bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental regulation of myeloid gene expression and demethylation during ex vivo culture of peripheral blood progenitor cells
Authors: Lubbert M , Brugger W , Mertelsmann R , Kanz L
Published: October 2019

Expression of tissue- and development-specific genes is coordinately regulated during maturation of hematopoietic precursor cells toward functional, end-stage peripheral blood (PB) cells. To study the expression and methylation of several myeloid-specific genes during in vitro differentiation of normal hematopoietic progenitor cells, we used a model of CD34+ selected PB progenitor cells (PBPCs). PBPCs from six patients with solid tumors were recruited by standard-dose chemotherapy and subsequent administration of recombinant granulocyte colony-stimulating factor (G-CSF). PBPCs were collected and CD34+ cells selected by immunoadsorption columns using a biotinylated anti-CD34 monoclonal antibody. Enriched cells contained between 78% and 90% (median, 84%) CD34+ cells as determined by fluorescence-activated cell sorting analysis. Cell preparations were cultured in the presence of interleukin-1 beta (IL-1 beta), IL-3, IL-6 and stem cell factor and with or without G-CSF for various time intervals up to 20 days. Genes for CD34 surface antigen, lysozyme (LZM) and myeloperoxidase (MPO) were examined by RNA and DNA analyses. A rapid and early downregulation of CD34 transcripts was observed, with concomitant, time-dependent upregulation of expression of both the LZM and MPO genes. These effects were enhanced in the presence of G-CSF. Analysis of the DNA methylation status at key sites within these genes showed a pattern of differentiation- and expression-associated demethylation of the LZM gene, which was also enhanced by G-CSF, and constitutive and unaltered demethylation at key regions of the CD34 and MPO genes. In conclusion, the genes for CD34, LZM, and MPO are regulated during in vitro culture of very immature PBPCs in the presence of stem cell factor, IL-1, IL-3, IL-6; their effects are enhanced by G-CSF.
Complete remission in severe aplastic anemia after high-dose cyclophosphamide without bone marrow transplantation
Authors: Brodsky RA , Sensenbrenner LL , Jones RJ
Published: October 2019

Severe aplastic anemia (SAA) can be successfully treated with allogeneic bone marrow transplantation (BMT) or immunosuppressive therapy. However, the majority of patients with SAA are not eligible for BMT because they lack an HLA-identical sibling. Conventional immunosuppressive therapy also has major limitations; many of its remissions are incomplete and relapse or secondary clonal disease is common. Cyclophosphamide is a potent immunosuppressive agent that is used in all BMT conditioning regimens for patients with SAA. Preliminary evidence suggested that high-dose cyclophosphamide, even without BMT, may be beneficial to patients with SAA. Therefore, 10 patients with SAA and lacking an HLA-identical sibling were treated with high-dose cyclophosphamide (45 mg/kg/d) for 4 consecutive days with or without cyclosporine. A complete response (hemoglobin level, > 13 g/dL; absolute neutrophil count, > 1.5 x 10(9)/L, and platelet count > 125 x 10(9)/L) was achieved in 7 of the 10 patients. One of the complete responders died from the acquired immunodeficiency syndrome 44 months after treatment with high-dose cyclophosphamide. The 6 remaining patients are alive and in continuous complete remission, with a median follow-up of 10.8 years (range, 7.3 to 17.8 years). The median time to last platelet transfusion and time to 0.5 x 10(9) neutrophils/L were 85 and 95 days, respectively. None of the complete responders has relapsed or developed a clonal disease. These results suggest that high-dose cyclophosphamide, even without BMT, may be more effective than conventional immunosuppressive therapy in restoring normal hematopoiesis and preventing relapse or secondary clonal disorders. Hence, further studies confirming the efficacy of this approach in SAA are indicated.
Retroviral transfer of the recombinant human erythropoietin receptor gene into single hematopoietic stem/progenitor cells from human cord blood increases the number of erythropoietin-dependent erythroid colonies
Authors: Lu L , Ge Y , Li ZH , Keeble W , Kabat D , Bagby GC , Broxmeyer HE , Hoatlin ME
Published: October 2019

To test whether an enforced expression of a lineage-specific cytokine receptor would influence the proliferation/differentiation of hematopoietic stem/progenitor cells, retroviral vectors containing the human erythropoietin receptor (hEpoR) gene were used to transduce the hEpoR gene into phenotypically sorted subsets of cells. CD34 , CD34++CD33-, and CD34++CD33+ populations of human cord blood, highly enriched for hematopoietic stem/progenitor cells, were sorted and plated as single cells per well in methylcellulose culture medium containing early acting growth factors in the presence or absence of Epo. The hEpoR gene was efficiently transduced into single high proliferative potential colony-forming cells (HPP-CFC) and multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit-erythroid [BFU- E]), and granulocyte-macrophage (colony-forming unit-granulocyte- macrophage [CFU-GM]) progenitor cells. As expected in cultures grown in the absence of Epo, no BFU-E or CFU-GEMM colonies grew. In the presence of Epo, the hEpoR-gene transduced cells formed significantly more CFU- GEMM and BFU-E colonies than did the controls. A significant decrease in HPP-CFC colonies was also observed under these conditions. Little or no effect of hEpoR gene transduction was apparent in the numbers of CFU- GM colonies formed in the presence or absence of Epo. All of the above results were similar whether the cell populations assessed were CD34 or their CD33- or CD33+ subsets plated in the presence of growth factors at 200 cells/mL or after limiting dilution at 2 cells/well. These results suggest that the profile of detectable stem/progenitors can be altered by retrovirus-mediated expression of the hEpoR gene.
CD14+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells induce secretion of interleukin-6 and G-CSF by marrow stroma
Authors: Mielcarek M , Roecklein BA , Torok-Storb B
Published: October 2019

