bluuebunny/crossref_metadata_2025 huggingface dataset:
Human CD4 lymphocytes specifically recognize a peptide representing the fusion region of the hybrid protein pml/RAR alpha present in acute promyelocytic leukemia cells
Authors: Gambacorti-Passerini C , Grignani F , Arienti F , Pandolfi PP , Pelicci PG , Parmiani G
Published: October 2019

Fusion proteins present in leukemic cells frequently contain a new amino acid at the fusion point. We tested whether a peptide (BCR1/25) encompassing the fusion region of the hybrid molecule pml/RAR alpha, which is selectively expressed by acute promyelocytic leukemia (APL) cells, can be recognized by human T lymphocytes in vitro. CD4+ lymphocytes, at both polyclonal and clonal level, recognized peptide BCR1/25 in an HLA-DR--restricted fashion on presentation by autologous antigen-presenting cell (APC) or by APC expressing the HLA-DR11 restricting molecule. Control peptides corresponding to the normal pml and RAR alpha proteins were not recognized. One clone (DEG5) also exerted a high and specific cytotoxicity against autologous cells pulsed with BCR1/25. The autologous DE LCL containing a transduced pml/RAR alpha fusion gene and expressing a bcr1 type of the pml/RAR alpha hybrid protein induced the proliferation of DE anti-BCR1/25 T cell clones. It is concluded that the bcr1 type-pml/RAR alpha fusion protein of APL contains an antigenic site, absent from the normal parent molecules and recognized by human CD4+ lymphocytes.
Effects of interleukin-3 and granulocyte-macrophage colony-stimulating factor on thrombopoiesis in congenital amegakaryocytic thrombocytopenia
Authors: Guinan EC , Lee YS , Lopez KD , Kohler S , Oette DH , Bruno E , Kozakewich H , Nathan DG , Hoffman R
Published: October 2019

Amegakaryocytic thrombocytopenia (AMT) is a rare and often fatal disorder of infancy and childhood presenting with isolated thrombocytopenia that progresses to marrow failure. The defect in thrombopoiesis is not well understood nor is the etiology of the progressive marrow failure. No standard modality of treatment exists. Here, we evaluated the capacity of marrow cells isolated from five patients with AMT and progressive marrow failure to generate megakaryocyte progenitor cells (CFU-MK). These in vitro studies demonstrated assayable numbers of CFU-MK from all patient bone marrows that responded in vitro to the addition of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or the combination of both. These findings suggest that the defect in AMT might be partially correctable by the administration of these cytokines. A Phase I/II trial of in vivo administration of these same hematopoietins in the identical patients was conducted in which no significant toxicity was observed. IL-3 but not GM-CSF administration resulted in improved platelet counts in two patients and decreased bleeding and transfusion requirement in the remaining three. No clinical benefit was observed when GM-CSF was administered after IL-3 pretreatment. Prolonged IL-3 administration has resulted in platelet increases in an additional two patients. In vitro responsiveness of CFU- MK to either cytokine did not predict the degree of clinical response. Although the optimal dose and schedule of IL-3 either alone or in combination remains to be established, this study suggests that IL-3 may contribute to the treatment of patients with AMT.
Mechanism of the chromosomal translocation t(14;18) in lymphoma: detection of a 45-Kd breakpoint binding protein
Authors: Jaeger U , Purtscher B , Karth GD , Knapp S , Mannhalter C , Lechner K
Published: October 2019

The translocation t(14;18) between the BCL-2 oncogene and the Ig heavy chain (IgH) gene provides the molecular basis for the development of follicular lymphomas. The illegitimate recombination occurs in early B cells. While V(D)J-recombinase is most likely involved on the chromosome 14 part, little is known about the mechanism of breakage on chromosome 18. We investigated the BCL-2 breakpoint regions for their structural vulnerability and protein binding capacity. We found that the major breakpoint region (mbr) contains an S1 nuclease-sensitive site and is the target of an endogenous nuclease present in early B cells. A 45 Kd nuclear protein (bp45) from early B cell extracts binds to a homopurine-homopyrimidine stretch (GGGAGGACGGGAGGAAGGCG) in the mbr, which is homologous to a recombinatorial element in Escherichia coli (CHI). The protein also binds to homologous sequences in the minor breakpoint cluster region (mcr) and in the IgH locus. The localization of the binding sites on both chromosomes as well as the tissue distribution of bp45 suggest that this protein-DNA interaction is involved in the translocation t(14;18). The DNA binding motif is also present at other translocation breakpoints indicating a more general role for this mechanism.
Elevated interleukin-8 serum concentrations in beta-thalassemia and graft-versus-host disease
Authors: Uguccioni M , Meliconi R , Nesci S , Lucarelli G , Ceska M , Gasbarrini G , Facchini A
Published: October 2019

