent platelets are important promoters of coagulation during the initial phase of thrombogenesis by providing assembly sites for the F.X activation complex.
The major histocompatibility complex region marked by HSP70-1 and HSP70- 2 variants is associated with clozapine-induced agranulocytosis in two different ethnic groups
Authors: Corzo D , Yunis JJ , Salazar M , Lieberman JA , Howard A , Awdeh Z , Alper CA , Yunis EJ
Published: October 2019

Genes of the major histocompatibility complex (MHC) have been associated with susceptibility to drug-induced adverse reactions. We previously found that clozapine-induced agranulocytosis (CA) is associated with the HLA-DRB1*0402, DRB4*0101, DQB1*0302, DQA1*0301 haplotype in Ashkenazi Jewish patients and with the HLA-DRB1*1601, DRB5*02, DQB1*0502, DQA1*0102 haplotype in non-Jewish patients. In the present study, we tested the hypothesis that the variants of the heat- shock protein 70 (HSP-70) encoded by the HSP-70 loci located within the MHC region and known to be involved in apoptosis and regulation of cell proliferation could play an important role in molecular mechanisms of CA. First, we analyzed HSP70–2 polymorphism in risk-associated haplotypes from HLA homozygous cells and normal individuals and confirmed that the HSP70–2 9-kb variant was associated invariably with DR4 (HLA-DRB1*0402, DQB1*0302) and DR2 (HLA-DRB1*01601, DQB1*0502, DQA1*0102 and HLA-DRB1*1501, DQB1*0602) haplotypes, which were the haplotypes found increased in Jewish and non-Jewish patients with CA, respectively. The 9.0-kb variant was also found to be associated with HLA-B44, DRB1*0401 and HLA-B44, DRB1*07 haplotypes. Second, in patients with CA (12 Ashkenazi Jewish and 20 non-Jewish patients), HSP70–1 A and HSP70–2 9.0-kb variants were associated with the MHC haplotypes found by us to be markers of susceptibility to CA. The clozapine-treated control group had an excess number of HSP70–1 C and HSP70–2 8.5-kb variants, consistent with genetic resistance to CA associated with those variants. This finding supports our hypothesis that a dominant gene within the MHC region (marked by HSP70–1 and HSP70–2), but not necessarily HLA, is associated with CA in two different ethnic groups.
Fas receptor (CD95)-mediated apoptosis is induced in leukemic cells entering G1B compartment of the cell cycle
Authors: Komada Y , Zhou YW , Zhang XL , Xue HL , Sakai H , Tanaka S , Sakatoku H , Sakurai M
Published: October 2019

Apoptotic cell death induced by cross-linking Fas receptor (FasR/CD95) has been investigated in human acute myelogenous leukemia (AML) cells. FasR-mediated growth inhibition and DNA fragmentation could be induced in certain cases of AML. Interestingly, when DNA synthesis and G1 -> S transition in the cell cycle were enhanced by interleukin-3 or granulocyte-macrophage colony-stimulating factor, Fas-insensitive blast cells acquired cellular susceptibility toward FasR-mediated growth inhibition. To further evaluate an association between the Fas-R- mediated action and a specific phase of the cell cycle, a FasR+ leukemic cell line, MML-1, was established from a patient with AML. The morphologic feature of dying cells and DNA fragmentation indicated that FasR cross-linking induced apoptotic cell death in MML-1 cells. Cell cycle arrest in G1A phase with the treatment of phorbol 12-myristate 13- acetate or thymidine rendered MML-1 cells resistant to FasR-mediated apoptosis without downregulation of surface FasR expression. However, S- phase arrest with 5-fluorouracil could neither enhance nor inhibit FasR- mediated apoptosis. Simultaneous DNA/RNA quantification analysis revealed the selective loss of cells in G1B compartment, accompanied by the increase of apoptotic nuclei in sub-G1 fraction. These findings suggested that FasR-mediated apoptotic signals could be transduced into cells in G1B compartment and G1A -> G1B transition might augment the induction of FasR-mediated apoptosis.
In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia
Authors: Klumper E , Pieters R , Veerman AJ , Huismans DR , Loonen AH , Hahlen K , Kaspers GJ , van Wering ER , Hartmann R , Henze G
Published: October 2019