The ability of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMCs) to induce secretion of cytokines in primary long-term marrow cultures (LTC) or in the human marrow stromal cell line HS23 was compared with that of marrow mononuclear cells. Equal numbers of G-PBMCs or marrow mononuclear cells were added to stromal cultures, supernatants were harvested at day 4 and levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, G-CSF, and tumor necrosis factor alpha (TNF alpha) were determined. G- PBMCs induced 21.4-fold higher levels of IL-6 and 12.5-fold higher levels of G-CSF in LTC cocultures compared with marrow mononuclear cells and induced 20.6-fold more IL-6 and 6.3-fold more G-CSF when added to HS23 cells. Experiments using sorted populations of CD20+, CD3+, and CD14+ cells showed that CD14+ cells within G-PBMCs were responsible for triggering the production of IL-6 and G-CSF. The effect did not require cell-cell contact and was inhibited when neutralizing antibodies to IL-1 alpha and IL-1 beta were used in combination. In these experiments, the greater stimulating ability of G-PBMCs is most likely attributable to the greater number of CD14+ cells in G-PBMCs (26.1+% +/- 2.3%) compared with marrow (2.5% +/- 0.8%), because equal numbers of CD14+ cells sorted from marrow and G-PBMCs showed comparable ability to induce IL-6 and G-CSF when placed directly on stromal cells.
All-trans-retinoic acid counteracts endothelial cell procoagulant activity induced by a human promyelocytic leukemia-derived cell line (NB4)
Authors: Falanga A , Marchetti M , Giovanelli S , Barbui T
Published: October 2019

Therapy with all-trans-retinoic acid (ATRA) can rapidly improve the coagulopathy of acute promyelocytic leukemia (APL). This study was designed to evaluate whether the APL cell line NB4 induces the procoagulant activity (PCA) of human endothelial cells (ECs) in vitro, and whether this property is modified after ATRA-induced NB4 maturation. EC monolayers were incubated for 4 hours at 37 degrees C with the conditioned media (CM) of NB4 treated with 1 mumol/L ATRA (ATRA-NB4-CM) or the vehicle (control-NB4-CM). EC lysates were tested for PCA. ATRA-NB4-CM induced significantly more PCA:tissue factor (TF) than control-NB4-CM (P .01). To identify the cause of TF induction, interleukin (IL)-1 beta antigen levels were measured in CM samples. ATRA-NB4-CM contained significantly more IL-1 beta than control-NB4-CM. EC PCA was significantly inhibited by an anti-IL-1 beta antibody. The addition to the media of 10 mumol/L ATRA counteracted the EC TF expression induced by NB4-CM. These data indicate that ATRA increases the promyelocyte-induced EC TF, partly through increased IL-1 beta production. However, ATRA can protect the endothelium from the procoagulant stimulus of leukemic cells.
Persistent activation of the tumor necrosis factor system in a subgroup of patients with common variable immunodeficiency--possible immunologic and clinical consequences
Authors: Aukrust P , Lien E , Kristoffersen AK , Muller F , Haug CJ , Espevik T , Froland SS
Published: October 2019

In patients with common variable immunodeficiency (CVI), we have previously defined a subgroup of patients (CVIHyper) characterized by decreased numbers of CD4+ lymphocytes in peripheral blood, splenomegaly, and persistent immune activation in vivo, particularly of monocytes/macrophages. To further characterize this hyperactivity, parameters of activation of the tumor necrosis factor (TNF) system (TNF alpha and soluble TNF receptors [sTNFRs]) were measured in 24 patients with CVI and 20 healthy controls. Patients with CVI had significantly higher serum levels of TNF alpha and both types of sTNFRs, with the highest levels in the CVIHyper subgroup. In vitro, peripheral blood mononuclear cells (PBMC) and purified monocytes from CVIHyper patients spontaneously released significantly higher levels, and, after lipopolysaccharide (LPS) stimulation, significantly lower levels of TNF alpha and soluble p75-TNFR than cells from both other CVI patients and healthy controls. CVIHyper patients also had significantly higher TNF alpha:sTNFRs ratios in both serum and in unstimulated PMBC supernatants. The present study demonstrates persistent in vivo activation of the TNF system in CVI, particularly in the CVIHyper subgroup. This activation may contribute to the pathogenesis of both clinical and immunologic manifestations in CVI.
Pharmacokinetics and immunomodulatory properties of intravenously administered recombinant human interleukin-10 in healthy volunteers
Authors: Huhn RD , Radwanski E , O'Connell SM , Sturgill MG , Clarke L , Cody RP , Affrime MB , Cutler DL
Published: October 2019

