bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical characteristics and treatment outcome of childhood acute lymphoblastic leukemia with the t(4;11)(q21;q23): a collaborative study of 40 cases [see comments]
Authors: Pui CH , Frankel LS , Carroll AJ , Raimondi SC , Shuster JJ , Head DR , Crist WM , Land VJ , Pullen DJ , Steuber CP
Published: October 2019

The t(4;11)(q21;q23) chromosomal abnormality was identified in 40 (2%) of 1,986 children with newly diagnosed acute lymphoblastic leukemia (ALL). This translocation was associated with female sex (63%), age less than 1 year (60%), hyperleukocytosis (median leukocyte count, 156.5 x 10(9)/L), CD10-/CD19+ B-precursor cell immunophenotype, and myeloid-associated antigen (CD15) expression (63%). Nearly all cases had at least some CD24- blast cells. The CD10-/CD15%/CD19+/CD24/+ phenotype was found in 20 of the 32 t(4;11) cases tested. None of the 40 cases had the cytogenetic finding of hyperdiploidy greater than 50, which is a favorable prognostic feature. For clinical comparison, the t(4;11) cases were divided into three groups according to age at diagnosis: less than 1 year (n = 24), 1 to 9 years (n = 8), and greater than or equal to 10 years (n = 8). Compared with older patients, infants were more likely to have initial central nervous system leukemia (P = .05) and less likely to have pre-B-cell ALL (P = .05). Complete continuous remission has been maintained in only 7 of 24 infants and 2 of 8 patients aged greater than or equal to 10 years, in contrast to 7 of 8 children in the intermediate age group (P = .048). These findings suggest that the t(4;11) is an adverse prognostic feature in these two age groups.
Abnormal megakaryocytopoiesis in the Belgrade laboratory rat
Authors: Rolovic Z , Basara N , Stojanovic N , Suvajdzic N , Pavlovic-Kentera V
Published: October 2019

The Belgrade laboratory (b/b) rat has a hereditary hypochromic microcytic anemia because of defective transmembrane iron transport into erythroblasts. The present study was prompted by our previous work in which we showed that the b/b rat has hypomegakaryocytic thrombocytopenia associated with increased megakaryocyte size. To define the basic mechanism underlying this abnormality in the b/b rat we have studied both megakaryocytopoiesis and granulopoiesis in anemic b/b rats, chronically transfused b/b rats, iron-treated b/b rats, and controls. We have found decreased concentrations of megakaryocyte and granulocyte progenitors in the marrow of b/b rats. Full correction of the severe anemia by chronic transfusion resulted in normalization of megakaryocyte progenitors, small acetylcholinesterase positive cells, megakaryocyte size, and platelet counts, along with granulocyte progenitors. In contrast, the partial correction of anemia obtained by iron treatment resulted in improvement, but not normalization, of these parameters. These findings indicate that abnormal megakaryocytopoiesis in the b/b rat can be best interpreted as a consequence of hypoxia because of the severe anemia. Because we have recently shown that the number of erythroid progenitors in b/b rats is also low, we propose that abnormal megakaryocytopoiesis in this animal is a reflection of an acquired stem cell disorder induced by the prolonged hypoxia resulting from the severe anemia.
Human immunodeficiency virus infection of megakaryocytic cells
Authors: Sakaguchi M , Sato T , Groopman JE
Published: October 2019

The human immunodeficiency virus (HIV) is capable of infecting certain cells of hematopoietic lineage, particularly monocyte-macrophages and T lymphocytes. Recently, the possibility that cells of megakaryocytic lineage are susceptible to HIV infection has been raised. We have characterized infection of the permanent megakaryocytic cell line CMK by HIV in vitro. CMK cells were easily infected by HIV type 2 (HIV-2), producing significant amounts of virus in culture. Infection appeared to be mediated by the CD4 surface antigen on CMK cells. Three different strains of HIV-1 were able to minimally infect CMK cells, suggesting there may be isolates of HIV tropic for megakaryocytes. Infection of CMK cells led to downregulation of the CD4 surface antigen but no discernable change in expression of megakaryocyte-associated proteins glycoprotein Ib and glycoprotein IIb/IIIa. These observations support the likelihood that megakaryocytes are susceptible to HIV infection, and cell lines of megakaryocytic origin may provide a useful model to study effects of the retrovirus on megakaryocyte function.
Expression of thrombomodulin by smooth muscle cells in culture: different effects of tumor necrosis factor and cyclic adenosine monophosphate on thrombomodulin expression by endothelial cells and smooth muscle cells in culture
Authors: Soff GA , Jackman RW , Rosenberg RD
Published: October 2019