Neutrophil chemotactic and functional defects occur in beta-thalassemia and in patients after bone marrow transplantation (BMT). Interleukin-8 (IL-8) is a novel chemotactic and activating peptide for neutrophils and can be detected in the circulation. IL-8 serum concentrations were evaluated in 30 beta-thalassemic patients before and after BMT. Serial samples from 16 patients were also studied. Fourteen sera from healthy children, 43 patients with chronic viral hepatitis, 16 patients on chronic transfusion treatment for various hematologic disorders, and 28 healthy adults were studied as controls. IL-8 was evaluated by an enzyme-linked immunosorbent assay. Patients with beta-thalassemia had higher IL-8 concentrations than did normal controls, patients with liver disease, and patients on chronic transfusion. beta-Thalassemic patients with severe liver siderosis and fibrosis had the highest IL-8 concentrations. After BMT in patients with successful engraftment, IL-8 concentrations decreased significantly. In contrast, in patients with acute graft-versus-host disease (GVHD), IL-8 concentrations were not statistically different from the concentrations found before BMT and were higher than in patients with no complications and patients with graft rejection. IL-8 may play a part in the immune dysregulation that occurs in beta-thalassemia and may be involved in the immune mechanisms leading to GVHD.
Severe congenital neutropenia: abnormal growth and differentiation of myeloid progenitors to granulocyte colony-stimulating factor (G-CSF) but normal response to G-CSF plus stem cell factor
Authors: Hestdal K , Welte K , Lie SO , Keller JR , Ruscetti FW , Abrahamsen TG
Published: October 2019

Several mechanisms have been proposed to explain the pathogenesis of severe congenital neutropenia (SCN); however, the mechanism(s) still remains unknown. In particular, clinical observations suggest that abnormal responsiveness of myeloid progenitors to hematopoietic growth factors (HGFs) is a possible mechanism. Therefore, to better define the status of hematopoietic progenitors in the bone marrow (BM) of patients with SCN, the responsiveness of myeloid progenitors to HGFs from two SCN patients was compared with the responsiveness of progenitors from healthy individuals. BM cells (BMCs) from the first SCN patient required higher (10- to 100-fold) concentrations of granulocyte colony- stimulating factor (G-CSF) to achieve maximal and half-maximal colony growth in vitro compared with BMCs from controls. In contrast, the dose- response of interleukin-3 (IL-3) and granulocyte-macrophage-CSF (GM- CSF) in colony formation was normal. Interestingly, IL-3, GM-CSF, and G- CSF at optimal doses showed reduced ability to induce neutrophil differentiation of BMCs from a SCN patient compared with BMCs from controls. Despite an abnormal responsiveness of mature myeloid progenitors to G-CSF in this SCN patient, myeloid progenitors responsive to the combination of stem cell factor (SCF) and G-CSF showed normal dose-response. In contrast to G-CSF alone, the combination of G-CSF and SCF induced the formation of neutrophils almost to the same extent compared with cultures of normal BMCs. Furthermore, also on BM progenitor cells obtained from the second patient with SCN, SCF highly synergized with G-CSF to promote neutrophil progenitor cell growth and differentiation in vitro. Thus, these results indicate that one mechanism of the pathogenesis in SCN patients is reduced responsiveness of neutrophil progenitor cells to G- CSF and that SCF can enhance the responsiveness of these cells to G-CSF.
Recombinant granulocyte colony-stimulating factor administration to healthy volunteers: induction of immunophenotypically and functionally altered neutrophils via an effect on myeloid progenitor cells
Authors: Kerst JM , de Haas M , van der Schoot CE , Slaper-Cortenbach IC , Kleijer M , von dem Borne AE , van Oers RH
Published: October 2019

We performed a detailed kinetic study on the in vivo effect of a single subcutaneous dose of granulocyte colony-stimulating factor (G-CSF; 300 micrograms) in four healthy individuals on the expression and function of neutrophil Fc gamma receptors (Fc gamma R). G-CSF did not induce Fc gamma RI (CD64) on circulating neutrophils. However, neutrophils newly formed in response to G-CSF were Fc gamma RI positive and were able to perform antibody-dependent cellular cytotoxicity in an Fc gamma RI- dependent way. Fc gamma RII (CD32) expression was not changed significantly. Fc gamma RIII (CD16, phosphatidylinositol-linked) expression, slightly increased immediately (30 minutes) postinjection, was found to be strongly decreased on the newly formed population. For comparison, we studied the expression of the PI-linked proteins leukocyte alkaline phosphatase (LAP) and CD14. Intracellular levels of LAP mirrored the biphasic expression pattern as membrane-bound Fc gamma RIII. In contrast, CD14 expression on neutrophils was initially constant, followed by high levels on the newly formed neutrophils. Soluble CD14 levels were found to be elevated transiently, whereas peak levels of soluble Fc gamma III were observed as late as 6 days postinjection. In conclusion, we have shown that G-CSF results in an immunophenotypically and functionally altered neutrophil population for an important part as a result of its effect on myeloid precursor cells.
C1-inhibitor-serine proteinase complexes and the biosynthesis of C1- inhibitor by Hep G2 and U 937 cells
Authors: Patston PA , Medcalf RL , Kourteva Y , Schapira M
Published: October 2019