Normal volunteers received single doses of recombinant human interleukin-10 (rhIL-10; n = 6 per group) or placebo (n = 3 per group) by intravenous injection to characterize pharmacokinetics, tolerability, and immunomodulatory effects. Dosages were 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 micrograms/kg. Dose-related adverse effects consisted of a mild-to-moderate flu-like syndrome characterized by fever with chills, headache, and myalgias at the highest dose. The mean terminal phase t1/2 ranged from 2.3 +/- 0.5 to 3.7 +/- 0.8 hours. Dose-related effects of rhIL-10 included transient increases of circulating neutrophils and monocytes and decreases of lymphocytes. rhIL-10 markedly suppressed, in a time- and dose-dependent manner, the synthesis of the inflammatory cytokines IL-1 beta and tumor necrosis factor alpha by whole blood stimulated ex vivo with bacterial lipopolysaccharide. Circulating numbers of CD14+/HLA-DR+ cells at 24 hours after the dose were increased in a dose-dependent manner. Effects on expression of HLA-DR by CD14+ cells were variable. There was no apparent effect on HLA-DR expression by CD20+ cells. The immunomodulatory effects of rhIL-10 merit further clinical investigation.
Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and granulocyte-macrophage colony-stimulating factor
Authors: Gazitt Y , Tian E , Barlogie B , Reading CL , Vesole DH , Jagannath S , Schnell J , Hoffman R , Tricot G
Published: October 2019

Peripheral blood stem cells (PBSCs) mobilized with high-dose chemotherapy and hematopoietic growth factors are now widely used to support myeloablative therapy of multiple myeloma and effect complete remissions in up to 50% of patients with apparent extension of event- free and overall survival. Because tumor cells are present not only in bone marrow, but also in virtually all PBSC harvests, it is conceivable that autografted myeloma cells contribute to relapse after autotransplants. In this study, the kinetics of mobilization of normal hematopoietic stem cells were compared with those of myeloma cells present in PBSC harvests of 12 patients after high-dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor administration. CD34+ and CD34+Lin-Thy+ stem cell contents were measured by multiparameter flow cytometry, and myeloma cells were quantitated by immunostaining for the relevant Ig light chain and by a quantitative polymerase chain reaction for the myeloma-specific CDRIII sequence. Results indicated marked heterogeneity in the percentages of mobilized stem cells among different patients (0.1% to 22.2% for CD34+ cells and 0.1% to 7.5% for CD34+Lin-Thy+ cells, respectively). The highest proportions of hematopoietic progenitor cells were observed early during apheresis, with 9 of 12 patients mobilizing adequate amounts of CD34+ cells for 2 autotransplants (> 4 x 10(6)/kg) within the first 2 days, whereas peak levels (percent and absolute numbers) of myeloma cells were present on days 5 and 6 (0.5% to 22.0%). During the last days of collection, mobilized tumor cells exhibited more frequently high labeling index values (1% to 10%; median, 4.4%) and an immature phenotype (CD19+). The differential mobilization observed between normal hematopoietic stem cells and myeloma cells can be exploited to reduce tumor cell contamination in PBSC harvests.
Epstein-Barr virus transmission from a blood donor to an organ transplant recipient with recovery of the same virus strain from the recipient's blood and oropharynx
Authors: Alfieri C , Tanner J , Carpentier L , Perpete C , Savoie A , Paradis K , Delage G , Joncas J
Published: October 2019

A previous study (Savoie et al, Blood 83:2715, 1994) identified eight transplant patients who acquired Epstein-Barr virus (EBV) infection during the peritransplant period. Three of these patients subsequently developed B-cell lymphoproliferative disease within 4 months of transplantation. Among these, there was a 16-year-old liver transplant patient who was negative for EBV at the time of transplant and who received an EBV-negative organ. After transplant, this patient was transfused with 9 U of packed red blood cells. Eight of the donors were EBV-positive and one was EBV-negative. We succeeded in obtaining spontaneous lymphoblastoid cell lines (LCLs) from the blood of three of these donors, one of whom also yielded a cord-blood line established with his throat-wash EBV. Blood from a fourth donor did not yield an LCL, but his throat washing did have transforming activity when inoculated onto cord-blood leukocytes. We initially could establish spontaneous LCLs only from the recipient's blood. However, a throat- wash sample taken 11 weeks later did show transforming activity. The recipient was shown to have acquired the EBV infection from one of eight EBV-seropositive blood donors. Analysis of fragment length polymorphisms after polymerase chain reaction amplification of the EBV BamHI-K fragment was used to establish strain identity. Western blot analysis for existence of size polymorphisms in three classes of Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, and EBNA-3) confirmed the DNA results. It is noteworthy that the blood donor responsible for transmitting his EBV strain to the recipient had experienced clinical infectious mononucleosis 15 months before donating blood. Our results may, thus, indicate a requirement for leukodepletion of blood destined for immunosuppressed EBV-negative patients. Finally, blood donors with a recent history of infectious mononucleosis should probably be identified so that their blood is not given to EBV-negative transplant patients.
Molecular and cellular basis for type I heparin cofactor II deficiency (heparin cofactor II Awaji)
Authors: Kondo S , Tokunaga F , Kario K , Matsuo T , Koide T
Published: October 2019