Thrombomodulin (TM), a critical component of the protein C anticoagulant pathway, has previously been localized to endothelial cells (EC), but not smooth muscle cells (SMC) of the blood vessel wall. We demonstrate that cultured rat, bovine, as well as human SMC, but not blood vessel wall smooth muscle tissue, possess significant functional levels of TM and TM mRNA. Cyclic adenosine monophosphate stimulates TM expression in cultured SMC, but not EC, while tumor necrosis factor suppresses TM expression in EC but not cultured SMC. We postulate that following acute or chronic EC injury, luminal SMC can express TM, and are therefore able to protect the damaged blood vessel from thrombosis.
Role of von Willebrand factor associated to extracellular matrices in platelet adhesion
Authors: Baruch D , Denis C , Marteaux C , Schoevaert D , Coulombel L , Meyer D
Published: October 2019

The respective role of plasmatic and endothelial extracellular matrix (ECM)-associated von Willebrand factor (vWF) in platelet adhesion was investigated at a high shear rate using a parallel-plate perfusion chamber. Incubation of the endothelial ECM with a monoclonal antibody (MoAb) to vWF, which specifically blocks vWF binding to platelet GP Ib (MoAb 322), inhibited 45% of platelet adhesion. Complete inhibition was achieved by incubating both plasma and endothelial ECM with MoAb 322 at concentrations that blocked only about 50% of adhesion when added separately. The effect of ECM-associated vWF was further demonstrated when a fibroblastic ECM, normally devoid of vWF, was coated with purified plasmatic vWF. Matrix associated-vWF was able to significantly enhance platelet adhesion in both the presence and the absence of plasmatic vWF. In contrast, this effect was not seen on endothelial ECM. Binding of exogenous vWF to the ECM was specific and dose dependent, reached the same value (500 ng/cm2) on both fibroblastic ECM and endothelial ECM, but exhibited a threefold-lower apparent dissociation constant (KD) on fibroblastic than on endothelial ECM. Our studies suggest that vWF deposited by endothelial cells in the ECM may be the most active form in platelet adhesion, whereas plasmatic vWF may only play a secondary role.
Syngeneic and allogeneic bone marrow engraftment after total body irradiation: dependence on dose, dose rate, and fractionation
Authors: Down JD , Tarbell NJ , Thames HD , Mauch PM
Published: October 2019

Murine bone marrow chimera models were used to assess the efficacy of host total body irradiation (TBI) given at different doses, dose rates, and fractionation schemes in providing for engraftment of syngeneic and allogeneic bone marrow. B6-Hbbd congenic and LP mice, respectively, were used as donors (10(7) bone marrow cells) for syngeneic and allogenic (H-2 compatible) transplantation in standard B6 recipients. Stable marrow chimerism was determined from host and donor stem cell- derived hemoglobin phenotypes (Hbbs and Hbbd) on gel electrophoresis at 3 months posttransplant. Partial engraftment of syngeneic marrow was seen at single doses as low as 2 Gy, with the donor component increasing steadily with increasing TBI dose to a level of 100% at 7 Gy. Immunologic resistance of the host appeared to prevent allogeneic engraftment until 5.5 Gy. A very steep radiation dose response was then observed so that the level of chimerism with 6 Gy and above became comparable with syngeneic engraftment. Low dose rate (5 cGy minute-1) and fractionated TBI required higher total doses for equivalent engraftment (radiation dose-sparing) in both syngeneic and allogenic bone marrow transplantation. This displacement in the dose-response curve on fractionation was seen with interfraction intervals of 3 and 6 hours. A further dose-sparing effect was observed on extending the interval to 18 and 24 hours, but only for allogeneic transplantation, and may therefore be related to recovery of immune-mediated graft resistance. The involvement of multiple target cell populations in determining allogenic engraftment rendered the application of the linear-quadratic model for radiation cell survival problematic in this case. The recovery in dose when low dose rate and 6-hour interfraction intervals were applied in either syngeneic or allogeneic BMT is consistent with appreciable sub-lethal damage repair in the primitive self-renewing stem cell population of the host marrow. These results contrast with the poor repair capacity of the 11-day spleen colony- forming units (CFUs) population after fractionated irradiation and support the notion that ablation of early stem cells in the pre-CFUs compartment is essential for long-term marrow engraftment.
Recombinant human granulocyte-macrophage colony-stimulating factor in combination with standard induction chemotherapy in de novo acute myeloid leukemia
Authors: Bettelheim P , Valent P , Andreeff M , Tafuri A , Haimi J , Gorischek C , Muhm M , Sillaber C , Haas O , Vieder L
Published: October 2019

