bluuebunny/crossref_metadata_2025 huggingface dataset:
The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: localization of interaction sites and physiologic relevance
Authors: Keijer J , Linders M , van Zonneveld AJ , Ehrlich HJ , de Boer JP , Pannekoek H
Published: October 2019

Plasminogen activator inhibitor 1 (PAI-1), an essential regulatory protein of the fibrinolytic system, harbors interaction sites for plasminogen activators (tissue-type [t-PA] and urokinase-type [u-PA]) and for fibrin. In this study, anti-PAI-1 monoclonal antibodies (MoAbs) were used to identify interaction sites of PAI-1 with these components. The binding sites of 18 different MoAbs were established and are located on five distinct “linear” areas of PAI-1. MoAbs, binding to two distinct areas of PAI-1, are able to prevent the inhibition of t-PA by PAI-1. In addition, two interaction sites for fibrin were identified on PAI-1. The area located between amino acids 110 and 145 of PAI-1 contains a binding site for both components and its significance is discussed in the context of the t-PA inhibition by fibrin-bound PAI-1. Subsequently, the MoAbs were used to assess the role of platelet-PAI-1 in clot-lysis. An in vitro clot-lysis system was used to demonstrate that clot-lysis resistance is dependent on the presence of activated platelets and that PAI-1 is a major determinant for lysis-resistance. We propose that, upon activation of platelets, PAI-1 is fixed within the clot by binding to fibrin and retains its full capacity to inhibit t-PA and u-PA.
Establishment and characterization of a new human leukemia cell line derived from M4E0
Authors: Yanagisawa K , Horiuchi T , Fujita S
Published: October 2019

A new human leukemia cell line, designated as ME-1, was established from the peripheral blood leukemia cells of a patient with acute myelomonocytic leukemia with eosinophilia (M4E0). This cell line has the characteristic chromosome abnormality of M4E0, inv(16) (p13q22). When cultured in RPMI 1640 medium containing 10% fetal calf serum, ME-1 cells were monoblastoid, but with the addition of cytokines such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, or medium conditioned by phytohemagglutinin-stimulated human peripheral leukocytes (PHA-LCM), the cells exhibited differentiation to macrophage-like cells. PHA-LCM also promoted eosinophilic-lineage differentiation of this cell line, although IL-5 did not do so. To elucidate the mechanism of proliferation and differentiation of ME-1 cells, we studied the effect of a potent inhibitor of protein kinase C, 1-(5-isoquinolinyl-sulfonyl)-2- methylpiperazine (H-7), on colony formation of ME-1 cells. H-7 inhibited colony formation of ME-1 cells by IL-3 or GM-CSF dose dependently, but had little inhibitory effect on colony formation by IL- 4. These results indicate that the proliferation and differentiation of ME-1 cells by IL-3 or GM-CSF were related to the activation of protein kinase C, while those by IL-4 involved other regulatory systems. ME-1 cells should be useful for studying the pathogenesis of M4E0 and the mechanisms of proliferation and differentiation of leukemic and normal progenitors by cytokines.
Expression of 5'-nucleotidase (CD73) related to other differentiation antigens in leukemias of B-cell lineage
Authors: Pieters R , Thompson LF , Broekema GJ , Huismans DR , Peters GJ , Pals ST , Horst E , Hahlen K , Veerman AJ
Published: October 2019

Ecto-5′nucleotidase (5′NT; CD73) expression was studied with a monoclonal antibody (7G2) and a radiochemical assay and compared with the expression of other antigens in B-cell-lineage leukemias on cells from 100 leukemic patients and two cell lines. A B-cell origin was confirmed by the expression of CD19 and HLA-DR. Four stages of B-cell leukemias were defined: stage I (pro-B) as CD10-, cytoplasmic mu- (c mu- ), surface Ig- (sIg-); stage II (cALL) as CD10+/c mu-/sIg-; stage III (pre-B) as CD10+ or -/c mu+/sIg-; and stage IV (B) as CD10-/c mu-/sIg+. A linear correlation was found between immunohistochemical and radiochemical determination of 5′NT (r = .86). 5′NT expression was low in T-cell leukemias and stage I, high in stages II and III, and low again in stage IV of B-cell leukemias. 5′NT expression was not related to c mu, CD20, CD21, CD22, CD34, and terminal deoxynucleotidyl transferase (TdT) expression, but was significantly related to CD10 and inversely related to kappa/lambda expression. However, the 5′NT activity in CD10+ leukemias (stages II and III) shows a very wide range. Within the group of CD10+ leukemias no differences were detected between 5′NT+ and 5′NT- cells in their expression of other B-cell antigens. We conclude that the place of 5′NT in leukemias corresponding to early stages of B-cell development has been characterized. 5′NT is expressed in CD10+ stages and decreases before the expression of sIgs. Future studies should make clear whether a high expression of this enzyme in CD10+ stages is a normal maturation phenomenon or a malignant phenomenon.
Rearrangement of the chromosome 11 bcl-1 locus in centrocytic lymphoma: analysis with multiple breakpoint probes
Authors: Williams ME , Meeker TC , Swerdlow SH
Published: October 2019