Heparin cofactor II (HCII) is a serine proteinase inhibitor in human plasma that rapidly inhibits thrombin in the presence of dermatan sulfate or heparin. To understand the molecular mechanism for HCII deficiency in a patient with reduced circulating HCII antigen, we studied a Japanese patient with type I HCII deficiency who suffered from angina pectoris and coronary artery disease. Polymerase chain reaction (PCR)-based sequence analysis showed that the propositus' gene for HCII (HCII Awaji gene) had a thymine insertion after codon (GAT) for Asp88 in exon II, resulting in a frameshift mutation. Consequently, the abnormal HCII Awaji protein was suggested to have an altered amino acid sequence from position 89 and terminate at 107, thus being composed of the NH2-terminal one fifth of normal HCII and dysfunctional for thrombin inhibition. The molecular weight and pI value of HCII Awaji were calculated to be 12,040 and 3.6, respectively, without posttranslational modification. Mutagenic PCR followed by the Tsp509I digestion showed that a half of the PCR products derived from the propositus and his sister was cleaved, suggesting that his sister also has the same mutant allele. Crossed-immunoelectrophoresis and Western blot analyses of plasma and urine from the the propositus and of plasma from his sister did not provide evidence for the existence of the abnormal HCII, suggesting that little truncated HCII was circulating in the patient's blood. However, stable expression assay using human kidney 293 cells transfected with the expression vector containing cDNA encoding wild-type or Awaji-type HCII showed that mutant as well as wild-type HCII was secreted into culture medium normally. These results suggest that the abnormal HCII Awaji protein is secreted normally, but rapidly degraded in the circulating blood.
Molecular characterization of the t(2;5) (p23; q35) translocation in anaplastic large cell lymphoma (Ki-1) and Hodgkin's disease
Authors: Yee HT , Ponzoni M , Merson A , Goldstein M , Scarpa A , Chilosi M , Menestrina F , Pittaluga S , de Wolf-Peeters C , Shiota M , Mori S , Frizzera G , Inghirami G
Published: October 2019

The precise cellular origin and the pathogenetic mechanism(s) leading to the neoplastic transformation of anaplastic large cell lymphoma (ALCL) and the Reed-Sternberg cell of Hodgkin's disease (HD) remains largely uncertain. Classical cytogenetic analysis has shown a unique translocation involving bands 2p23 and 5q35 bands in a variable number of ALCLs. It has been recently shown that the nucleophosmin/B23 (NPM) gene (5q35) and a novel anaplastic lymphoma kinase (ALK; 2p23) are the fused genes of t(2;5). To investigate the presence and the precise frequency of NPM-ALK gene products among ALCL and HD cases, a large and well-characterized panel of ALCL (n = 49) and HD (n = 72) cases was studied using multiple strategies including reverse transcriptase- polymerase chain reaction (RT-PCR), Southern blot analysis, and immunohistochemistry. Overall, 6 (3 T and 3 null) of 49 ALCL and 3 (2 nodular sclerosis and 1 mixed cellularity) of 72 HD showed the presence of NPM-ALK transcripts by RT-PCR. NPM-ALK gene rearrangements were detected in all RT-PCR, NPM-ALK-positive ALCL by Southern blot analysis. Furthermore, in all the available cases we were able to show the presence of ALK-related protein using a specific polyclonal antiserum recognizing the cytoplasmic domain of ALK by immunohistochemistry. Our data show that NPM-ALK gene transcripts are identified in a subpopulation of ALCL, almost exclusively in T or null cell in origin, and in rare cases of HD. These findings show that some HD may be closely related to ALCL, giving us new insights on the pathogenesis and possibly biologic evolution of HD.
Graft-versus-myeloma effect: proof of principle
Authors: Tricot G , Vesole DH , Jagannath S , Hilton J , Munshi N , Barlogie B
Published: October 2019

The presence of a graft-versus-tumor effect has been well established in leukemia but not in multiple myeloma. A 40-year-old patient with myeloma refractory to standard chemotherapy and autologous transplantation received a matched unrelated T-cell-depleted transplant after conditioning with fractionated total-body irradiation, thiotepa, and cyclophosphamide. This procedure resulted in a transient and incomplete response with evidence of rapidly progressive disease within 2.5 months posttransplantation. The patient then received a small number of donor peripheral blood (PB) mononuclear cells (CD3 cells 1.2 x 10(6)/kg) without any further cytotoxic therapy. A complete remission was attained, lasting now for more than 14 months. The procedure was associated with severe acute and subsequently limited chronic graft- versus-host disease (GVHD). This report provides the first direct evidence of a graft-versus-myeloma effect after allogenic transplantation.
Deletion variants within the NF-kappa B activation domain of the LMP1 oncogene prevail in acquired immunodeficiency syndrome-related large cell lymphomas and human immunodeficiency virus-negative atypical lymphoproliferations
Authors: Knecht H , Raphael M , McQuain C , Rothenberger S , Pihan G , Camilleri-Broet S , Bachmann E , Kershaw GR , Ryan S , Kittler EL , Quesenberry PJ , Schlaifer D , Woda BA , Brousset P
Published: October 2019