The biosynthesis of the serpin alpha 1-proteinase inhibitor is regulated by a feedback mechanism whereby complexes between alpha 1- proteinase inhibitor and serine proteinases bind to liver cells and monocytes, a reaction that activates alpha 1-proteinase-inhibitor gene transcription. Such a mechanism may form the basis for the development of new therapeutic strategies for serpin deficiency states with reduced levels of otherwise normally functioning serpins. This issue was addressed for C1-inhibitor, the missing serpin in hereditary angioedema. C1-inhibitor biosynthesis by Hep G2 hepatoma cells was assessed by enzyme-linked immunosorbant assay, by metabolic labeling followed by immunoprecipitation, and by Northern blotting. C1-inhibitor biosynthesis was stimulated by gamma-interferon (100 U/mL) but not by cell exposure to C1-inhibitor-kallikrein (1 mumol/L), C1-inhibitor-C1s (1 mumol/L), and C1-inhibitor-plasmin complexes (1 mumol/L) or to reactive site-cleaved C1-inhibitor (1 mumol/L). Moreover, radioiodinated C1s-C1-inhibitor complex did not bind to Hep G2 cells. C1-inhibitor-kallikrein complex was also without effect on C1-inhibitor mRNA in U 937 cells. Therefore, the proposed mechanism, by which serpin- enzyme complex or reactive site-cleaved serpin binding to a specific receptor provides a signal for the stimulation of the biosynthesis of that serpin, is not operative for the biosynthesis of C1-inhibitor by Hep G2 or U 937 cells.
Immunoglobulin levels and monoclonal gammopathies in children after bone marrow transplantation
Authors: Gerritsen EJ , van Tol MJ , Lankester AC , van der Weijden-Ragas CP , Jol-van der Zijde CM , Oudeman-Gruber NJ , Radl J , Vossen JM
Published: October 2019

Bone marrow graft recipients suffer profound immunodeficiency during at least 3 months after transplantation. B-cell reconstitution following allogeneic bone marrow transplantation (BMT) in children was studied longitudinally by quantification of Ig (sub)class levels in serum and by investigation of numbers and characteristics of homogeneous Ig components (H-Ig); ie, monoclonal gammopathies (MG). For the latter purpose, a sensitive immunoblotting technique capable of detecting H-Ig of a concentration as low as 0.5 microgram/mL was used. Sera of 40 children grafted for a variety of diseases were investigated and followed up for 5 years. It was found that Ig (sub)classes reached normal levels from 3 months after BMT onward. The sequential increase of the different Ig isotypes was in accordance with that seen in normal ontogeny. This was especially clear following BMT for severe congenital immunodeficiency. H-Ig appeared from as early as 6 weeks after BMT in increasing numbers, beginning within IgM, IgG3, and IgG1, and afterward within other isotypes. After an initial increase of serum Ig levels, “overshooting” occurred accompanied by high frequency of H-Ig. H-Ig were still present at 5 years after BMT, when Ig levels normalized. Our data indicate that B-cell reconstitution after allogeneic BMT recapitulates normal ontogeny but in a clonally dysregulated fashion; that is, with overexpression of some clones and underexpression of others.
Enzyme therapy in Gaucher disease type 1: dosage efficacy and adverse effects in 33 patients treated for 6 to 24 months
Authors: Pastores GM , Sibille AR , Grabowski GA
Published: October 2019

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent genetic disease among the Ashkenazi Jews (q approximately 0.047). The disease results from inherited defects of acid beta-glucosidase and the accumulation of the substrate, glucosylceramide, in cells of monocyte/macrophage origin. The therapeutic response to macrophage-targeted (alpha-mannosyl-terminated) alglucerase (Ceredase, at 60 to 15 IU/kg every 2 weeks) was analyzed in 33 patients (age range, 2 to 63 years; 15 splenectomized) with extensive Gaucher disease over periods of 6 to 24 months. The efficacy of several different doses and dosage reductions was evaluated. In patients with anemia (n = 30) and/or thrombocytopenia (n = 19), hemoglobin levels and platelet counts increased by 0% to 178% and 15% to 155%, respectively, within 3 to 12 months. In patients with splenomegaly (n = 17) and/or hepatomegaly (n = 28), liver and spleen volumes decreased in 6 months from 7% to 64% and 8% to 84% by 12 months, respectively. Hematologic and visceral improvements were noted at any doses between 60 and 15 IU/kg every 2 weeks. Furthermore, these positive initial therapeutic responses were persistent throughout therapy, with doses reduced by 50%. Pulmonary Gaucher disease did not improve clinically in 3 patients. Unrelated cirrhotic (n = 2), cholestatic (n = 1), or renal disease (n = 1) did not influence the rate of patient improvement. Two of five patients who developed serum antibodies against alglucerase during the first 6 to 12 months of therapy had mild antibody reactions. This study shows similar regression of clinical Gaucher disease manifestations with enzyme therapy, using doses between 30 and 60 IU/kg every 2 weeks. Therapeutic efficacy was not diminished after 50% to 75% dose reductions or in the presence of anti-enzyme antibodies.
Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemia cell line, UT-7
Authors: Komatsu N , Yamamoto M , Fujita H , Miwa A , Hatake K , Endo T , Okano H , Katsube T , Fukumaki Y , Sassa S
Published: October 2019

UT-7 is a human leukemic cell line capable of growing in interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo) (Komatsu et al, Cancer Res 51:341, 1991). To study the effect of Epo on proliferation and differentiation of UT-7, we maintained the UT-7 cell culture for more than 6 months in the presence of Epo. As a result, a subline, UT-7/Epo, was established. The growth of UT-7/Epo could be supported by Epo but not by GM-CSF or IL-3. UT- 7/Epo showed a greater level of heme content and ratio of benzidine- positive staining cells than did UT-7. Butyric acid promoted the synthesis of hemoglobin in UT-7/Epo, but not UT-7. Further, the mRNA concentrations of the c-myb oncogene and GM-CSF receptor beta-subunit were decreased substantially in UT-7/Epo cells. These findings showed that UT-7/Epo cells had progressed further in erythroid development than UT-7 cells, and suggested that long-term culture in Epo had promoted this differentiation. Whereas availability of the Epo receptor (Epo-R) for binding of Epo was reduced in UT-7/Epo cells compared with UT-7 cells, the Epo-R showed a similar affinity for Epo. This observation suggested that change(s) in postreceptor signaling step might be involved in the establishment and maintenance of the UT-7/Epo phenotype.
Adsorption and conversion of prothrombin on a rotating disc
Authors: Willems GM , Giesen PL , Hermens WT
Published: October 2019