Centrocytic lymphoma is a B-cell non-Hodgkin's lymphoma (NHL) composed of lymphocytes resembling cleaved follicular center cells (centrocytes). Previous studies have suggested an association between t(11;14) chromosomal translocations and bcl-1 rearrangement in centrocytic and related intermediate lymphocytic lymphomas. To further characterize the association between bcl-1 and centrocytic lymphoma, Southern blot analysis was performed on samples from 23 patients using four separate bcl-1 breakpoint probes spanning 63 kb of the chromosome 11 bcl-1 locus. Rearrangements were identified in six patients with the major translocation cluster (MTC) probe and in another six with probe p94PS, located about 24 kb 5′ of MTC. Eleven of these 12 cases showed comigration of rearranged bcl-1 and Ig heavy chain-joining genes, consistent with the t(11;14) chromosomal translocation. No rearrangements were observed with the bcl-1 locus probes p210 or p11EH located 5′ of p94PS, nor with bcl-2 or c-myc oncogene probes. No bcl-1 rearrangements were identified in B-cell follicular NHL (15), small noncleaved cell (Burkitt's and non-Burkitt's) NHL (8), T-cell NHL (4), multiple myeloma (14), and pre-B-cell acute lymphoblastic leukemia (9). One of 23 B-cell NHL of large cell type and one of 19 chronic lymphocytic leukemias or small lymphocytic NHL had MTC rearrangement. Thus, bcl-1 rearrangement occurred at MTC or p94PS in 12 of 23 centrocytic lymphomas (52%), confirming a nonrandom association and suggesting a pathogenetic role for the bcl-1 locus in this immunohistologic subtype of NHL.
Differential transcriptional and posttranscriptional regulation of gene expression of the colony-stimulating factors by interleukin-1 and fetal bovine serum in murine fibroblasts
Authors: Falkenburg JH , Harrington MA , de Paus RA , Walsh WK , Daub R , Landegent JE , Broxmeyer HE
Published: October 2019

Colony-stimulating factors (CSF) are important factors in the proliferation and differentiation of hematopoietic progenitor cells (HPC), and in the survival and activation of mature blood cells. Interleukin-1 (IL-1) combined with fetal bovine serum (FBS) strongly induces the expression of macrophage-CSF (M-CSF), granulocyte-CSF (G- CSF), and granulocyte-macrophage-CSF (GM-CSF) in fibroblasts. Here, we report on the regulation of CSF gene expression in murine fibroblasts following IL-1 and FBS stimulation. We demonstrate that 10T1/2 murine fibroblasts induced by FBS or IL-1 accumulate M-CSF messenger RNA (mRNA). G-CSF mRNA expression was induced by IL-1, and not by FBS. For GM-CSF expression, induction with both FBS and IL-1 was required. Blocking studies with actinomycin-D showed that active transcription is essential for accumulation of all three CSF mRNAs. After blocking protein synthesis with cycloheximide, IL-1- or FBS-induced M-CSF expression and IL-1 plus FBS-induced GM-CSF expression still occurred and was increased. IL-1-induced G-CSF expression was completely prevented in these cells by pretreatment with cycloheximide, illustrating that, for this effect, intermediate protein synthesis was required. The half-lives of M-CSF transcripts were not substantially altered by addition of IL-1, FBS, or FBS plus IL-1. Using nuclear run- on assays, we demonstrated that the transcription rate of M-CSF was increased up to 20-fold by the addition of FBS, IL-1, or FBS plus IL-1. After blocking protein synthesis with cycloheximide, IL-1-or FBS- induced increase in M-CSF transcription rate was also observed. GM-CSF transcription increased up to fourfold after induction with FBS or IL- 1. G-CSF transcription rate was not altered by FBS or IL-1. Our results indicate that M-CSF expression induced by FBS or IL-1 in these fibroblasts is primarily regulated at the transcriptional level. GM-CSF expression appears to be regulated both transcriptionally and posttranscriptionally, and G-CSF expression is regulated mainly at the posttranscriptional level.
Acute leukemia after a primary myelodysplastic syndrome: immunophenotypic, genotypic, and clinical characteristics [see comments]
Authors: San Miguel JF , Hernandez JM , Gonzalez-Sarmiento R , Gonzalez M , Sanchez I , Orfao A , Canizo MC , Lopez Borrasca A
Published: October 2019

We studied the nature of blast cells in 41 patients with acute leukemia following a previous primary myelodysplastic syndrome (MDS) by a combined multiparameter analysis including morphologic, immunophenotypic, and molecular genetic (Igs, T-cell receptor (TCR)- beta, -gamma, and -delta and the major breakpoint cluster region [M- bcr]) investigations. In addition, the clinical and hematologic characteristics according to the immunophenotype of blast cells were analyzed. Our results show that, although the granulocytic and/or monocytic lineages are those most commonly involved in these acute leukemias, other cell components, including the megakaryocytic and lymphoid, may be present (12% and 15% of the cases, respectively). Moreover, both morphologic and phenotypic studies show the frequent coexistence of two or three cell populations. Interestingly, in all cases the lymphoblastic component constantly displayed an early B phenotype (CD19+, CD10-, TdT+). Upon analyzing whether the type of MDS conditioned any differences in the immunophenotype of blast cells, we observed that, although the lymphoid lineage may be involved in all MDS subgroups, some differences emerge within the myeloid leukemic transformations. Thus, the refractory anemias with excess of blasts (RAEB) and RAEB in transformation displayed a significantly higher incidence of myeloblastic and megakaryoblastic transformations, while in the RA, RA with ring sideroblasts and chronic myelomonocytic leukemia, the granulo-monocytic phenotype predominated. In addition, our results show that the clinical and hematologic characteristics of these patients may be partially related to the immunophenotype of the blast cells. Ig heavy chain gene rearrangements were found in two of 19 patients analyzed (11%), one with a hybrid leukemia (lymphoid-myeloid) and the other with a granulo-monocytic phenotype. Two other hybrid transformations analyzed were in germline configuration. Gamma and delta gene rearrangements were found in 21% and 37% of these acute transformation, respectively. The TCR-beta and M-bcr were in germline configuration in all 19 cases studied. In summary, immunophenotype and molecular studies point to a pluripotent stem cell with preferential myeloid commitment as the target cell of leukemias following a primary MDS.
Phase I trial of recombinant human macrophage colony-stimulating factor in patients with invasive fungal infections
Authors: Nemunaitis J , Meyers JD , Buckner CD , Shannon-Dorcy K , Mori M , Shulman H , Bianco JA , Higano CS , Groves E , Storb R
Published: October 2019