This sequencing study of 17 acquired immunodeficiency syndrome-related lymphomas (9 primary brain, 8 systemic) and 8 human immunodeficiency virus-negative atypical lymphoproliferations expressing large amounts of the latent membrane protein 1 (LMP1) of Epstein-Barr virus was performed to characterize the carboxy terminal NF-kappa B activation domain of LMP1 at the molecular level in an immunocompromised host. In- frame deletions within the NF-kappa B activation domain were identified in all but 2 primary brain lymphomas, 4 systemic lymphomas, and 4 atypical lymphoproliferations, ie, in 60% of cases. In addition, non silent point mutations (range 1 to 5, mean 3.3) were detected in all cases. Although all changes occurred within the first 100 nucleotides of the carboxy terminal NF-kappa B activation domain--a critical sequence for the protein half-life--not a single point mutation was found in the remaining 62 nucleotides, necessary for malignant transformation. Such a clustering of nonrandom sequence variations, associated with a high oncoprotein expression in immunocompromised hosts, suggests that this part of the LMP1 oncogene behaves as a hypervariable region with natural selection of growth-promoting variants through prolongation of the protein half-life.
Hematopoietic stem cells are not direct cytotoxic targets of natural killer cells
Authors: Aguila HL , Weissman IL
Published: October 2019

Bone marrow (BM) transplants from one individual to an irradiated histoincompatible individual of the same species are rejected. In mice, the primary host barrier cells that recognize bone marrow grafts bearing hematopoietic histocompatibility antigens bear surface markers of natural killer (NK) lymphocytes. Because of the innate ability of NK cells to kill susceptible targets, it has been proposed that the cytotoxic bone marrow graft rejection. To test this hypothesis, we purified hematopoietic stem cells from mice and incubated them with purified populations of actively cytotoxic allogeneic and semisyngeneic NK cells, followed by analysis of the ability of the treated hematopoietic stem cells (HSCs) to rescue lethally irradiated syngeneic animals. Such rescue was unimpaired. Also, HSC allografts were transplanted into transgenic mice deficient in NK and killer T-cell cytotoxicity generated by expressing diphtheria toxin A chain under the control of granzyme A promoter. Allogeneic HSCs were susceptible to allogeneic restriction in these mice, implying that the effector functions of NK marker-positive cells do not require NK cell cytotoxicity.
Generation of human dendritic cells/Langerhans cells from circulating CD34+ hematopoietic progenitor cells
Authors: Strunk D , Rappersberger K , Egger C , Strobl H , Kromer E , Elbe A , Maurer D , Stingl G
Published: October 2019

Human Langerhans cells (LC) are CD1a+ dendritic cells (DC) that function as potent antigen-presenting cells for primary and secondary immune responses. Limitations in DC/LC numbers, imposed by difficult and tedious isolation procedures, have so far precluded their use as immunogens in the generation and/or augmentation of host responses against various pathogens. Therefore, we have developed a procedure for the generation of human DC/LC from CD34+ hematopoietic progenitor cells (HPC) isolated (mean: 0.7 x 10(6)/ buffy coat and 2.6 x 10(6)/leukapheresis product) and purified ( > 95%) from the peripheral blood of healthy adults. In vitro stimulation of these cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha led to their vigorous proliferation and differentiation resulting in the emergence of CD45+/CD68+/CD3-/CD19- /CD56- leukocytes some of which (mean: 12%) express CD1a and exhibit anti-CD4 and anti-major histocompatibility complex (MHC) class II reactivity. These CD1a- leukocytes include (1) LC as evidenced by the presence of Birbeck granules (BG), (2) CD14+ monocytes, and (3) Birbeck granule-negative cells with a dendritic morphology. Addition of interleukin (IL)-4 to the cytokine cocktail interfered with the development of monocytes and led to a reduction in the overall yield but, on the other hand, resulted in an increased percentage of CD1a+ cells (mean: 24%) among all cells generated. In vitro generated CD1a+, but not CD1a- HPC-derived cells are potent stimulators of the primary mixed leukocyte reaction and, as such, promising candidates for vaccination purposes.
The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro
Authors: Rusten LS , Lyman SD , Veiby OP , Jacobsen SE
Published: October 2019

The present studies investigated the effects of the recently cloned flt3 ligand (FL) on the in vitro growth and differentiation of primitive and committed subsets of human CD34+ bone marrow (BM) progenitor cells. FL alone was a weak growth stimulator of CD34+ BM cells, but synergistically and directly enhanced colony formation in combination with interleukin (IL) 3, granulocyte colony-stimulating factor (G-CSF), CSF-1, granulocyte macrophage (GM) CSF stem cell factor (SCF), and IL-6. FL and SCF were equally effective in stimulating colony formation in combination with IL-3. However, the tri-factor combination of FL + IL-3 + SCF stimulated 2.3-fold and 2.5-fold more colonies than FL + IL-3 and SCF + IL-3, respectively. These additional recruited progenitors appeared to be predominantly located in a primitive (CD71-) subset of the CD34+ progenitors, as 4.5-fold more colonies were formed by CD34+CD71- cells in response to FL + IL-3 + SCF than to FL + IL-3 or SCF + IL-3. Similar findings were observed in serum-containing and serum-deprived cultures. Whereas FL did not enhance burst-forming unit-erythroid (BFU-E) colony formation of CD34+ BM cells in the presence of serum, a low number of BFU-E colonies were formed in response to FL plus erythropoietin (Epo) under serum-deprived conditions. In addition, FL both in serum-containing and serum-deprived cultures stimulated colony formation of more committed myeloid progenitors in CD34+CD71+ BM cells. Thus, FL potently stimulates the growth of primitive and more committed human BM progenitor cells.
The acylation of megakaryocyte proteins: glycoprotein IX is primarily myristoylated while glycoprotein Ib is palmitoylated
Authors: Schick PK , Walker J
Published: October 2019