Cellular drug resistance is thought to be an important cause of the poor prognosis for children with relapsed or refractory acute lymphoblastic leukemia (ALL), but it is unknown when, to which drugs, and to what extent resistance is present. We determined in vitro resistance to 13 drugs with the MTT assay. Compared with 141 children with initial ALL, cells from 137 children with relapsed ALL were significantly more resistant to glucocorticoids, L-asparaginase, anthracyclines, and thiopurines, but not to vinca-alkaloids, cytarabine, ifosfamide, and epipodophyllotoxins. Relapsed ALL cells expressed the highest level of resistance to glucocorticoids, with a median level 357- and >24-fold more resistant to prednisolone and dexamethasone, respectively, than initial ALL cells, whereas the resistance ratios for the other drugs differed from 0.8- to 1.9-fold, intraindividual comparisons between initial and relapsed samples from 16 children with ALL showed that both de novo and acquired drug resistance were involved. Specific in vitro drug-resistance profiles were associated with high-risk relapsed ALL groups. In vitro drug resistance was also related to the clinical response to chemotherapy in relapsed/refractory childhood ALL. We conclude that drug resistance may explain the poor prognosis for children with relapsed/refractory ALL. These day may be helpful to design alternative treatment regimens for relapsed childhood ALL.
The instability of the membrane skeleton in thalassemic red blood cells
Authors: Yuan J , Bunyaratvej A , Fucharoen S , Fung C , Shinar E , Schrier SL
Published: October 2019

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta- thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha- globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.
Radioimmunotherapy of interleukin-2R alpha-expressing adult T-cell leukemia with Yttrium-90-labeled anti-Tac [see comments]
Authors: Waldmann TA , White JD , Carrasquillo JA , Reynolds JC , Paik CH , Gansow OA , Brechbiel MW , Jaffe ES , Fleisher TA , Goldman CK
Published: October 2019

Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotropic virus-I. It is an aggressive leukemia with a median survival time of 9 months; no chemotherapy regimen appears successful in inducing long-term disease- free survival. The scientific basis of the present study is that ATL cells express high-affinity interleukin-2 receptors identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference, we administered anti-Tac armed with Yttrium-90 (90Y) to 18 patients with ATL initially (first 9 patients) in a phase I dose-escalation trial and subsequently (second group of 9 patients) in a phase II trial involving a uniform 10-mCi dose of 90Y-labeled anti- Tac. Patients undergoing a remission were permitted to receive up to eight additional doses. At the 5- to 15-mCi doses used, 9 of 16 evaluable patients responded to 90Y anti-Tac with a partial (7 patients) or complete (2 patients) remission. The responses observed represent improved efficacy in terms of length of remission when compared with previous results with unmodified anti-Tac. Clinically meaningful (> or = grade 3) toxicity was largely limited to the hematopoietic system. In conclusion, radioimmunotherapy with 90Y anti- Tac directed toward the IL-2R expressed on ATL cells may provide a useful approach for treatment of this aggressive malignancy.
Aggregation of mammalian cells expressing the platelet glycoprotein (GP) Ib-IX complex and the requirement for tyrosine sulfation of GP Ib alpha
Authors: Dong JF , Hyun W , Lopez JA
Published: October 2019