A phase I dose escalation trial of recombinant human macrophage colony- stimulating factor (rhM-CSF) in combination with conventional antifungal therapy was conducted in 24 marrow transplant recipients with invasive fungal infection. Daily doses ranged from 100 to 2,000 micrograms/m2/d. Toxicity, such as constitutional symptoms, directly ascribed to rhM-CSF was not observed; however, transient, dose-related thrombocytopenia was observed. Patients who received 2,000 micrograms/m2/d of rhM-CSF had a mean reduction in platelet count of 61,000/mm3 during the rhM-CSF infusion period, which was significant when compared with patients who received lower doses of rhM-CSF (P = .008). Fourteen of the 16 patients who received rhM-CSF after undergoing allogeneic bone marrow transplantation had no change in the severity of graft-versus-host disease (GVHD) while receiving rhM-CSF. One had an increase in the severity of GVHD and one had a decrease. There were no effects on neutrophil, monocyte, or lymphocyte counts. Six patients had resolution of their infections, 12 were not evaluable for response, and six did not respond. Ten patients survived 100 days after initiation of rhM-CSF and 14 died. Further trials with rhM-CSF to assess antifungal activity are indicated.
Enzyme replacement therapy for Gaucher disease
Authors: Beutler E , Kay A , Saven A , Garver P , Thurston D , Dawson A , Rosenbloom B
Published: October 2019

Four patients with moderately severe type I Gaucher disease were treated with commercially available mannose terminated glucocerebrosidase (Ceredase; Genzyme, Boston, MA) for up to 13 months. The enzyme was administered at the rate of three to four times weekly at one fourth the total recommended dosage, greatly decreasing the cost. Marked regression of hepatomegaly and improvement in liver function tests, peripheral blood counts, and serum angiotensin- converting enzyme levels were documented. The two patients with pulmonary involvement manifested improvement in pulmonary function tests. Skeletal disease remained unchanged.
Phase I-II trial of pentoxifylline for the prevention of transplant- related toxicities following bone marrow transplantation [published erratum appears in Blood 1992 Jun 15;79(12):3397] [see comments]
Authors: Bianco JA , Appelbaum FR , Nemunaitis J , Almgren J , Andrews F , Kettner P , Shields A , Singer JW
Published: October 2019

Disease relapse and transplant related toxicities have limited the application of bone marrow transplantation (BMT) in the treatment for hematologic malignancies. Because elevated levels of tumor necrosis factor alpha (TNF-alpha) have been correlated with the development of transplant related complications, we conducted a phase I-II trial of pentoxifylline (PTX), a xanthine derivative capable of down-regulating TNF-alpha production, in patients with hematologic malignancies undergoing BMT. Thirty consecutive adult patients (median age, 34) were entered and received either an allogeneic (n = 26) or autologous (n = 4) BMT. Patients were enrolled at increasing dose levels (1,200, 1,600, and 2,000 mg/d) from day -10 through day +100 posttransplant. PTX was well tolerated with no significant adverse side effects noted at any of the dose levels administered. The actuarial day 100 survival for these 30 patients was 90% (95% confidence interval 79% to 100%). When compared with a good risk control group, PTX recipients experienced less mucositis (3.7 +/- 1.1 v 18.7 +/- 1.1 days, P = .004), less hepatic venocclusive disease (10% v 65%, P = .001), a lower incidence of renal insufficiency (3% v 65%, P = .0003), required less days of total parenteral nutrition (TPN) (24.0 +/- 1.3 v 35.0 +/- 2.4, P = .001) and were discharged from the hospital earlier than controls (day 26.0 +/- 1.8 v 37.0 +/- 3.8, P = .01). In addition the incidence of graft-versus-host disease (GVHD) greater than or equal to grade II was also reduced among the PTX recipients (35% v 68%, P = .03). PTX at doses in excess of 1,200 mg/d further reduced the severity of mucositis, and TPN requirements resulting in earlier hospital discharge than patients receiving 1,200 mg/d of PTX. In this study oral administration of PTX in doses up to 2,000 mg/d was well tolerated and associated with a reduction in morbidity and mortality in patients undergoing BMT. Prospective randomized trials are currently in progress to test these preliminary observations.
Use of recombinant human interleukin-2 in conjunction with syngeneic bone marrow transplantation in mice as a model for control of minimal residual disease in malignant hematologic disorders
Authors: Ackerstein A , Kedar E , Slavin S
Published: October 2019