Based on in vitro data suggesting that recombinant human granulocyte- macrophage colony-stimulating factor (rhGM-CSF) is capable of stimulating acute myeloid leukemia (AML) blast cells to become more sensitive to cell-cycle-specific drugs we conducted a phase I/II study in de novo AML patients (pts). rhGM-CSF (250 micrograms/m2/d, continuous intravenous infusion) was administered in 18 pts suffering from de novo AML in combination with standard induction chemotherapy (3 + 7 = daunorubicin 45 mg/m2 days 1 through 3, cytosine-arabinoside [Ara- C] 200 mg/m2 continuous infusion days 1 through 7). GM-CSF was started 48 or 24 hours before chemotherapy (prephase) in 14 pts. In four pts with high white blood cell counts (WBC) rhGM-CSF was started after chemotherapy-induced cell reduction (WBC less than 30,000/mm3). During prephase GM-CSF induced an increase in neutrophil and blast cell counts in 13 of 14 and 10 of 14 pts, respectively. In vivo recruitment of leukemic cells into drug-sensitive phases of the cell cycle could be demonstrated by multiparameter cell-cycle analyses in peripheral blood (n = 7) and bone marrow (n = 4) specimens. On day 14, complete aplasia was evident in 17 of 18 pts. GM-CSF was administered until recovery from chemotherapy-induced myelosuppression (absolute neutrophil counts, [ANC] greater than 500/mm3). Fifteen pts (83%) achieved complete remission, 12 did so with one cycle. A shorter duration of neutropenia was evident in these pts compared with historical controls (n = 39), (ANC greater than 500/mm3, day 22.5 +/- 3.4 v 25.2 +/- 3.7, P less than .05). Three pts achieved complete remission after a second cycle (same combination of rhGM-CSF and 3 + 7). Two pts died during bone marrow aplasia because of invasive pulmonary aspergillosis. Clinical side effects possibly related to GM-CSF, mainly fever, diarrhea, and weight gain were mild and tolerable (World Health Organization toxicity grade less than or equal to 2). Together, rhGM-CSF recruits kinetically quiescient AML cells in vivo to enter drug-sensitive phases of the cell cycle and promotes early myeloid recovery from aplasia after exposure to standard induction chemotherapy for AML.
Monoclonal antibody-purged autologous bone marrow transplantation therapy for multiple myeloma
Authors: Anderson KC , Barut BA , Ritz J , Freedman AS , Takvorian T , Rabinowe SN , Soiffer R , Heflin L , Coral F , Dear K
Published: October 2019

Eleven patients with plasma cell dyscrasias underwent high-dose chemoradiotherapy and anti-B-cell monoclonal antibody (MoAb)-treated autologous bone marrow transplantation (ABMT). The majority of patients had advanced Durie-Salmon stage myeloma at diagnosis, all were pretreated with chemotherapy, and six had received prior radiotherapy. At the time of ABMT, all patients demonstrated good performance status with Karnofsky score of 80% or greater and had less than 10% marrow tumor cells. Eight patients had residual monoclonal marrow plasma cells and 10 patients had paraprotein. Following high-dose melphalan and total body irradiation (TBI) there were seven complete responses, three partial responses, and one toxic death. Granulocytes greater than 500/mm3 were noted at a median of 21 (range 12 to 46) days posttransplant (PT) and untransfused platelets greater than 20,000/mm3 were noted at a median of 23 (12 to 53) days PT in 10 of the 11 patients. Natural killer cells and cytotoxic/suppressor T cells predominated early PT, with return of B cells at 3 months PT and normalization of T4:T8 ratio at 1 year PT. Less than 5% polyclonal marrow plasma cells were noted in all patients after transplant. Three of the seven complete responders have had return of paraprotein, two with myeloma, and have subsequently responded to alpha 2 interferon therapy. Eight patients are alive at 18.9 (8.9 to 43.1) months PT and four remain disease-free at 12.3, 17.5, 18.9, and 29 months PT. This preliminary study confirms that high-dose melphalan and TBI can achieve high response rates without unexpected toxicity in patients who have sensitive disease, and that MoAb-based purging techniques do not inhibit engraftment. Although the follow-up is short- and long-term outcome to be determined, relapses post-ABMT in these heavily pretreated patients suggest that ABMT or alternative treatment strategies should be evaluated earlier in the disease course.
Cyclosporine therapy for advanced Langerhans cell histiocytosis [see comments]
Authors: Mahmoud HH , Wang WC , Murphy SB
Published: October 2019