The glycoprotein (GP) Ib-IX complex mediates platelet aggregation in response to high shear forces by binding von Willebrand factor (vWF) in the plasma. We investigated the possibility that the complex could mediate a similar phenomenon if expressed in nonhematopoietic cells. When agitated on a tabletop shaker, CHO and L cells expressing the full complex formed large aggregates in the presence of vWF and the modulator ristocetin. When the rate of agitation was increased, aggregation occurred without added ristocetin and appeared to require only the application of a physical force. The aggregation was homophilic and temperature-dependent and required a functional ligand- binding subunit of the GP Ib-IX complex, GP Ib alpha. Posttranslational tyrosine sulfation of GP Ib alpha was required for aggregate formation and stability. Thus, aggregation of cells expressing the GP Ib-IX complex is a unique example of a ligand-receptor interaction induced by mechanical forces and demonstrates an important biological role for sulfation of tyrosine residues.
The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction
Authors: Wang LM , Michieli P , Lie WR , Liu F , Lee CC , Minty A , Sun XJ , Levine A , White MF , Pierce JH
Published: October 2019

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3- dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin- like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL- 13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.
Evolutionarily conserved alternative pre-mRNA splicing regulates structure and function of the spectrin-actin binding domain of erythroid protein 4.1
Authors: Winardi R , Discher D , Kelley C , Zon L , Mays K , Mohandas N , Conboy JG
Published: October 2019

A developmental alternative splicing switch, involving exon 16 of protein 4.1 pre-mRNA, occurs during mammalian erythropoiesis. By controlling expression of a 21-amino acid peptide required for high- affinity interaction of protein 4.1 with spectrin and actin, this switch helps to regulate erythrocyte membrane mechanical stability. Here we show that key aspects of protein 4.1 structure and function are conserved in nucleated erythroid cells of the amphibian Xenopus laevis. Analysis of protein 4.1 cDNA sequences cloned from Xenopus erythrocytes and oocytes showed that tissue-specific alternative splicing of exon 16 also occurs in frogs. Importantly, functional studies with recombinant Xenopus erythroid 4.1 demonstrated specific binding to and mechanical stabilization of 4.1-deficient human erythrocyte membranes. Phylogenetic sequence comparison showed two evolutionarily conserved peptides that represent candidate spectrin-actin binding sites. Finally, in situ hybridization of early embryos showed high expression of 4.1 mRNA in ventral blood islands and in developing brain structures. These results demonstrate that regulated expression of structurally and functionally distinct protein 4.1 isoforms, mediated by tissue-specific alternative splicing, has been highly evolutionarily conserved. Moreover, both nucleated amphibian erythrocytes and their enucleated mammalian counterparts express 4.1 isoforms functionally competent for spectrin-actin binding.
Therapy for ongoing graft-versus-host disease induced across the major or minor histocompatibility barrier in mice with anti-CD3F(ab')2-ricin toxin A chain immunotoxin
Authors: Vallera DA , Taylor PA , Panoskaltsis-Mortari A , Blazar BR
Published: October 2019

A new pharmacologic agent, anti-CD3F(ab')2-ricin toxin A chain (RTA), was synthesized for the purpose of targeting T cells and as a means of treating established graft-versus-host disease (GVHD). The Fc region of anti-CD3 monoclonal antibody (MoAb) was removed to prevent its ability to activate T cells. The resulting F(ab')2 fragments were conjugated to deglycosylated RTA (dgRTA), a catalytic and potent phytotoxin. The resulting immunotoxin (IT) was potent (greater than 95% inhibition) and selective in inhibiting T-cell mitogenesis in vitro. In vivo, the IT depleted 80% of T cells in mice receiving bone marrow (BM) transplants. Transplantation in an aggressive acute GVHD model using C57BL/6 donor cells and H-2 disparate B10.BR recipients resulted in an infiltration of CD3-expressing cells and a median survival time (MST) of 20 to 30 days. A 5-day course of anti-CD3F(ab')2-RTA (30 micrograms/d intraperitoneally) beginning 7 days after GVHD induction was beneficial in treating established GVHD in these mice, as evidenced by significantly prolonged survival (MST, greater than 80 days), superior mean weight values, and improved clinical appearance. Neither intact anti-CD3, unconjugated anti-CD3 F(ab')2 fragments, nor a mixture of anti-CD4 and anti-CD8 MoAbs (which are highly effective in prophylactic models) were as effective. F(ab')2 fragments made from anti-Lyt-1 (the murine homologue of human anti-CD5) linked to RTA were also not effective, despite the fact that both anti-CD3F(ab')2-RTA and anti-Lyt- 1F(ab')2-RTA had similar half-lives of about 9 hours. The IT also increased MST in two aggressive models of GVHD across non-H-2 minor histocompatibility barriers, indicating that the usefulness of anti- CD3F(ab')2-dgRTA is not limited to a single-strain combination. This agent should be further investigated as an alternative to current strategies for treating steroid refractory GVHD.
DLA-identical bone marrow grafts after low-dose total body irradiation: effects of high-dose corticosteroids and cyclosporine on engraftment
Authors: Yu C , Storb R , Mathey B , Deeg HJ , Schuening FG , Graham TC , Seidel K , Burnett R , Wagner JL , Shulman H
Published: October 2019