Unlike allogeneic bone marrow transplantation (BMT), autologous BMT is not accompanied by immune-mediated graft-versus-leukemia (GVL) effects; hence, the relapse rate observed after autologous BMT in malignant hematologic disorders is higher than that observed after allogeneic BMT. Autologous BMT represents a much safer medical procedure available for many patients in need in situations where allogeneic BMT is not feasible or risky. The present experiments were designed to investigate whether it might be possible to combine the therapeutic benefits of autologous BMT with additional immunotherapy after BMT. The tumor model used for investigating GVL effects was the murine B-cell leukemia (BCL1), a spontaneous, nonimmunogenic, highly lethal leukemia of BALB/c origin. BALB/c mice inoculated with 10(3) BCL1 leukemia cells were treated on day-1 with cyclophosphamide 100 mg/kg and transplanted with normal syngeneic BM cells on day 0. High-dose recombinant interleukin-2 (rIL-2) (100,000 Cetus units x 3/day intraperitoneally x 5 consecutive days) was initiated on day +1, +7, or +21 after BMT. Kinetics of lymphocyte reconstitution after syngeneic BMT indicated a steep increase in the absolute number of peripheral blood lymphocytes on days 17 through 24. All experimental groups were observed for relapse. Mice receiving no rIL-2 therapy relapsed and died within 50 days after BMT, whereas mice receiving rIL-2 showed long-term disease-free survival. Optimal time for administration of rIL-2 was noted at 3 weeks post-BMT, with 90% of the mice surviving with no evidence of disease for more than 1 year. Similarly, when 10(4) BCL1 cells were given 1 day after syngeneic BMT to simulate minimal residual disease after syngeneic BMT, rIL-2 therapy administered at 14 days post-BMT seemed effective in prolonging disease-free survival in contrast to the same regimen given at 1 day after BMT. Our data suggest that immunotherapy with rIL-2 should be further investigated as a new immunotherapeutic tool for decreasing the relapse rate after BMT for hematologic malignancies.
On the target specificity of plasminogen activator inhibitor 1: the role of heparin, vitronectin, and the reactive site
Authors: Keijer J , Linders M , Wegman JJ , Ehrlich HJ , Mertens K , Pannekoek H
Published: October 2019

Plasminogen activator inhibitor 1 (PAI-1) is the fast-acting inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA, u- PA) and is an essential regulatory protein of the fibrinolytic system. In the presence of either the protein vitronectin or the glycosaminoglycan heparin, PAI-1 is also an efficient inhibitor of thrombin. To assess whether these cofactors turn PAI-1 into a general protease inhibitor or whether their influence is restricted to thrombin, the second-order association rate constants between PAI-1 and the human plasma proteases t-PA, u-PA, plasmin, thrombin, Factor Xa (FXa), and Factor XIIa (FXIIa) in the absence and in the presence of either vitronectin or heparin are determined. In addition, the role of the PAI-1 reactive site P3 to P3′ residues for the specificity of inhibition was studied by using PAI-1 reactive site mutants. Our results show that: (1) Heparin exclusively increases the rate of inhibition of thrombin by PAI-1, whereas in the presence of heparin the rate of inhibition of the other proteases is not altered; (2) Vitronectin is an obligatory cofactor for the inhibition of thrombin by PAI-1. In addition, vitronectin moderately increases the rate of inhibition by PAI-1 of u-PA and of plasmin, but does not alter the rate of inhibition of t-PA, FXa, or FXIIa; (3) Apart from the important role of the P1 residue, no consensus can be presented on the nature of other residues within the P3 to P3′ region with regard to target protease specificity.
Endothelial cells express the interleukin-1 receptor type I
Authors: Boraschi D , Rambaldi A , Sica A , Ghiara P , Colotta F , Wang JM , de Rossi M , Zoia C , Remuzzi G , Bussolino F
Published: October 2019

Interleukin-1 (IL-1) profoundly affects a number of functions of vascular cells. Two distinct IL-1 receptors (IL-1R) are expressed on different cell types: the 80 Kd IL-1RI on T cells and fibroblasts, and the 68 Kd IL-1RII on B cells and myelomonocytic cells. The presence and functionality of IL-1R on vascular cells has been investigated by using polyomatransformed mouse endothelial cell (EC) lines (sEnd.1 and tEnd.1). These cells expressed specific and saturable binding sites for IL-1 (1,273 sites per cell with kd 9.5 x 10(-11) mol/L for sEnd.1, and 771 sites per cell with kd 8.5 x 10(-11) mol/L for tEnd.1, with radioiodinated IL-1 alpha as ligand). Binding of IL-1 was also evident at single cell level by autoradiography. By cross-linking studies, the molecular weight of the IL-1 binding protein on EC was approximately 80 Kd. This was confirmed by the presence in EC of mRNA for the 80 Kd IL- 1RI. The IL-1RI on EC was apparently functional, since EC responded to IL-1 with IL-6 mRNA expression and IL-6 bioactivity production. These results were extended to human EC and vascular smooth muscle cells, which were also found to express mRNA for IL-1RI.
Analysis at the clonal level of T-cell phenotype and functions in severe aplastic anemia patients
Authors: Viale M , Merli A , Bacigalupo A
Published: October 2019

The aim of this study was to analyze at the clonal level the phenotype and functions of T cells from patients with severe aplastic anemia (SAA). For this purpose we studied 175 T-cell clones obtained from peripheral blood (PB) and bone marrow (BM) of four SAA patients and 97 clones from two healthy controls. The percentage of CD8+ T-cell clones obtained from the patients' PB and BM was higher, but not significantly (P = .07 and P = .14, respectively), than that obtained in controls. A higher proportion of T-cell clones from SAA patients exhibited lectin- dependent cytolytic activity and especially natural killer-like activity when compared with controls (PB: P less than .01, P less than .05; BM: P less than .05, P less than .01, respectively). Lymphokine release was tested before and after mitogen stimulation. A number of patients' clones were able to release interferons (IFNs) spontaneously (PB: 28.6% v 0%, P less than .05; BM: 28.6% v 0%, P less than .10). After mitogen stimulation, patients' BM T-cell clones produced IFNs in greater proportions (90.9% v 46.7%, P less than .01) and in greater quantities (PB: 25.5 arbitrary units [AU]/mL v 5.7 AU/mL, P less than .03; BM: 26 AU/mL v 9.1 AU/mL, P = .011) as compared with controls. Tumor necrosis factor (TNF) activity was not found in supernatants of unprimed T-cell clones. After mitogen stimulation, PB T-cell microcultures produced TNF alpha in greater proportions (97.9% v 72.2%, P less than .01) and, also in this case, in greater quantities (PB: 7.2 AU/mL v 1.5 AU/mL, P = .007; BM: 9.9 AU/mL v 1.5 AU/mL, P = .003) than controls. In conclusion, T-cell clones from SAA patients exhibit predominantly a CD8+ phenotype, a greater cytotoxic activity, and can be shown to produce greater quantities of suppressor lymphokines when compared with controls.
Heterogeneity of the molecular basis of hereditary pyropoikilocytosis and hereditary elliptocytosis associated with increased levels of the spectrin alpha I/74-kilodalton tryptic peptide
Authors: Floyd PB , Gallagher PG , Valentino LA , Davis M , Marchesi SL , Forget BG
Published: October 2019

Hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis are closely related, congenital disorders of the red blood cell usually associated with defective spectrin self-association and abnormal limited tryptic digestion of the N-terminal of domain of spectrin. Enhanced cleavage by trypsin of spectrin from affected individuals at arginyl residue 45* and lysyl residue 48* frequently yields increased amounts of an alpha 1/74-Kd fragment at the expense of the normal alpha 1/80-Kd parent fragment. Limited tryptic digestion of three unrelated individuals with HPP showed the alpha 1/74 defect. To ascertain the molecular defect responsible for the abnormality, the structure of exon 2 of the alpha-spectrin gene was examined. Genomic DNA from the subjects was amplified by the polymerase chain reaction using primers flanking exon 2. Restriction endonuclease digestion of amplified products showed the loss of the HindIII site at codons 47 and 48 in one allele of subject 1 and abolished the AhaII site at codons 27 and 28 in one allele of subjects 2 and 3. Nucleotide sequence analysis of subcloned amplified DNA from the HPP subjects showed three novel amino acid substitutions. In subject 1 (a black individual), a single base substitution (AAG----AGG) at codon position 48 changes amino acid residue lysine to arginine. In subject 2 (a white individual), a single base substitution (CGT----AGT) at codon 28 changes arginine to serine. In subject 3 (a black individual), a different base substitution at position 28 (CGT----CTT) changes arginine to leucine. These mutations occur at positions of the alpha l domain where other mutations have also been described, indicating that the normal residues at these positions play an important role in spectrin dimer self-association and thus, in membrane stability.
Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease
Authors: Reusser P , Riddell SR , Meyers JD , Greenberg PD
Published: October 2019

The high rate of severe cytomegalovirus (CMV) disease after bone marrow transplantation (BMT) is related to the profound immunodeficiency posttransplant. Because cytotoxic T lymphocytes (CTL) have been implicated in resistance to viral infections, we examined the restoration of the CMV-specific CTL response in 20 patients who received bone marrow from HLA-matched, CMV-seropositive donors. Blood specimens were obtained from patients at 1, 2, and 3 months after BMT and from the donors on a single occasion. Peripheral blood mononuclear cells were cocultured with CMV-infected donor-derived fibroblasts for 2 weeks and then tested for cytotoxicity against CMV-infected and uninfected autologous or HLA-mismatched fibroblasts. Cytolytic activity was detected in all 20 donors, with specificity for autologous CMV- infected targets demonstrable in 17 (median CMV-specific lysis at an effector:target ratio of 15:1 was 32%, range 18% to 83%). Specific lysis was mediated by CD8+, class I-restricted T cells, as shown by inhibition with anti-class I monoclonal antibody and by selective depletion of effector cells. By contrast, CMV-specific CTL were detected in only 10 of 20 patients after BMT (median lysis 29% at 3 months post-BMT). None of these 10 patients developed CMV pneumonia, whereas 6 of the 10 patients with an undetectable CMV-specific CTL response after BMT died with CMV pneumonia. These results demonstrate that restoration of CMV-specific CTL responses may require an extended time period after BMT in some patients, and that such patients are at increased risk of developing severe CMV disease. Approaches to reconstitute CMV immunity in BMT patients by adoptive transfer of CMV- specific CD8+ CTL clones derived from the bone marrow donor are now being pursued.
A clinical and experimental study on all-trans retinoic acid-treated acute promyelocytic leukemia patients
Authors: Chen ZX , Xue YQ , Zhang R , Tao RF , Xia XM , Li C , Wang W , Zu WY , Yao XZ , Ling BJ
Published: October 2019

Fifty patients with acute promyelocytic leukemia (APL) have been treated with all-trans retinoic acid (RA). In vitro induced differentiation of primarily cultured bone marrow cells from the patients, colony-forming unit granulocyte-macrophage (CFU-GM) and L-CFU colony-forming assays, and karyotype analysis were performed over the treatment course. The very high bone marrow complete remission (CR) rate (94%) suggested that all-trans RA was superior to conventional chemotherapeutic regimens for the treatment of APL. The leukemic clone was reduced by RA-induced terminal differentiation and loss of proliferation capacity of leukemic cells. Relapse after CR in about 40% of patients was the major reason for the failure of the RA treatment. Patients who relapsed after a chemotherapy-maintained CR could be effectively reinduced to second CR by RA. However, if relapse occurred after a CR maintained by both RA and chemotherapy, the sensitivity of newly emerged leukemic clones to RA was greatly reduced. Therefore, it is suggested that RA should be replaced by conventional chemotherapy as soon as CR is achieved. Laboratory studies proved valuable in selecting cases for RA therapy and in predicting therapeutic effects and prognosis.
Enzymic control of the expression of the X determinant (CD15) in human myeloid cells during maturation: the regulatory role of 6- sialytransferase
Authors: Skacel PO , Edwards AJ , Harrison CT , Watkins WM
Published: October 2019