In most flow systems, the rate of protein transfer from bulk solution to a macroscopic surface is site-dependent. In studies on surface- mediated protein conversion, this hampers the comparison of a proposed expression for the conversion process, such as the Michaelis-Menten equation, which actually measured overall conversion rates. However, the rotating disc is a classical example of a uniformly accessible surface and therefore was used for a quantitative analysis of prothrombin conversion by the phospholipid-bound factor Xa/factor Va complex (prothrombinase complex). A simple design of a rotating disc, adapted for ellipsometric measurement of protein adsorption, is presented. Agreement between experiment and theory was obtained for the influence of rotation velocity on the initial, transport-limited, adsorption rates of lysozyme, prothrombin, and fibrinogen. After coverage of the disc with a 20% phosphatidylserine/80% phosphatidylcholine bilayer and preadsorption of factor Va, addition of excess factor Xa and prothrombin resulted in effective conversion of prothrombin. For high (10 fmol.cm-2) surface coverage of prothrombinase, the rate of conversion equals the transport limited adsorption rate of prothrombin. For low (0.1 to 0.5 fmol.cm-2) surface concentrations of prothrombinase, the conversion rate dropped below the transport limit and the intrinsic kinetic parameters could be estimated at Km = 7.1 +/- 1.2 nM and kcat = 25 +/- 1.0 s-1 (20 degrees C). At these low surface activities of prothrombinase, the effect of the rotation rate (6 to 225 rad.s-1) on prothrombin conversion could be explained by the rotation-rate dependent prothrombin transport. This indicates that the fluid shear rate has no drastic influence on the intrinsic kinetics of prothrombin conversion.
Absence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death
Authors: Tamaru Y , Miyawaki T , Iwai K , Tsuji T , Nibu R , Yachie A , Koizumi S , Taniguchi N
Published: October 2019

bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death (apoptosis). There is now increasing evidence that regulation of bcl-2 expression is a determinant of life or death in normal lymphocytes. We have recently described that activated (CD45RO+) CD4+ and CD8+ T cells in acute infectious mononucleosis (IM) undergo apoptotic cell death on culturing, indicating an activation-driven cell death of mature T cells. In this work, we examine bcl-2 expression by activated T cells in acute IM using a flow-cytometric analysis with an anti-bcl-2 monoclonal antibody (MoAb). It was consistently observed that most T cells from acute IM patients displayed only much less bcl-2, while normal T cells expressed bcl-2 relatively strongly. Multicolor analysis showed that bcl-2- lacking T cells in acute IM were restricted to the CD45RO+ (activated) populations of CD4+, as well as CD8+ T cells. In contrast, the relatively intense levels of bcl-2 were expressed in both CD45RO+ and CD45RO- T-cell populations from normal subjects. This marked difference in bcl-2 expression of CD45RO+ T cells between acute IM and normal controls was also confirmed by Western blot analysis. Activated (CD45RO+) T cells with low bcl-2 expression, but not bcl-2-expressing CD45RO- T cells, in acute IM patients were found to die easily when cultured without added growth factors. However, in normal individuals, both CD45RO+ and CD45RO- T cells were relatively stable on culturing. These findings suggest that lack of bcl-2 expression by activated (CD45RO+) T cells in acute IM might be associated with their susceptibility to programmed cell death.
Distinct ongoing Ig heavy chain rearrangement processes in childhood B- precursor acute lymphoblastic leukemia
Authors: Steenbergen EJ , Verhagen OJ , van Leeuwen EF , von dem Borne AE , van der Schoot CE
Published: October 2019

Acute lymphoblastic leukemia (ALL) is thought to arise from the clonal expansion of a single transformed precursor cell. However, an oligoclonal Ig heavy chain (IgH) rearrangement pattern has been observed in 30% of ALL patients and was shown to be the result of ongoing rearrangement events. The extent and nature of these ongoing rearrangement processes in individual patients has so far remained obscure. We performed a detailed analysis of leukemic VHDJH rearrangements in three children with B-precursor ALL at diagnosis and one B-lymphoid blast crisis of a child with Ph+ chronic myeloid leukemia at diagnosis and relapse. The children were selected because they presented with multiple IgH rearrangements on Southern blot analysis. Polymerase chain reaction analysis of leukemic cells from two B-precursor ALL patients showed exclusively two groups of related sequences resulting from VH gene replacement events. Most VH gene replacements involved 3′ located acceptor VH genes. Analysis of cells from the other B-precursor ALL patient showed exclusively related sequences as a result of VH gene joinings to a pre-existing DJH rearrangement. In the B-lymphoid blast crisis, a single germline precursor cell had generated multiple unrelated rearrangements and additional groups of related rearrangements resulting from VH to DJH joinings. Direct proof for the VH to DJH joining mechanism was obtained by amplification of the expected preexisting DJH rearrangements. Our findings suggest that the pattern of ongoing rearrangements in an individual patient reflects the IgH rearrangement status of the precursor cell at the time of malignant transformation. Sequence analysis of VHDJH rearrangements at diagnosis may therefore allow a prediction of the reliability of complementarity determining region 3 probes for the detection of minimal residual disease.
N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome
Authors: Paquette RL , Landaw EM , Pierre RV , Kahan J , Lubbert M , Lazcano O , Isaac G , McCormick F , Koeffler HP
Published: October 2019