Previous studies found that marrow allografts from DLA-identical littermates resulted in survival of 60% of recipient dogs after an otherwise lethal dose of 450 cGy of total body irradiation (TBI), either because of successful allografts or autologous recovery after rejection of the allografts. Forty percent of dogs died with marrow aplasia after allograft rejection. The current study asked whether allogeneic engraftment could be enhanced and survival improved by treating allograft recipients with high doses of corticosteroids or with cyclosporine (CSP), administered either before or after transplantation. Five dogs in group 1 received corticosteroids beginning on day -5 and ending on day 32 after transplant. The starting dose was 12.5 mg of prednisone per kilogram orally twice daily. All five dogs rejected their allografts; three died early with marrow aplasia and two showed endogenous marrow recovery. Nine dogs received CSP from day -6 to day -1 before transplantation at a dose of 20 mg/kg/d intravenously administered in divided doses. All nine dogs rejected the marrow allograft; six died with marrow aplasia and three survived with endogenous marrow recovery. Seven dogs received CSP after transplantation at a dose of 30 mg/kg/d orally from day -1 to day 35. All seven had sustained allografts (two mixed chimeras and five complete donor-type chimeras) and became healthy long-term survivors without graft-versus-host disease. These results extend previous observations and confirm that grafts of marrow from DLA-identical littermates improved survival of dogs exposed to low but otherwise lethal doses of TBI. Additional therapy with high-dose corticosteroids administered peritransplantation and posttransplantation or CSP administered before transplantation neither enhanced the rate of allogeneic engraftment nor improved survival; however, CSP administered after transplantation resulted in successful allografts and event-free survival in all cases.
Leukocyte rolling in vivo is mediated by P-selectin glycoprotein ligand- 1
Authors: Norman KE , Moore KL , McEver RP , Ley K
Published: October 2019

Leukocyte rolling, an early and important step in the inflammatory response, is mediated by the selectin family of adhesion molecules. The selectins bind with low affinity to sialylated and fucosylated glycans such as sialyl Lewisx (sLex), but bind with high affinity to only a few specific glycoproteins on cell surfaces. One such glycoprotein is P- selectin glycoprotein ligand-1 (PSGL-1). The relative contributions of low- and high-affinity ligands to leukocyte rolling in vivo are unknown. We show here that a monoclonal antibody to PSGL-1 (PL1) dramatically reduces rolling of human polymorphonuclear neutrophils (PMN) and promyelocytic HL-60 cells in venules of acutely exteriorized rat mesentery. Control PMN and HL-60 cell rolling flux fractions were 39% +/- 3% and 33% +/- 5%, which were reduced by PL1 to 7% +/- 2% and 6% +/- 2%, respectively. Similar reductions were seen with F(ab) fragments of PL1. PL1-treated PMN rolled at significantly higher mean velocities than untreated PMN owing to intermittent rather that continuous interactions. These findings show that interaction of P- selectin with PSGL-1 is required for rolling of myeloid cells in mesenteric venules at physiologic shear stress in vivo.
Characterization of hematopoietic progenitors from human yolk sacs and embryos
Authors: Huyhn A , Dommergues M , Izac B , Croisille L , Katz A , Vainchenker W , Coulombel L
Published: October 2019