To establish the basis for the reduced expression of the X determinant on leukemic blasts and the changes in antigenic expression that occur during myeloid maturation, the presence on myeloid cells of X and related structures was examined in conjunction with studies on the activities of the glycosyltransferases involved in their biosynthesis. Expression of X and sialyl-X was weak on blasts in comparison with neutrophils despite the presence of the requisite precursor structures. Much higher levels of 3-fucosyltransferase activity were found in blasts than in neutrophils when nonsialylated substrates were used, but, whereas the enzyme in neutrophils reacted equally well with 3′- sialylated and nonsialylated acceptors, the enzyme in blasts showed a marked preference for nonsialylated substrates. 6′-Sialyltransferase activity was strong in blasts but was not detectable in neutrophils, whereas a much lower level of 3′-sialyltransferase activity was present in both blasts and neutrophils. Dimethyl sulfoxide-induced maturation of HL60 cells was associated with (1) a decrease in both 6′- sialyltransferase and 3-fucosyltransferase activities, (2) a change in the substrate specificity of 3-fucosyltransferase towards that found in mature cells, and (3) increased cell surface expression of sialyl-X. These results suggest that the reduced expression of X in myeloblasts is related to the presence of the strong 6′-sialyltransferase, which uses the precursor substrate at the expense of the 3-fucosyltransferase and prevents the synthesis of X and sialyl-X. The developmental regulation of the levels of 3′- and 6′-sialyltransferases, and the level and specificity of the 3-fucosyltransferases, therefore controls the expression of X and its degree of sialylation.
Modulation of erythropoiesis by novel human bone marrow cytochrome P450- dependent metabolites of arachidonic acid
Authors: Abraham NG , Feldman E , Falck JR , Lutton JD , Schwartzman ML
Published: October 2019

In the hematopoietic system the adherent stromal cells produce cytokines necessary for proliferation and differentiation of hematopoietic cells. In the present study, we showed the ability of adherent stromal cells to generate novel metabolites of arachidonic acid via the NADPH-cytochrome P450-dependent monooxygenase system. These metabolites were recovered in the incubation media, suggesting their release from cells. The formation of arachidonic acid metabolites was inhibited by 7-ethoxyresorufin and SKF-525A, but not by indomethacin or BW-755C. By using two-step high-pressure liquid chromatography (HPLC), bone marrow-adherent stromal cells and incubation media showed the presence of metabolites in a peak eluted at 19 to 20 minutes. The isolated HPLC peak was used to measure its effect on colony-forming unit-erythroid (CFU-E) growth and compare it with that of synthetic cytochrome P450 arachidonate metabolites, 19- and 20- hydroxyeicosatetraenoic (HETE) acid. These bone marrow cytochrome P450 arachidonic acid metabolites at picomolar concentration potentiated erythropoietin (Epo)-induced CFU-E growth by fourfold to sixfold. Addition of 19- and 20-HETE to the bone marrow culture resulted in a potentiating effect on CFU-E number in a dose-dependent manner. 20-HETE was much more potent in stimulating CFU-E growth than 19-HETE at a similar concentration of 10(-11) mol/L. The potentiating effect of 20- HETE resulted in a shifting to the left of the dose-response curve to Epo. To substantiate the finding of an active NADPH-dependent cytochrome P450-metabolizing system, we further examined the ability of adherent cells to metabolize exogenous pharmacologic compounds such as benzo(a)pyrene, a substrate for the heme-cytochrome P450 system, aryl hydrocarbon hydroxylase. The adherent stromal cytochrome P450 metabolizes benzo(a)pyrene at comparable levels to blood vessel endothelial cells. These novel observations underscore the importance of adherent stromal cytochrome P450 to metabolize endogenous substrates, including arachidonic acid, to compounds that may interact in a paracrine manner with Epodependent hematopoietic cells.
Suppression of chronic myelogenous leukemia colony growth by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors: a novel application for inhibitors of IL-1 activity
Authors: Estrov Z , Kurzrock R , Wetzler M , Kantarjian H , Blake M , Harris D , Gutterman JU , Talpaz M
Published: October 2019

In this study, we investigated the role of interleukin-1 beta (IL-1 beta) in the malignant evolution of chronic myelogenous leukemia (CML) and the functional activity of IL-1 inhibitors. Bone marrow (BM) and peripheral blood (PB) low-density cells from 38 CML patients were studied in the colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte colony culture assay. Samples from patients with early stage, interferon-alpha (IFN)-sensitive disease formed hematopoietic colonies in the presence of fetal calf serum (FCS), erythropoietin (Epo), and one of the following: granulocyte-macrophage colony- stimulating factor (10 ng/mL), IL-3 (15 ng/mL), both, or phytohemagglutinin-conditioned medium. The addition of IL-1 beta augmented IFN-sensitive CML colony growth in a dose-dependent manner at concentrations of 10 to 100 U/mL. In sharp contrast, addition of the above growth factors did not augment the colony growth-promoting effect of FCS and Epo in samples from IFN-resistant patients; further, adherent cell fractionation or T-lymphocyte depletion attenuated the “autonomous” colony growth. Lysates of 2.5 x 10(7) low-density cells from each of six IFN-resistant and six IFN-sensitive CML patients and three normal volunteers were tested for intrinsic IL-1 beta content in an enzyme-linked immunosorbent assay and yielded a mean of 610 pg, 54.6 pg, and 49.4 pg of IL-1 beta, respectively (P less than .045). Interestingly, both soluble IL-1 receptors (sIL-1R) and IL-1 receptor antagonist (IL-1RA) at concentrations of 5 to 100 ng/mL (sIL-1R) and 10 to 500 ng/mL (IL-1RA) inhibited CML colony growth in a dose-dependent fashion, with maximal inhibition of 64% and 65%, respectively. A similar effect was noted with the use of anti-IL-1 beta neutralizing antibodies. These data implicate IL-1 beta in CML disease progression and suggest that the inhibitory effects of molecules such as sIL-1R and IL-1RA could conceivably be the basis of a novel therapeutic strategy against this disorder.
Alpha-thalassemia resulting from deletion of regulatory sequences far upstream of the alpha-globin structural genes
Authors: Romao L , Osorio-Almeida L , Higgs DR , Lavinha J , Liebhaber SA
Published: October 2019