Prompted by evidence that Langerhans cell histiocytosis (LCH) is a nonmalignant disorder of immune regulation, we used cyclosporine (12 mg/kg/d orally) to treat three young children with advanced multisystem LCH. All three patients had partial responses to cyclosporine within 2 months of therapy, as evidenced by complete resolution of organ dysfunction and regression of the majority of lesions. Complete responses were attained by adding relatively nontoxic chemotherapy (ie, prednisone and vinblastine). Toxicity from cyclosporine comprised mild and reversible elevations of the serum creatinine and blood urea nitrogen. These results indicate that further evaluation of cyclosporine for the treatment of patients with advanced LCH is warranted.
The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562
Authors: Donovan-Peluso M , Acuto S , O'Neill D , Hom A , Maggio A , Bank A
Published: October 2019

We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3′ enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3′ enhancer is erythroid-specific but not developmental stage- or gene-specific.
Rapid simultaneous detection of multiple retroviral DNA sequences using the polymerase chain reaction and capillary DNA chromatography
Authors: Sunzeri FJ , Lee TH , Brownlee RG , Busch MP
Published: October 2019

The polymerase chain reaction (PCR) technique is a powerful new tool for amplifying target DNA, thus allowing for sensitive detection of specific nucleic acid sequences. One important potential use of PCR involves screening the donated blood supply for transfusion-transmitted viruses. Realization of this goal has been limited by (1) the requirement for multiple, discrete PCR reactions to amplify and detect target sequences of more than one virus, and (2) the lack of a rapid, nonhazardous means for specific detection of one or more PCR-amplified products. We report the simultaneous amplification of three distinct target sequences without discernable loss in sensitivity toward any single target sequence. We also demonstrate very rapid separation and detection of PCR-amplified viral DNA through the use of automated capillary DNA chromatography. Amplified DNA peaks were initially identified by scanning the capillary effluent at ultraviolet wavelengths, while discrimination of human immunodeficiency virus type 1 and human T-cell leukemic virus type I PCR-amplified DNA was accomplished through use of virus-specific, fluorescently labeled primers and probes. These results indicate progress toward an automated system for screening the blood supply for nucleic acid sequences of multiple pathogens.
Philadelphia chromosome and monosomy 7 in childhood acute lymphoblastic leukemia: a Pediatric Oncology Group study
Authors: Russo C , Carroll A , Kohler S , Borowitz M , Amylon M , Homans A , Kedar A , Shuster J , Land V , Crist W
Published: October 2019

During an 8-year period, 3,638 children from institutions of the Pediatric Oncology Group (POG) were diagnosed with acute lymphoblastic leukemia (ALL). Fifty-seven patients had Philadelphia chromosome- positive (Ph1) ALL. Blast cells obtained at diagnosis from 13 of these 57 cases (23%) were also found to have partial or complete monosomy 7 (- 7). This subgroup of children with Ph1/-7 ALL was comprised primarily of older males with early B-lineage ALL. Bone marrow specimens from six Ph1/-7 patients were studied further using the polymerase chain reaction and primers that flank the ALL, and chronic myelogenous leukemia breakpoints to determine the molecular characteristic of the 9;22 translocation. Rearrangements were detected in RNA from bone marrow and/or peripheral blood cells of six patients, although four were in hematologic remission at the time of the analysis. Five cases showed the ALL breakpoint, while one child with Ph1/-7 showed the chronic myelogenous leukemia breakpoint. The induction failure rate was much higher in this subgroup (31%) as compared with Ph1-negative cases, and the projected duration of event-free survival reflected the aggressive nature of this subgroup because no children are projected to remain in remission at 2 years. ALL with both the 9;22 translocation and -7 appears to represent a unique and previously undescribed subgroup of childhood ALL associated with a particularly adverse outcome. Leukemic transformation in such patients may involve the interaction of a dominant oncogene (Ph1) and a tumor suppressor gene (- 7).
MYC rearrangement and translocations involving band 8q24 in diffuse large cell lymphomas
Authors: Ladanyi M , Offit K , Jhanwar SC , Filippa DA , Chaganti RS
Published: October 2019