Hematopoiesis first arises in the extraembryonic yolk sac, and it is generally believed that yolk sac-derived stem cells migrate and seed the fetal liver at approximately week 6 of development in humans. Recently, the identification at day 8.5 to 9 of multipotential stem cells in intraembryonic sites different from the liver suggests that the establishment of hematopoiesis might be more complex than initially believed. In an attempt to understand initial steps of hematopoiesis during human ontogeny, we characterized clonogenic myeloid progenitor cells in human yolk sacs and corresponding embryos at 25 to 50 days of development. Most erythroid colonies derived from the yolk sacs differed from adult marrow-derived progenitors in that they also contained cells of the granulomacrophagic lineage, suggesting that they were pluripotent and exhibited a different response to cytokines. Furthermore, a subclass of nonerythroid progenitors generated very large granulomacrophagic colonies, some of which generated secondary erythroid colonies on replating. Analysis of the distribution of progenitors revealed that in contrast to erythroid progenitors, whose numbers were equally distributed between the yolk sac and the embryo, 80% of the nonerythroid progenitors were found in the embryo at stages II and III. Interestingly, a high proportion of nonerythroid progenitors (including high proliferative potential cells) was present in colony assays initiated with cells remaining after the liver has been removed. These findings were validated in colony assays established with CD34+ cells purified from extraembryonic yolk sacs and intraembryonic tissues. Increased knowledge about the biology of hematopoietic stem cells early in life may help to further understanding of the mechanisms associated with the restriction in proliferative and differentiative potential observed in the adult hematopoietic stem cell compartment.
The protein tyrosine kinase JAK2 is activated in neutrophils from patients with severe congenital neutropenia
Authors: Rauprich P , Kasper B , Tidow N , Welte K
Published: October 2019

Severe congenital neutropenia (SCN; or Kostmann syndrome) is an autosomal recessive disorder characterized by a maturation arrest of myelopoiesis at the level of promyelocytes. Myeloid precursor cells from patients with SCN require pharmacological dosages of recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF; Filgrastim; Amgen, Thousand Oaks, CA) to differentiate to normal neutrophils. Thus, it is hypothesized that the underlying defect responsible for SCN is based on an abnormal G-CSF-induced signal transduction pathway. Because JAK2, a nonreceptor tyrosine kinase, is involved in the signaling pathway of G-CSF, we examined the expression and activity of JAK2 in neutrophils from SCN patients during r-metHuG-CSF treatment. The immunoprecipitated JAK2 protein showed increased tyrosine phosphorylation in neutrophils from SCN patients as compared with that in neutrophils from healthy donors, suggesting that this kinase is activated. In vitro kinase assays of immunoprecipitated JAK2 confirmed that neutrophils from SCN patients show an increased autophosphorylation of JAK2 in comparison with that of neutrophils from healthy volunteers. These findings suggest that JAK2 is activated in SCN patients.
Verotoxin-1 promotes leukocyte adhesion to cultured endothelial cells under physiologic flow conditions
Authors: Morigi M , Micheletti G , Figliuzzi M , Imberti B , Karmali MA , Remuzzi A , Remuzzi G , Zoja C
Published: October 2019