We describe an alpha-thalassemia determinant in which alpha-globin expression is silenced by a deletion located 27 kb 5′ to the transcription start site of the alpha 2-globin gene. This alpha- thalassemic determinant, (alpha alpha)MM, is a member of a newly described group of thalassemic mutations resulting from deletion of locus-controlling sequences critical to globin gene expression.
Myeloablative chemoradiotherapy and autologous bone marrow infusions for treatment of neuroblastoma: factors influencing engraftment
Authors: Graham-Pole J , Gee A , Emerson S , Gallo J , Lee C , Luzins J , Janssen WE , Pick T , Worthington-White D , Elfenbein G
Published: October 2019

Bone marrow harvested from cancer patients for autologous bone marrow reinfusion (ABMR) after myeloablative treatment may be injured, in both its proliferating and stromal cell pools, by either previous treatment or manipulation at the time of harvest. We have examined the relative effects of seven covariates on hematologic recovery after ABMR in children with neuroblastoma (NBL) using univariate and step-up analysis. We measured recovery by times to achieve (1) white blood cell counts greater than 1,000/microL; (2) absolute neutrophil counts greater than 500/microL; and (3) platelet counts greater than 20,000/microL without transfusion. In univariate analysis, recovery was significantly associated with the amount of prior chemotherapy and the interval between last chemotherapy and marrow harvest. Patient sex, the number of granulocyte-macrophage colonies infused, harvest-to-freeze interval, and use of purging were marginally associated. After adjusting for potential confounders in a multivariate model, the amounts of chemotherapy and granulocyte-macrophage colonies infused were independently significant predictors of time to total white blood cell count recovery; chemotherapy courses and chemo-to-harvest interval were predictors of neutrophil count recovery; and sex, use of purging, and harvest-to-freeze interval were marginal predictors of platelet recovery. The speed of hematologic recovery after ABMR seems to depend mainly on pre-existing factors and marginally on manipulation of the marrow after harvest. These factors may affect both proliferating and stromal cell pools.
Hepatitis C virus infection and chronic liver disease in children with leukemia in long-term remission
Authors: Locasciulli A , Gornati G , Tagger A , Ribero ML , Cavalletto D , Cavalletto L , Masera G , Shulman HM , Portmann B , Alberti A
Published: October 2019

Antibody to the recently identified hepatitis C virus (HCV) was investigated in sera of 50 leukemic children who had chronic liver disease (CLD), observed for 1 to 12.6 years after therapy withdrawal. All patients were tested for anti-HCV at regular intervals: Ortho- enzyme-linked immunosorbent assay (ELISA) test was performed in all cases. Reactive sera were also tested by recombinant immunoblotting assay to define the specificity of the results obtained by ELISA. Twelve cases (24%) were persistently positive (group A), 11 (22%) were transiently anti-HCV+ positive (group B), and 27 (54%) were negative. Mean SGPT peak during follow-up was significantly higher in group A (P = .014, A v B and P less than .00001, A v C). SGPT normalized off- therapy in 1 of 12 cases (group A), 10 of 11 (group B), and 19 of 27 (group C) (P = .0004, A v B and P = .012, A v C). Accordingly, liver histology, available in 37 patients, showed signs of chronic hepatitis in all patients in group A while most patients in group B and C had less severe liver lesions. These results indicate that HCV plays a significant role in the etiology of chronic hepatitis in leukemic patients and that persistent anti-HCV activity correlates with a more severe CLD, which could jeopardize the final prognosis of children cured of leukemia.
P53 gene mutations in acute myeloid leukemia with 17p monosomy
Authors: Fenaux P , Jonveaux P , Quiquandon I , Lai JL , Pignon JM , Loucheux-Lefebvre MH , Bauters F , Berger R , Kerckaert JP
Published: October 2019

We looked for mutations of exons 5 to 8 of the P53 gene in 10 patients with acute myeloid leukemia (AML) and 17p monosomy, and 36 patients with AML and no cytogenetic abnormalities of 17p. DNA was analyzed by polymerase chain reaction, single-strand conformation polymorphism analysis, and nucleotide sequencing. Four of the 10 patients with 17p monosomy showed point mutation, single-nucleotide deletion, or insertion in exons 7 or 8. By contrast, only 1 of the 36 patients with AML and no cytogenetic abnormalities of 17p showed a mutation of the P53 gene in exons 5 to 8 (P less than .01). These results suggest that alterations of the P53 gene may have a role in leukemogenesis in some cases of AML. The fact that P53 gene mutations occurred more often in patients with 17p monosomy seems to support the “recessive” model of tumor suppressive activity of the P53 gene rather than the “dominant” model, in which alteration of only one allele is sufficient for the development of malignancy.
Human platelet-specific antigen, Siba, is associated with the molecular weight polymorphism of glycoprotein Ib alpha
Authors: Ishida F , Saji H , Maruya E , Furihata K
Published: October 2019