To evaluate the clinical significance of N-ras mutations in the myelodysplastic syndrome (MDS) archival bone marrow samples from 252 patients were studied for the presence of N-ras exon I mutations using polymerase chain reaction amplification and differential oligonucleotide hybridization. Subsequently, clinical information about these patients was obtained and analyzed. Of 220 evaluable patients, 20 (9%) had point mutation of N-ras involving codon 12. Individuals with N- ras mutation had a significantly shorter survival period than those who were N-ras negative (P = .02). An increased risk of acute myelogenous leukemia (AML) was also found in patients with N-ras mutations (P = .005). N-ras mutations were not associated with any French-American- British (FAB) subtype, with the presence of increased myeloblasts, or with chromosomal aberrations in the bone marrow. However, the presence of increased bone marrow blasts was strongly associated with poor survival rate and risk of AML (P < .001 for each). After stratifying for the percentage of blasts, N-ras mutations remained significantly associated with shorter survival period (P = .04) and increased risk of AML (P = .02). Bone marrow cytogenetic abnormalities, particularly when multiple abnormalities were present, were significantly associated with a poor prognosis (P < .001). In conclusion, N-ras mutation, although relatively infrequent in MDS, is associated with short survival period and increased probability of developing AML.
Allogeneic bone marrow transplantation for 93 patients with myelodysplastic syndrome
Authors: Anderson JE , Appelbaum FR , Fisher LD , Schoch G , Shulman H , Anasetti C , Bensinger WI , Bryant E , Buckner CD , Doney K
Published: October 2019

We treated 93 patients with myelodysplastic syndrome using cyclophosphamide and either total body irradiation (n = 88) or busulfan (n = 5) followed by marrow transplantation. Sixty-five marrow donors were genotypically HLA-identical siblings and 28 were other family members or unrelated donors. Before transplantation all patients had either severe neutropenia or thrombocytopenia or had greater than 5% blasts in the marrow or peripheral blood. The probabilities of disease- free survival, relapse, and non-relapse mortality at 4 years were 41%, 28%, and 43%, respectively. Multivariate analysis revealed that younger age and shorter disease duration were significantly associated with improved disease-free survival and decreased non-relapse mortality. Relapse was seen only in patients with excess blasts at the time of transplantation (51% at 4 years). Patients younger than age 40 and without excess blasts had a 4-year disease-free survival of 62%. This study confirms that allogeneic marrow transplantation can cure some patients with myelodysplasia. Because of the favorable outcome in younger patients without excess blasts, we recommend that transplantation be considered early for patients younger than age 40, before disease progression or development of life-threatening cytopenias. For older patients and those with excess blasts, changes in the transplant procedure will be necessary to improve outcome.
Fibrinogen potentiates the effect of interleukin-3 on early human hematopoietic progenitors
Authors: Zhou YQ , Levesque JP , Hatzfeld A , Cardoso AA , Li ML , Sansilvestri P , Hatzfeld J
Published: October 2019

The effect of human fibrinogen on the proliferation of purified SBA- CD34+ human bone marrow progenitors was investigated in clonal cultures. Fibrinogen alone or in combination with erythropoietin had no significant effect. However, in the presence of recombinant human interleukin-3 (IL-3), fibrinogen increased significantly in a dose- dependent manner the number of mixed and burst-forming unit-ethrocyte-- derived colonies, whereas the number of other colonies did not significantly change. In the presence of fibrinogen, low concentrations of IL-3 (0.17 U/mL) produced three times more mixed colonies than without fibrinogen, reaching the number of colonies obtained with optimal concentrations of IL-3 (1.67 U/mL). Fibrinogen fragment D had the same effect in the presence of IL-3 as intact fibrinogen, whereas fibrinogen fragment E and human collagen IV did not. This effect was not mediated by integrins, because peptides or monoclonal antibodies that block fibrinogen binding on integrins alpha IIb beta 3, alpha v beta 3 (RGD-peptides), alpha m beta 2 (OKM-1), and alpha x beta 2 (HC1/1) did not affect the observed mitogenic effect. The mitogenic effect of fibrinogen and its D fragment was not mediated by induction of IL-6 or granulocyte--colony-stimulating factor secretion, because it was not inhibited by blocking antisera against these two growth factors. Our results indicate that fibrinogen potentiates the effect of IL-3 on primitive hematopoietic progenitors and suggest that the mitogenic effect of fibrinogen could be mediated via a specific mitogenic receptor that does not belong to the integrin family.
Unusual deletions within the immunoglobulin heavy-chain locus in acute leukemias
Authors: Dyer MJ , Heward JM , Zani VJ , Buccheri V , Catovsky D
Published: October 2019