The acylation of megakaryocyte proteins was studied with special emphasis on the myristoylation and palmitoylation of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at different phases of maturation. Cells were incubated with [3H]myristate, [3H]palmitate, or [3H]acetate to study endogenous protein acylation. Cycloheximide was used to distinguish between cotranslational and posttranslational acylation and hydroxylamine to distinguish between thioester and amide linkages. After incubations, delipidated proteins or GPIb complex subunits, immunoprecipitated with PG-1, AN-51 or FMC-25 monoclonal antibody, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by fluorography. Radiolabeled fatty acids bound to GPIX and GPIb were also analyzed by high pressure liquid chromatography (HPLC) and scintillation spectrometry. With [3H]myristic acid and [3H]acetate, GPIX was found to be a major myristoylated protein in megakaryocytes and CHRF-288 cells. Myristic acid was linked to GPIX by an amide bond, and this process occurred cotranslationally. With [3H]acetate, GPIb was primarily palmitoylated, but with [3H]myristate, GPIb was acylated with about equal mounts of myristic acid and palmitic acids. Both fatty acids were linked to GPIb by thioester bonds, and acylation was posttranslational. The myristoylation of GPIX while the palmitoylation of GPIb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation may have different functions relevant to the assembly of the GPIb complex in megakaryocytes.
Alpha-granule membrane mirrors the platelet plasma membrane and contains the glycoproteins Ib, IX, and V
Authors: Berger G , Masse JM , Cramer EM
Published: October 2019

We have recently shown that several components from the platelet plasma membrane were also present at different rates in the alpha-granule membrane. This is the case for the glycoprotein (GP) IIb-IIIa (CD41), CD36, CD9, PECAM1, and Rap1b, while the GPIB-IX-V complex was considered to escape the rule. In this investigation, we studied the subcellular localization of GPIb, GPIX, and GPV in the resting platelets of normal subjects, patients with Bernard-Soulier syndrome, patients with Gray platelet syndrome, and human cultured megakaryocytes. Ultra-thin sections of the cells were labeled with antibodies directed against glycocalicin, GPIb, GPIX, and GPV. We have shown that a significant and reproducible labeling for the three GPs was associated with the alpha-granule membrane, accounting for approximately 10% of the total labeling. Furthermore, GPIb labeling appears Willebrand factor (vWF). After thrombin activation, vWF remained close to the limiting membrane of the open canalicular system (OCS), suggesting an early association of both receptor and ligand. Plasma membrane and alpha-granule labeling was virtually absent from the Bernard-Soulier platelets (characterized by a GPIb deficiency), thus proving the specificity of the reaction. In Gray platelets (storage granule deficiency syndrome), the small residual alpha- granules were also occasionally labeled for GPIb, GPIX, and GPIX. Cultured megakaryocytes that displayed the classical GPIb distribution, eg, demarcation and plasma membranes, exhibited also a discrete labeling associated to the alpha-granules. In conclusion, this study shows that, evenly for these three GPs, the alpha-granule membrane mirrors the plasma membrane composition. This might occur through an endocytotic process affecting each plasma membrane protein to a different extent and could have a physiologic relevance in further presentation of a receptor bound to its alpha-granule ligand to the platelet surface.
Common Epstein-Barr virus (EBV) type-1 variant strains in both malignant and benign EBV-associated disorders
Authors: Schuster V , Ott G , Seidenspinner S , Kreth HW
Published: October 2019