The configuration of the MYC gene in diffuse large cell lymphomas (DLCL) with translocations involving band 8q24 [t(8q24)] has not been systematically studied. We collected cytogenetic and clinical data on 171 consecutive cases of DLCL, including cleaved, noncleaved, and immunoblastic types, of which 96 had DNA available and 124 had abnormal karyotypes. The cases with DNA available were evaluated for MYC rearrangement (MYC-R) by Southern hybridization of EcoRI-digested tumor DNA using an exon-1 probe, a combination of probe and enzyme known to detect over 85% of breaks in sporadic Burkitt's lymphoma. In cases studied at diagnosis, MYC-R, t(8;14)(q24;q32), or other t(8q24) were not prognostically significant. Among the 124 cases with karyotypic abnormalities, seropositivity for human immunodeficiency virus was significantly more common in cases with a t(8q24) (72%) than in cases without it (9%) (P less than .05). Of the four cases with an MYC -R, two had a t(8;14), one had a t(7;8;14)(p15;q24;q32), and one had a t(8;?)(q24;?) and a del(8)(q24). In the three previous cases with translocations involving 8q24 and 14q32, comigration of the rearranged MYC band with either the J region or the switch-mu region of the Ig heavy chain gene could not be demonstrated, leaving the 14q32 breakpoint undefined at the molecular level. Among the remaining 72 cases where both an abnormal karyotype and molecular data were available, 11 had a t(8q24), either t(8;14) or t(8;22)(q24;q11), in the absence of an MYC-R. In these cases, the 8q24 break was presumably located outside of the EcoRI MYC fragment. All 15 cases with a t(8q24) were also screened for point mutations in the PvuII site in the first exon of MYC; two cases that were not MYC-R showed loss of this restriction site. These results indicate that in most DLCL with t(8;14) or other t(8q24), the 8q24 breakpoint lies away from the MYC gene; in a minority of these cases, point mutations in regulatory noncoding regions were detected.
Interphase cytogenetic analysis detects minimal residual disease in a case of acute lymphoblastic leukemia and resolves the question of origin of relapse after allogeneic bone marrow transplantation
Authors: Anastasi J , Thangavelu M , Vardiman JW , Hooberman AL , Bian ML , Larson RA , Le Beau MM
Published: October 2019

We used in situ hybridization with a probe for the X chromosome to study interphase cells of bone marrow and peripheral blood specimens from a male patient with acute lymphoblastic leukemia characterized by hyperdiploidy, including trisomy X. In a posttreatment bone marrow specimen, which was interpreted as a regenerating bone marrow morphologically and which demonstrated a normal karyotype cytogenetically, trisomy X was found in 16 of 1,000 interphase cells. This finding indicated the presence of leukemic cells that were undetected by conventional morphologic and cytogenetic techniques (ie, minimal residual disease). Cytogenetic studies of a relapse specimen obtained after a sex-mismatched bone marrow transplant showed only a normal female karyotype in each of 40 metaphase cells, suggesting that the relapse occurred in donor cells. However, interphase analysis demonstrated trisomy X in more than 80% of interphase cells and indicated that the relapse was of the original clone and was not a transformation of donor cells. This case illustrates that interphase analysis can be useful as an adjunct to conventional cytogenetic analysis in the detection of minimal residual disease and in the analysis of interphase cells that are not accessible to routine cytogenetic methods. It also illustrates that previously reported instances of relapse of leukemia in donor cells could have been incorrect if supported by cytogenetic data alone.
Epstein-Barr virus infection precedes clonal expansion in Burkitt's and acquired immunodeficiency syndrome-associated lymphoma [see comments]
Authors: Neri A , Barriga F , Inghirami G , Knowles DM , Neequaye J , Magrath IT , Dalla- Favera R
Published: October 2019