We have investigated the structure of the Ig heavy (IGH) chain locus in 309 cases of acute leukemia. Seventy-one cases of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) were analyzed: in six cases deletion of joining (JH) segments in the presence of cytogenetically normal chromosome 14 was observed. Similar deletions were seen in 1 out of 8 cases of biphenotypic acute leukemia analyzed: this case exhibited t(9:22)(q34;q11) and coexpressed both myeloid and B cell differentiation antigens. Five of the 7 cases analyzed had deleted the JH segments from both chromosomes. Because these deletions may have contributed to the pathogenesis of the disease we have attempted to define their boundaries. Using probes that map both 5′ and 3′ of JH, the 3′ (centromeric) boundary of the deletions was mapped to an approximately 30-kb central region of the 60 kb between C delta and C gamma 3 in 10 of the 12 deleted chromosomes. In the remaining two chromosomes, the 3′ boundary mapped to S mu. The 5′ (telomeric) boundary could not be defined. However, three cases with biallelic deletion of JH showed biallelic deletion of the most proximal variable (VH) (VH6 and VH5-B2) genes, indicating that the deletions spanned over 500 kb. VH5-B1 and VH5-B3 were retained in germline configuration and no gross deletions were observed using a VH3 subgroup-specific probe, indicating that the 5′ boundary mapped within the VH locus. Unusual deletions of the portion of the IgH locus including JH segments and the C mu and C delta genes may occur in acute leukemias with immunophenotypic evidence of commitment to the B cell differentiation pathway. The possible consequences of the deletions remain to be determined. However, the clustering of the centromeric boundary of the deletions to S mu and to a region between the C delta-C gamma 3 genes, a known “hot spot” for recombination, may indicate the operation of a distinct pathogenic mechanism.
Demonstration of Epstein-Barr virus replication in Reed-Sternberg cells of Hodgkin's disease
Authors: Brousset P , Knecht H , Rubin B , Drouet E , Chittal S , Meggetto F , Saati TA , Bachmann E , Denoyel G , Sergeant A
Published: October 2019

Epstein-Barr virus (EBV) is detectable in approximately 40% of cases of Hodgkin's disease (HD). The viral genomes remain latent but positive staining with anti-ZEBRA antibody in a small fraction of Reed-Sternberg (RS) cells of some cases of HD would suggest possible activation of EBV replication within these cells. We report the investigation of 40 cases of EBV-associated HD (including 5 human immunodeficiency virus [HIV]- positive cases) using anti-ZEBRA antibodies. Positive staining was found in only three (HIV-negative) cases. One of these three cases showed approximately 1% of ZEBRA-positive tumor cells, whereas the other two cases showed rare positive cells. In the case with 1% ZEBRA- positive cells, a strong signal was obtained with anti-EA-R antibody and BHLF1 oligoprobes, which indicated early gene expression. EBV replication could be shown in this case by nonisotopic in situ DNA-DNA hybridization, which showed markedly increased numbers of EBV genomes in a few RS cells. Viral replication was confirmed using reverse transcriptase and polymerase chain reaction that detected transcripts from the BLLF1 gene encoding for the membrane antigen gp350/220. EBV replication in RS cells seems to be an exceptional event but may provide clues to mechanisms of control of viral latency and assume clinical implications in the future.
Lipoxin A4 induces hyperadhesiveness in human endothelial cells for neutrophils
Authors: Lerner R , Heimburger M , Palmblad J
Published: October 2019

Lipoxin A4 (LXA4), but not lipoxin B4, induced in vitro a dose- dependent, slowly emerging hyperadhesiveness in human umbilical vein endothelial cells (HUVEC), leading to a 1.9-fold increase in the binding of neutrophils (polymorphonuclear neutrophil granulocytes [PMN]). The maximal response to LXA4 occurred at 1 nmol/L and after 30 minutes of treatment of HUVEC. These response kinetics were intermediate in comparison with those of fast-acting inducers of HUVEC adhesivity (eg, thrombin, leukotriene B4 [LTB4] or platelet activating factor [PAF]), needing 5 to 15 minutes, or to the slow inducer interleukin-1 (IL-1 beta), which requires hours. The maximal LXA4 effect was slightly lower than that of LTB4 (100 nmol/L) and thrombin (1 U/mL), and less than that of PAF (100 nmol/L) or IL-1 beta (2.5 U/mL) (2.2-, 2.0-, 2.4-, or 13.6-fold increases, respectively). The LXA4 effect was inhibited by the PAF receptor antagonist WEB-2086; however, it could not be blocked by pertussis toxin. LXA4 conferred a slow, sustained increase in HUVEC cytosolic calcium ion concentrations, whereas thrombin did so rapidly and transiently. LXA4 also caused PMN to become hyperadhesive. Thus, this novel effect of LXA4 on HUVEC appears to be associated with endogenous PAF expression and slow increases of cytosolic calcium concentrations but not pertussis- sensitive G proteins.
Dynamics of GATA transcription factor expression during erythroid differentiation
Authors: Leonard M , Brice M , Engel JD , Papayannopoulou T
Published: October 2019