Hemolytic uremic syndrome (HUS), which is the most common cause of acute renal failure in infants and small children, is caused by verotoxin (VT)-producing Escherichia coli infection. Endothelial injury determines microvascular thrombosis and evidence is available from recent studies that suggests that leukocyte activation participates in endothelial damage. We studied here the effect of VT-1 on leukocyte adhesion to vascular endothelium under physiologic flow conditions. Human umbilical vein endothelial cells (HUVECs) were incubated for 24 hours with VT-1 (0.1, 1, and 10 pmol/L) and then exposed to a total leukocyte suspension in a parallel plate flow chamber under laminar flow conditions (1.5 dynes/cm2). Adherent cells were counted by digital image processing. Results showed that VT-1 dose-dependently increased the number of adhering leukocytes to HUVECs as compared with unstimulated cells. The adhesive response elicited by VT-1 was comparable to that of interleukin-1 beta (IL-1 beta), one of the most potent inducers of endothelial cell adhesiveness. Exposure of HUVECs to VT-1 did not affect the number of rolling leukocytes, which was similar to that of control values. To examine the role of adhesion molecules in VT-1-induced leukocyte adhesion, HUVECs were incubated with mouse monoclonal antibodies against E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) before adhesion assay. Functional blocking of E-selectin, ICAM-1, and VCAM-1 on endothelial cells significantly inhibited VT-1-induced increase in leukocyte adhesion. In some experiments, before VT-1 incubation, HUVECs were pretreated for 24 hours with tumor necrosis factor alpha (TNF alpha; 100 U/mL), which is known to increase VT receptor expression on HUVECs. The number of adhering leukocytes on HUVECs exposed to TNF alpha and VT-1 significantly increased as compared with HUVECs incubated with VT-1 and TNF alpha alone. These results suggest that VT-1 modulates leukocyte-endothelium interaction, thus increasing leukocyte adhesion and upregulating adhesive proteins on endothelial surface membrane.
Relevance of marrow fibrosis in bone marrow transplantation: a retrospective analysis of engraftment
Authors: Soll E , Massumoto C , Clift RA , Buckner CD , Appelbaum FR , Storb R , Sale G , Hackman R , Martin P
Published: October 2019

A retrospective study compared posttransplant engraftment parameters in 203 patients with myelofibrosis (MF) with those in a population of 203 matched controls without MF. There were no significant differences between these groups in the proportions of patients who died without achieving engraftment and in the disease-free survival distributions. Furthermore, comparisons between the two groups of patients reaching the respective endpoints showed no differences in the time distributions for reaching 0.5 or 1.0 x 10(9)/L granulocytes, but the time to platelet transfusion independence was 3 days longer in patients with MF. In further analysis, results for 33 patients with severe MF were compared with those of their respective controls. The proportions of patients with severe MF who died without reaching these engraftment endpoints and the disease-free survival distributions in the two groups were similar. Among patients who reached the respective engraftment endpoints, there was no statistically significant difference in the pace of granulocyte recovery. In patients with severe MF, there was a 7- day delay in the time to reach platelet transfusion independence and a 2-day delay in the time to reach red blood cell independence, but the differences were not statistically significant. The present results do not substantiate concerns raised by earlier studies. MF may delay the time to reach platelet independence by approximately 3 days and may increase platelet transfusion requirements, but no other perturbation of hematopoietic reconstitution was apparent.
High expression of latent membrane protein 1 of Epstein-Barr virus and BCL-2 oncoprotein in acquired immunodeficiency syndrome-related primary brain lymphomas
Authors: Camilleri-Broet S , Davi F , Feuillard J , Bourgeois C , Seilhean D , Hauw JJ , Raphael M
Published: October 2019