In the present study, Epstein-Barr virus (EBV) isolates from 18 malignant tumors (angioimmunoblastic lymphadenopathy [AILD], n = 4; Hodgkin's disease [HD], n = 3; pleomorphic T-cell non-Hodgkin's lymphoma [T-NHL], n = 1; B-cell non-Hodgkin's lymphoma [B-NHL], n = 8; gastric carcinoma, n = 2) as well as from 10 tonsils of EBV- seropositive children and from peripheral blood mononuclear cells of 12 children with uncomplicated infectious mononucleosis (IM) and of a boy with severe chronic active EBV infection were genotyped in the EBV nuclear antigen-2 (EBNA-2) gene. A total of 40 of 41 isolates harbored EBV type 1; in 1 specimen (tonsil), only EBV type 2 was found. Further molecular characterization of EBV type-1 wild-type isolates in the EBNA- 2 gene and in the 40-kb distant EBV-encoded small RNAs (EBER) region showed that different groups of stable EBV type-1 variant strains exist in vivo both in benign and malignant lymphatic tissue. Group 1 is composed of EBV type-1 isolates (B-NHL, n = 3; T-NHL, n = 1; HD, n = 1; IM, n = 4) that showed a B95–8-like DNA sequence pattern in both viral genes. Group 2 isolates (HD, n = 1; AILD, n = B-NHL, n = 1; tonsils of EBV-seropositive children, n = 9; IM, n = 20 showed a nucleotide change at position 49095 in the EBNA-2 gene, leading to an amino acid substitution (Pro-->Ser), and EBV type-2 sequences in the EBER region. EBV type-1 isolates that fall into group 3 (AILD, n = 3; HD, n = 1; B- NHL, n = 4; gastric carcinoma, n = 2; IM, n = 6; severe chronic active EBV infection, n = 1) were characterized by typical nucleotide changes and a 3-bp insertion (CTC; extra Leu residue) in the EBNA-2 gene and an EBV type-2-specific sequence pattern in the EBER region. These EBV type- 1 variant strains may represent the most prevalent circulating EBV type- 1 strains in the exposed population and seem not to be restricted to a certain EBV-associated disease or tumor type. However, analysis of more EBV isolates from benign and malignant lesions must show whether more EBV type-1 substrains exist in vivo.
The Tie receptor tyrosine kinase is expressed by human hematopoietic progenitor cells and by a subset of megakaryocytic cells
Authors: Batard P , Sansilvestri P , Scheinecker C , Knapp W , Debili N , Vainchenker W , Buhring HJ , Monier MN , Kukk E , Partanen J , Matikainen MT , Alitalo R , Hatzfeld J , Alitalo K
Published: October 2019

Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting- gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.
Hemodynamic forces modulate the effects of cytokines on fibrinolytic activity of endothelial cells
Authors: Kawai Y , Matsumoto Y , Watanabe K , Yamamoto H , Satoh K , Murata M , Handa M , Ikeda Y
Published: October 2019

We investigated the effects of hemodynamic force on fibrinolytic activity of cultured human umbilical vein endothelial cells stimulated by cytokines, using a modified cone-plate viscometer in which well- controlled and -defined shear forces were generated. Treatment of the cells with interleukin (IL)-beta or tumor necrosis factor alpha (TNFalpha) under static conditions had no effect on tissue plasminogen activator (t-PA) secretion, while release of plasminogen activator inhibitor 1 (PAI-1) increased. When cells were exposed to increasing shear stress up to 24 dynes/cm2, levels of t-PA and t-PA/PAI-1 complex significantly increased relative to shear stress, while total PAI-1 and active PAI-1 secretion decreased gradually. In the presence of IL-1beta or TNFalpha, the increase in production of t-PA and the t-PA/PAI-1 complex was further augmented. Dot blot hybridization analysis of cultured cells in similar experimental conditions using t-PA and PAI-1 cDNA probes revealed no t-PA mRNA in 3 microg total RNA from static endothelial cells under resting or cytokine-stimulated conditions, but abundant t-PA mRNA was detected in cells subjected to a shear force of 18 dynes/cm2, and the increase was further augmented by addition of cytokines. In contrast, PAI-1 mRNA was detected in resting and cytokine- stimulated, nonsheared endothelial cells, but levels decreased after exposure to shear stress, even in the presence of cytokines. These results indicate a role for hemodynamic forces in regulating fibrinolytic activity with or without cytokine stimulation.
Target-induced death by apoptosis in human lymphokine-activated natural killer cells
Authors: Taga K , Yamauchi A , Kabashima K , Bloom ET , Muller J , Tosato G
Published: October 2019

Activated human natural killer (NK) cells undergo rapid apoptotic cell death after ligand binding to the Fc receptor (CD16). We examined whether human NK cells die after engagement in cytolytic functions. Peripheral blood NK cells, with and without prior activation in vitro with interleukin-2 (IL-2), were tested for the occurrence of cell death after incubation with K562, the prototype NK-sensitive target cell. A proportion (15.2%) of NK cells that were stimulated for 3 days with IL- 2 and then incubated for 4 hours with K562 cells showed rapid cell death, but NK cells not stimulated with IL-2 did not. This cell death was found to involve nuclear condensation and fragmentation and DNA cleavage, all of which are characteristic of apoptosis. These data indicate that a proportion of activated human NK cells undergo apoptosis as they engage in target cell lysis. Target-induced NK cell death may represent an important mechanism for regulation of inflammatory processes involving NK cells.
Frequent expression of the cell death-inducing gene Bax in Reed- Sternberg cells of Hodgkin's disease
Authors: Rigal-Huguet F , Gopas J , Prinsloo I , Pris J , Delsol G , Reed JC , Schlaifer D , Brousset P , Benharroch D , Krajewski S , Laurent G , Meggetto F
Published: October 2019