Although the formation of terminally differentiated erythroid cells has been shown to require the presence of a functional GATA-1 gene in vivo, the role of this transcription factor and other members of the GATA family at earlier stages of erythroid differentiation is unclear. In this report, the expression of GATA-1, GATA-2, and GATA-3 has been examined in enriched peripheral blood progenitors before and after culture in a well-characterized liquid culture system. In addition primary leukemic cells as well as several erythroleukemic and nonerythroid cell lines were analyzed for GATA factor expression. The results show that the profile of GATA factor expression in erythroid cells is distinct from that of myeloid or lymphoid lineages. Erythroleukemic cell lines express little or no GATA-3, but high levels of GATA-1 and GATA-2. When they are induced to display the terminal erythroid phenotype, little change in the level of GATA-1 is detected but a significant decline in the levels of GATA-2 is observed commensurate with the degree of maturation achieved by the cells. Enrichment of erythroid progenitors from peripheral blood leads to selection of cells that express both GATA-1 and GATA-2. As the enriched populations are cultured in suspension in the presence of multiple cytokines, the levels of both GATA-1 and GATA-2 initially increase. However, in cultures containing only erythropoietin, which show exclusive erythroid differentiation, the levels of GATA-1 continue to increase, whereas GATA-2 expression declines as erythroid maturation progresses. In contrast, cultures lacking Epo (ie, with interleukin-3 and kit ligand) display limited progression towards both the myeloid and erythroid pathways, and high levels of expression of both GATA-1 and GATA-2 are maintained. Despite the initial upregulation of GATA-1 expression in the latter cultures, terminal erythroid differentiation does not occur in the absence of erythropoietin. These results indicate that GATA-1 upregulation is associated with both the initiation and the maintenance of the erythroid program, but that these two processes appear to be under separate regulatory control. Thus, the dynamic changes in the levels of different GATA factors that occur during primary erythroid differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.
Selectin-mediated rolling of neutrophils on immobilized platelets
Authors: Buttrum SM , Hatton R , Nash GB
Published: October 2019

Interaction between neutrophils and platelets at the site of vascular damage or in ischaemic tissue may promote thrombosis and/or vascular occlusion. To study this interaction, we have developed a novel technique that allows visualization of adhesion of flowing neutrophils to immobilized, activated platelets. The total number of adherent neutrophils decreased with increasing wall shear stress in the range 0.05 to 0.4 Pa. Although a proportion of the adherent neutrophils were stationary, most were rolling with a velocity greater than 0.4 micron/s. The percentage of rolling cells increased with increasing wall shear stress, but the mean rolling cell velocity was nearly independent of shear stress. Adhesion of neutrophils was nearly abolished by treatment of the platelets with antibody to P-selectin, or by treatment of neutrophils with either neuraminidase, dextran sulfate, or EDTA. Studies with a series of antibodies to L-selectin (TQ-1, Dreg- 56, LAM1–3, and LAM1–10) suggested that this molecule was one neutrophil ligand for rolling adhesion. Thus, sialylated carbohydrate on neutrophils appears essential for P-selectin-mediated adhesion, and a proportion of this ligand may be presented by L-selectin. Treatment of the neutrophils with N-formyl-methionyl-leucyl-phenylalanine decreased the number of rolling cells, and increased the rolling velocity, possibly due to shedding of neutrophil ligand(s) and/or cell shape change. In vivo, immobilized platelets could play an important role in promoting attachment of neutrophils to vessel walls, eg, by slowing neutrophils so that integrin-mediated immobilization could occur.
Immunolocalization of beta 1 integrins in platelets and platelet- derived microvesicles
Authors: Wencel-Drake JD , Dieter MG , Lam SC
Published: October 2019

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.
Tumor-infiltrating lymphocytes derived from select B-cell lymphomas secrete granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha in response to autologous tumor stimulation
Authors: Schwartzentruber DJ , Stetler-Stevenson M , Rosenberg SA , Topalian SL
Published: October 2019

Tumor infiltrating lymphocytes (TIL) were cultured from 17 B-cell lymphoma specimens derived from patients with predominantly low-grade malignancies. Specimens included 15 lymph-node biopsies, 1 malignant pleural effusion, and PBL from 1 patient with circulating lymphoma cells. The phenotypic and proliferative characteristics of TIL cultured in interleukin-2 (IL-2) were studied, as well as cytolysis and cytokine secretion in response to autologous tumor. Flow cytometry of fresh tumor suspensions showed that 50% of cells (median) were malignant B cells and 36% were infiltrating T lymphocytes. After culture for approximately 1 month, TIL were 75% +/- 8% CD3+ (mean +/- SEM), 47% +/- 8% CD4+ and 35% +/- 7% CD8+. TIL proliferation was modest in most cases: the median maximum expansion was 32-fold in 25 days. Lysis of autologous tumor in 4-hour 51Cr release assays was mediated by 2 of 12 TIL studied, but was nonspecific. However, these same two TIL, when cocultured with various tumor stimulators, preferentially secreted tumor necrosis factor-alpha and granulocyte-macrophage colony- stimulating factor after autologous tumor stimulation; unstimulated TIL secreted undetectable or barely detectable levels of these cytokines. In one TIL culture, cytokines were secreted by purified CD4+ TIL but not by CD8+ cells, and secretion was completely abrogated by the anti- major histocompatibility complex (MHC) class II antibody IVA12. Thus, although specific cytokine secretion by lymphoma TIL in response to autologous tumor was observed, it occurred in fewer than 20% of patients studied.
Efficient killing of chronic B-lymphocytic leukemia cells by superantigen-directed T cells
Authors: Wallgren A , Festin R , Gidlof C , Dohlsten M , Kalland T , Totterman TH
Published: October 2019