Nearly all primary brain lymphomas in acquired immunodeficiency syndrome (AIDS) patients are associated withEpstein-Barr virus (EBV). The role of EBV in lymphomagenesis is not totally elucidated. One possible mechanism is the overexpression of the BCL-2 oncoprotein, because the latent membrane protein 1 (LMP1) has been reported to transactivate the bcl-2 gene in vitro. To study the interrelationship beetween LMP1 and BCL-2 in vivo, we have analyzed and compared their expression in 11 AIDS-related primary brain lymphomas and 57 AIDS- related systemic lymphomas by immunoperoxidase technique on frozen sections. In AIDS-related primary brain lymphoma, LMP1 and BCL-2 were expressed in all cases but 1. All positive cases exhibited morphologic immunoblastic features. In contrast, the only negative case was histologically close to Burkitt's lymphoma. In systemic lymphomas, LMP1 was expressed in 21 cases, whereas BCL-2 was positive in only 3 cases, all of which were extranodal. These results indicate that, in addition to the histologic type, the role of EBV genes and BCL-2 expression in lymphomatous cells differ as a function of their localization. In AIDS- related primary brain lymphomas, this correlation between LMP1 and BCL- 2 overexpression may have a major implication in lymphomagenesis.
Positive and negative regulation of granulocyte-macrophage colony- stimulating factor promoter activity by AML1-related transcription factor, PEBP2
Authors: Takahashi A , Satake M , Yamaguchi-Iwai Y , Bae SC , Lu J , Maruyama M , Zhang YW , Oka H , Arai N , Arai K
Published: October 2019

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter contains a consensus sequence for the polyomavirus enhancer binding-protein 2 (PEBP2) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM- CSF promoter. PEBP2 alpha A1, alpha B1, and alpha B2 proteins bound the PEBP2 site within the mouse GM-CSF promoter. PEBP2 alpha A1 and alpha B1 enhanced the expression of the GM-CSF promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13- acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained PEBP2 site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human PEBP2 alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of PEBP2 in the GM-CSF promoter activity, the present findings suggested the importance of the relative ratio of different PEBP2 isoforms in regulating the levels of the promoter activity.
Clinical events in the first decade in a cohort of infants with sickle cell disease. Cooperative Study of Sickle Cell Disease [see comments]
Authors: Gill FM , Sleeper LA , Weiner SJ , Brown AK , Bellevue R , Grover R , Pegelow CH , Vichinsky E
Published: October 2019

Within the Cooperative Study of Sickle Cell Disease, 694 infants with confirmed sickle cell disease were enrolled at less than 6 months of age. Information about the nature and frequency of complications was collected prospectively over a 10-year period. Painful crises and acute chest syndrome were the most common sickle cell-related events in homozygous sickle cell anemia (SS), hemoglobin SC disease (SC), and S beta thalassemia patients (overall incidence in SS patients of 32.4 and 24.5 cases per 100 person-years, respectively). Bacteremia occurred most frequently in SS children under 4 years of age and in SC patients less than 2 years of age. The mortality rate was low in this cohort compared with that found in previous reports. Twenty children, all with Hb SS, died (1.1 deaths per 100 person-years among SS patients). Infection, most commonly with Streptococcus pneumoniae and Hemophilus influenzae, caused 11 deaths. Two children died of splenic sequestration, 1 of cerebrovascular accident, and 6 of unclear causes. Two patients underwent cholecystectomies, and 17 underwent splenectomies after one or more splenic sequestration crises. The experience of this cohort should reflect closely the true clinical course of those children with Hb SS and Hb SC disease who are observed in sickle cell centers in the United States.
Iodine-mediated inactivation of lipid- and nonlipid-enveloped viruses in human antithrombin III concentrate
Authors: Highsmith F , Xue H , Chen X , Benade L , Owens J , Shanbrom E , Drohan W
Published: October 2019

Human plasma-derived protein concentrates intended for clinical use must be treated for viral inactivation to ensure patient safety. This study explored the use of liquid iodine for inactivation of several lipid- and nonlipid-enveloped viruses in an antithrombin III (AT-III) concentrate. Iodine at levels of 0.01% to 0.02% caused between 43% and 94% loss of AT-III activity, as well as degradation of AT-III as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. However, addition of up to 0.1% human albumin protected the AT-III against both inactivation and fragmentation. At albumin levels sufficient to retain greater than 75% of AT-III activity, greater than 6 logs of sindbis, encephalomyocarditis, and vesicular stomatitis viruses, greater than 4 logs of pseudorabies, and greater than 3 logs of human immunodeficiency virus were inactivated. Except with sindbis virus, this represented complete inactivation of all the viruses spiked into the AT-III concentrate.
Molecular cloning of cDNA encoding a novel platelet-endothelial cell tetra-span antigen, PETA-3
Authors: Fitter S , Tetaz TJ , Berndt MC , Ashman LK
Published: October 2019