The expression of a cell death-inducing gene, Bax, was investigated in 52 cases of Hodgkin's disease in parallel with Epstein-Barr virus and was compared with the immunodetection of other apoptosis-regulating proteins, Mcl-1, Bcl-2, and Bcl-x. Bax immunostaining was found in 92% of the cases, among them 28% with a strong signal in more than 75% of the Reed-Sternberg cells. Mcl-1 was positive in 80% of the cases, whereas Bcl-2 and Bcl-x were found in 53% and 88% of the cases, respectively. Of 48 (89%) Bax-positive tumors, 43 were found to express apoptosis-inhibiting proteins such as Mcl-1 or Bcl-2. With the exception of 1 case, all Bax-positive tumors also expressed either Bcl- 2, Bcl-x, Mcl-1, or combinations of these anti-apoptotic proteins. No correlation was found between Bax expression and the presence of apoptotic cells as detected by morphology and the in situ 3′ OH-DNA end- labeling technique. Our findings show that the apoptosis-inducing gene Bax expression is frequently expressed in Hodgkin's disease, providing a potential explanation for the good chemoresponses generally obtained for patients with this neoplastic disorder.
Functional inactivation in mice of the gene for the interleukin-3 (IL- 3)-specific receptor beta-chain: implications for IL-3 function and the mechanism of receptor transmodulation in hematopoietic cells
Authors: Nicola NA , Robb L , Metcalf D , Cary D , Drinkwater CC , Begley CG
Published: October 2019

The receptors for granulocyte-macrophage colony-stimulating factor (GM- CSF) and interleukin-3 and -5 (IL-3, IL-5) share a common signaling subunit (beta c). However, in the mouse, IL-3 can also use an alternative IL-3-specific receptor beta-chain (beta IL-3). To assess the relative contributions of beta c and beta IL-3 to IL-3 receptor formation and function, mice were generated in which the beta IL-3 gene was functionally inactivated by replacement of exons 9–13 with a neomycin resistance cassette. Bone marrow cells from these mice displayed a lower affinity IL-3 receptor than normal and were hyporesponsive to IL-3, but the mice displayed no obvious hematopoietic abnormalities. The data suggested that beta c and beta IL-3 are normally coexpressed on IL-3-responsive cells and have identical qualitative signaling capacities. Receptor transmodulation studies on bone marrow cells from wild-type, beta c -/-, and beta IL-3 -/- mice showed that the previously described hierarchical pattern of transmodulation was dependent on the relative numbers of both beta IL-3 and beta c receptor chains and also provided evidence for an unexpected interaction between beta c chains and G-CSF and M-CSF receptors.
Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro
Authors: Helgason CD , Sauvageau G , Lawrence HJ , Largman C , Humphries RK
Published: October 2019

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.
Evidence for oligoclonal diversification of the VH6-containing immunoglobulin repertoire during reconstitution after bone marrow transplantation
Authors: Nasman I , Lundkvist I
Published: October 2019

Patients who have undergone bone marrow transplantation (BMT) remain immunodeficient for months to years posttransplantation. To evaluate the basic molecular events underlying reconstitution of the humoral immune response, we have performed a detailed nucleotide sequence analysis of VH6 containing rearrangements in circulating B cells from two BM donor/recipient pairs. Our results show that the third complementarity determining region (CDR3) diversity is much lower early after transplantation, compared with that of the donors, and that the clonal variability remains low for 3 months. Repertoire diversification follows an oligoclonal pattern where B lymphopoiesis appears to occur in waves up to 6 months posttransplantation. The repertoire among donated marrow cells is not reflected in peripheral blood lymphocytes from the transplanted patients. There is differential D gene utilization among both donor and patient samples, whereas JH gene usage is biased toward JH4, 5, and 6. One month after transplantation the vast majority of the sequenced clones are functional and contain a high frequency of replacement mutations in the CDRs of the VH6 gene. We conclude that Ig gene expression is very restricted early after BMT and that development of the B-cell repertoire appears to follow a wavelike pattern.
Rearrangement of the MLL gene confers a poor prognosis in childhood acute lymphoblastic leukemia, regardless of presenting age
Authors: Behm FG , Raimondi SC , Frestedt JL , Liu Q , Crist WM , Downing JR , Rivera GK , Kersey JH , Pui CH
Published: October 2019

MLL gene rearrangements are associated with an extremely poor prognosis in infants with acute lymphoblastic leukemia (ALL), but little is known about their clinical significance in older children. Therefore, we studied 45 cases of childhood ALL with abnormalities of chromosome 11q23 for rearrangement of the MLL gene to determine if this feature confers a uniformly poor prognosis. MLL gene rearrangements were detected in all 18 cases with the common t(4;11), t(9;11) or t(11;19) translocations, whereas only 5 of 12 patients with either unbalanced or uncommon balanced translocations demonstrated a rearrangement. Abnormalities of the MLL gene were not detected in any of the 15 cases with a deletion or inversion of the chromosomes 11q23 region. The presence of an MLL rearrangement was significantly associated with age less than 1 year (P < .001), leukocyte count –>50 x 10(9)/L (P = .003), and the absence of leukemic cell CD10 expression (P < .001). In a stratified statistical analysis adjusted for age and treatment protocol, MLL gene rearrangement was correlated with an inferior treatment outcome (P = .028). The 4-year event-free survival estimate (+/-SE) was 10% +/-6.5% for cases with a rearranged MLL gene and 64% +/-19.2% for other cases. When infants were excluded from the analysis, MLL rearrangement was still significantly associated with a poor outcome (P = .02), and remained so with the exclusion of t(4;11)-positive cases (P = .05). Thus, regardless of presenting age, MLL gene rearrangement identifies a high-risk subgroup of patients who are not likely to be cured with conventional treatment.