In vitro studies have indicated that chronic lymphocytic leukemia of B- cell origin (B-CLL) is resistant to cytotoxic effector lymphocytes such as natural killer and lymphokine activated killer (LAK) cells. We show here that B-cell cells are sensitive to Staphylococcal enterotoxin (SE) A-directed T-cell killing. Activation of the target cells by phorbol ester (tetradecanoyl phorbol acetate, [TPA]) greatly enhances their sensitivity to lysis. In SE-dependent cellular cytotoxicity (SDCC), members of the SE superantigen family form a bridge between T cells and target cells expressing major histocompatability complex class II molecules. Binding of SEA to the T-cell-receptor V beta region induces a strong cytotoxic capacity and cytokine production. Cells from 9 B-CLL patients were cultured in the presence or absence of TPA and used as targets in a 4-hour SDCC assay using an allogeneic T-cell line as effector. At an effector:target cell ratio 30:1, 70% to 80% of TPA- induced B-CLL cells were killed. Even at the effector:target ratio of 3:1, 47% +/- 6% of TPA-activated B-cell cells were lysed compared with 13% +/- 2% of resting cells (P < .001). A T-cell line established from a B-CLL patient killed autologous tumor cells as efficiently as allogeneic effectors. SEA-directed T cells were far more lytic to B-CLL cells compared with LAK cells or lectin (phytohemagglutinin-directed T cells. Mechanisms of SDCC lysis were investigated. Effector plus target cell supernatants contained high levels of tumor necrosis factor (TNF)- alpha and interferon-gamma, but these supernatants were not directly toxic to B-CLL cells in short term culture. High concentrations of recombinant TNF-alpha or TNF-beta had no lytic effect. Addition of neutralizing anti-TNF-alpha and anti-TNF-beta antibodies into the SDCC assay did not inhibit SEA-directed T-cell killing. TPA-activated B-CLL cells showed a 1.2- to 13-fold increased expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA)-1, and LFA-3, whereas expression of HLA class II molecules increased up to 5 times. The expression of CD72, CD40, and BB-1/B7 increased 1.8 to 4.5 times. The role of these surface molecules in SDCC was analyzed in blocking experiments with monoclonal antibodies. Antibodies to ICAM-1, CD18, and HLA-DR abolished the cytotoxicity, and a substantial reduction was seen with antibody to CD72.(ABSTRACT TRUNCATED AT 400 WORDS)
Hodgkin's lymphoma-derived tissue serially transplanted into severe combined immunodeficient mice
Authors: Kapp U , Wolf J , Hummel M , Pawlita M , von Kalle C , Dallenbach F , Schwonzen M , Krueger GR , Muller-Lantzsch N , Fonatsch C
Published: October 2019

Hodgkin (H) and Reed-Sternberg (RS) cells are considered to be the malignant cell population in Hodgkin's disease (HD). To date, their analysis has been hampered by their scarcity in primary tumors, poor growth in vitro, and lack of an animal model. To establish an in vivo system for the characterization of the malignant cells in HD, tumor biopsy samples from 13 HD patients were transplanted beneath the renal capsule or into the liver of severe combined immunodeficient (SCID) mice. HD-derived tissue from three patients gave rise to human tumors in SCID mice. Three different histologic patterns were observed: (1) lymphoproliferative disease (LPD), (2) anaplastic large cell lymphoma (ALCL), (3) Hodgkin-like lesions (HDLL). Immunohistochemical analysis showed that the tumors consisted of activated B cells (CD30+, CD20+). Epstein-Barr virus (EBV)-encoded transcripts were found in 80% to 100% of the tumor cells, although H and RS cells in the primary tumors of two patients were EBV-. All tumors examined (3 of 3) and the majority (6 of 10) of cell lines recultured in vitro had an abnormal karyotype. Southern blot analysis of the human Ig heavy chain gene showed that monoclonal or oligoclonal tumors of different B-cell origin grew in the SCID mice from the same germ line-configurated primary biopsy specimen. Our data suggest that the human cells in the SCID mice have either been derived from EBV superinfected H and RS cells or from EBV-infected bystander cells. If the latter is true, then these bystander cells must be genetically abnormal. The genetic defect would be either aneuploidy or instable euploidy. In either case, the cells might proliferate into malignant aneuploid HDLL or ALCL under the influence of EBV and the special environment encountered in the SCID mice.
Tyrosine phosphorylation of P160 BCR by P210 BCR-ABL
Authors: Lu D , Liu J , Campbell M , Guo JQ , Heisterkamp N , Groffen J , Canaani E , Arlinghaus R
Published: October 2019

It is well established that the chimeric BCR-ABL gene formed by joining parts of the BCR and ABL genes plays a key role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias. We report that simultaneous expression of P210 BCR-ABL and P160 BCR in simian COS-1 cells yielded stable complexes of these two proteins, and induced phosphorylation of P160 BCR on tyrosine residues in vivo. Tyrosine phosphorylation of a deletion mutant encoding 553 amino acids of BCR N- terminal sequences was also detected when it was coexpressed with P210 BCR-ABL. We propose that tyrosine phosphorylation of P160 BCR by P210 BCR-ABL and their stable physical interaction may perturb normal BCR functions and that these alterations are directly involved in the pathologic processes found in Ph chromosome-associated leukemias.