Platelet-endothelial cell tetra-span antigen (PETA-3) was originally identified as a novel human platelet surface glycoprotein, gp27, which was detected by a monoclonal antibody (MoAb), 14A2.H1. Although this glycoprotein is present in low abundance on the platelet surface, MoAb 14A2.H1 stimulates platelet aggregation and mediator release. We now report isolation of a cDNA clone encoding PETA-3 from a library derived from the megakaryoblastic leukemia cell line MO7e. The clone encodes an open reading frame of 253 amino acids that displays 25% to 30% amino acid sequence identity with several members of the newly defined Tetraspan, or Transmembrane 4 superfamily. These proteins consist of four conserved putative transmembrane domains with a large divergent extracellular loop between the third and fourth membrane-spanning regions. PETA-3 has a single consensus sequence for N-linked glycosylation located in this extracellular loop. A single PETA-3 RNA transcript (1.6 kb) was detected in RNA isolated from MO7e cells, bone marrow stromal cells, the C11 endothelial cell line, and several myeloid leukemia cell lines. No transcript was detected in the lymphoblastoid cell lines MOLT-4 and BALM-1. This pattern correlates well with previous protein expression data. Northern blot analysis of RNA from a range of human tissues indicated that the transcript was present in most tissues, the notable exception being brain.
The effects of low molecular weight and standard heparin on calcium loss from fetal rat calvaria
Authors: Shaughnessy SG , Young E , Deschamps P , Hirsh J
Published: October 2019

Osteoporosis is a well-recognized complication of long-term heparin use. However, the mechanisms by which heparin can influence bone metabolism are unclear. We report here that unfractionated heparin stimulates the process of bone resorption and that the low molecular weight heparins (LMWHs), enoxaparin, fragmin, logiparin, and ardeparin produce significantly less calcium loss than unfractionated heparin. To assess calcium loss from bone, we quantified the release of 45Ca into the culture medium of fetal rat calvaria. 45Ca release was increased in a dose-dependent manner by the addition of either unfractionated heparin or the LMWHs; but more than 50-fold higher LMWH concentrations were required to obtain an equivalent effect to unfractionated heparin. Thus, at concentration > or = 2 micrograms/mL (0.35 anti-Xa units/mL), unfractionated heparin stimulated 45Ca release 1.53 +/- 0.06 fold. 45Ca release was increased to a similar extent by the addition of either 10(- 7) mol/L parathyroid hormone (PTH) or 10(-6) mol/L 1,25 dihydroxyvitamin D3 (1,25 Vit D3). In contrast to unfractionated heparin, LMWH concentrations > or = 100 micrograms/mL (> or = 14.0 anti- Xa units/mL) were required before maximum isotope release was observed. At concentrations well above therapeutic levels, the LMWHs stimulated 45Ca release by only 1.25 /+- 0.01-fold. Heparins with high and low antithrombin III affinities stimulated 45Ca release equally well. Both size and sulfation were found to be major determinants of heparin's ability to promote isotope release. Thus, the ability of defined heparin fragments to stimulate 45Ca release correlated with their molecular weight, and after N-desulfation the ability of heparin to induce isotope release was greatly diminished. Dermatan sulfate had no effect on 45Ca release. We conclude that size and sulfation are major determinants of heparin's ability to promote bone resorption and that the risk of heparin-induced osteoporosis may be reduced by the use of LMWH preparations.