Platelet-specific antigen Sib(a) has been highly implicated in the pathogenesis of refractoriness to human leukocyte antigen (HLA)-matched platelet transfusions in Japan. We provide evidence that the Sib(a) antigen is located on the glycoprotein (GP) Ib alpha and has a close association with the molecular weight (mol wt) polymorphism of GPIb. In modified antigen-capture ELISA (MACE), anti-Sib(a) antibody reacted only with GPIb/IX held by a murine anti-GPIb/IX monoclonal antibody (MoAb). The reactivity of anti-Sib(a) antibody to Sib(a)-positive (Sib(a+)) platelets was abolished after they were treated with Serratia marcescens protease. Platelets from 50 healthy volunteers were semiquantitatively phenotyped for Sib(a) antigen by MACE and divided into three distinct groups: strongly positive, positive, and negative. They were also analyzed by sodium dodeyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and periodic acid-silver staining for mol wt polymorphism of GPIb, phenotyped as A, B, C, or D. Without exception, Sib(a+) platelets showed larger phenotypes (A or B). Removal of sialic acid from Sib(a+) platelets did not reduce the binding of anti-Sib(a). Finally, anti-Sib(a) antibody specifically immunoprecipitated A and B phenotypes of GPIb from Sib(a+) platelets. Thus, Sib(a) antigen evidently is located in the region of glycocalicin that is present only on the A and B phenotypes of GPIb.
Chloroquine attenuates hemorrhagic shock-induced suppression of Kupffer cell antigen presentation and major histocompatibility complex class II antigen expression through blockade of tumor necrosis factor and prostaglandin release
Authors: Ertel W , Morrison MH , Ayala A , Chaudry IH
Published: October 2019

Hemorrhagic shock suppresses the ability of Kupffer cells (KC) to present antigen and express the major histocompatibility complex class II (Ia) antigen. These alterations are concomitant with an enhanced release of cytokines (tumor necrosis factor [TNF], interleukin-1 [IL- 1], IL-6) and prostaglandin E2 (PGE2) by KC after hemorrhagic shock. The aim of this study was to determine whether chloroquine (CQ) administration in vivo before or after hemorrhage affects the altered cytokine and PGE2 release by KC as well as the capacity of KC to present antigen and express Ia. To study this, C3H/HeN mice were bled to and maintained at a mean arterial blood pressure of 35 mm Hg for 60 minutes, followed by fluid resuscitation. Chloroquine (10 mg/kg body weight) was injected intramuscularly 2 hours before or during resuscitation following shock. The administration of CQ led to a significant reduction in the hemorrhage-induced elevation of TNF, IL-6, and PGE2 release by KC; however, IL-1 secretion was not affected by CQ. In addition, CQ treatment abolished the hemorrhage-induced increase in circulating TNF and IL-6. These changes in cytokine and PGE2 release following CQ administration correlated with a significant enhancement of the antigen-presenting capacity of KC. No differences were observed between pretreatment and posttreatment with CQ. Our data indicate that CQ selectively inhibits the release of TNF, IL-6, and PGE2 by KC, while IL-1 secretion was unaffected. Because the reduction of these inflammatory mediators was concomitant with a significant improvement of KC capacity to present antigen and express Ia, we propose that TNF, IL-6, and PGE2 play a pivotal role in the induction of posthemorrhage immunosuppression. Furthermore, the data suggest that the suppression of KC functions occurs during or after resuscitation, because posttreatment with CQ was as effective as pretreatment. Additional studies indicated that the survival of animals after hemorrhage and sepsis was significantly increased by posttreatment of hemorrhaged mice with CQ. Thus, CQ, because of its unique ability to selectively inhibit the release of inflammatory cytokines and prostaglandins, represents a potent immunomodulating agent in the treatment of conditions associated with increased cytokine release and for decreasing the mortality from sepsis after hemorrhage.
Clonal granulocytes and bone marrow cells in the cellular phase of agnogenic myeloid metaplasia
Authors: Kreipe H , Jaquet K , Felgner J , Radzun HJ , Parwaresch MR
Published: October 2019

Myelofibrosis with myeloid metaplasia (MMM) belongs to the group of myeloproliferative syndromes. It is characterized by a sustained proliferation of megakaryocytes and increased medullary reticulin fibers. Until now the cellular phase at onset of the disease has not been analyzed for clonality of the hematopoietic cells. In this study we used X-linked restriction length polymorphism (RFLP) analysis to investigate the clonality of granulocytes and bone marrow cells from the cellular phase and advanced stages of the disease. In each of 12 heterozygous females, monoclonality of granulocytes or total bone marrow cells could be demonstrated. These results show that the cellular phase represents a monoclonal, and hence a probably neoplastic, proliferation of a pluripotent stem cell. The monoclonality of granulocytes present at the onset of disease should allow analysis of DNA of these easily accessible peripheral cells for the detection of specific clonal aberrations.
Recombinant human stem cell factor, a c-kit ligand, stimulates hematopoiesis in primates
Authors: Andrews RG , Knitter GH , Bartelmez SH , Langley KE , Farrar D , Hendren RW , Appelbaum FR , Bernstein ID , Zsebo KM
Published: October 2019

Recombinant human stem cell factor (SCF) is homologous with recombinant rat SCF (rrSCF) and is a ligand for c-kit. We determined the influence of SCF on hematopoiesis in vitro and in vivo in baboons. In vitro, SCF alone stimulated little growth of hematopoietic colony-forming cells from baboon marrow, but did increase the number of colonies formed in response to erythropoietin (Epo), interleukin-3 (IL-3), and granulocyte- macrophage colony-stimulating factor (GM-CSF). In vivo, SCF caused an increase in the peripheral blood of the number of erythrocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In marrow, it caused an increase in marrow cellularity and in the absolute number of colony-forming unit-granulocyte-monocyte (CFU-GM) and burst- forming unit-erythroid (BFU-E) in marrow following infusion of SCF. The in vivo stimulation of multiple lymphohematopoietic lineages corroborates previous in vitro studies and suggests a potentially important clinical role for SCF.