The Epstein-Barr virus (EBV) is associated with distinct forms of human lymphoid malignancies, including the endemic (eBL) and sporadic forms of Burkitt's lymphoma (sBL) and acquired immunodeficiency syndrome- associated non-Hodgkin lymphoma (AIDS-NHL). However, whether EBV has a pathogenetic role in these tumors or is a passenger virus has not been conclusively demonstrated. One element to distinguish between these two possibilities is to determine whether EBV infection has preceded and, thus, possibly contributed to clonal expansion, or whether infection has occurred after clonal expansion and thus is unlikely to contribute to pathogenesis. Toward this end we analyzed the structure of the heterogeneous genomic termini of EBV as markers of clonal infection in a panel of eBL (11 cases), sBL (9 cases), and AIDS-NHL (10 cases) biopsies. We show that EBV termini are uniformly clonal in sBL, eBL, and AIDS-NHL, strongly suggesting that EBV infection has preceded and, thus, most likely contributed to clonal expansion in these malignancies.
Early occurrence of human cytomegalovirus infection after bone marrow transplantation as demonstrated by the polymerase chain reaction technique
Authors: Einsele H , Steidle M , Vallbracht A , Saal JG , Ehninger G , Muller CA
Published: October 2019

Twenty-eight patients undergoing bone marrow transplantation (BMT) were followed-up at weekly intervals from day -10 to discharge from hospital after BMT for human cytomegalovirus (HCMV) infection using polymerase chain reaction (PCR), slot-blot hybridization, and conventional virus culture. High specificity of the PCR assay applied could be shown by failure to amplify DNA extracted from a wide range of other viruses frequently infecting marrow transplant recipients. The PCR technique allowed us to diagnose viremia and viruria in 20 (83%) of 24 seropositive patients after BMT, whereas culture assays showed 16 (67%) of 24 of these patients to be viruric and 9 (37%) of 24 cases to be viremic. Slot-blot hybridization showed a frequency of viruria and viremia in 12 (50%) of 24 seropositive patients. By application of PCR techniques, HCMV detection could be achieved even in the very early posttransplant period. HCMV was detected in five patients even before the onset of clinical symptoms of acute graft-versus-host disease. Analysis by PCR techniques of 33 organ biopsy specimens from patients after BMT showed the presence of HCMV in 13 of 14 liver samples obtained from patients with HCMV viremia; three liver specimens from patients without viremia were negative by all the techniques applied. HCMV could also be demonstrated in postmortem lung biopsy specimens from all patients (n = 10) with interstitial pneumonia.
Preparation and characterization of an intravenous solution of IgG from human immunodeficiency virus-seropositive donors
Authors: Cummins LM , Weinhold KJ , Matthews TJ , Langlois AJ , Perno CF , Condie RM , Allain JP
Published: October 2019

An intravenous solution of 99% pure globulin (hyperimmune IgG, HIVIG) was obtained from pooled plasma of selected human immunodeficiency virus (HIV-1)-seropositive asymptomatic donors with greater than 400 CD4+/microliters cells per microliter and a high titer of antibody to HIV-1 p24 protein. HIVIG had high titers of antibody to p24, glycoprotein 41 (gp41), and gp120, group-specific neutralizing activity, and binding to the gp120 hypervariable loop region. It inhibited syncytia formation. At low concentration, it enhanced viral production of HIV-1 in infected peripheral blood monocytes but was inhibitory at higher concentration. HIVIG directed group-specific antibody-dependent cellular cytotoxicity against HIV-infected targets. For a period of 6 to 28 months, plasma donors kept stable antibody titers and had a 1.0% decrease in CD4+ cells per month. One gram per kilogram HIVIG injected in two juvenile chimpanzees was well tolerated and did not transmit HIV, as measured by negative cell culture, IgM immune response to HIV proteins, and polymerase chain reaction. The mean half-life of HIV-1 p24 antibody was 15 days. These preliminary data suggest that HIVIG is a safe product suitable for clinical trial in HIV-1-infected individuals.
Monitoring of specific antibodies to human immunodeficiency virus structural proteins: clinical significance
Authors: Allain JP , Laurian Y , Einstein MH , Braun BP , Delaney SR , Stephens JE , Daluga CK , Dahlen SJ , Knigge KM
Published: October 2019

Levels of antibodies to six major structural proteins of human immunodeficiency virus type 1 (gp120, gp41, p66, p31, p24, and p17) were assessed in serial samples from 22 persons with severe hemophilia (16 asymptomatic and 6 who developed acquired immunodeficiency syndrome [AIDS] or AIDS-related complex) with an automated dot blot assay using purified recombinant antigens. High and sustained levels of antibody to gp120, gp41, and p31 were found in all patients irrespective of their clinical condition for 4 to 6 years after seroconversion. In contrast, immune response to p66 and p17 was significantly lower in symptomatic patients. Over time, the levels of these two antibodies, as well as anti-p24, decreased and tended to become undetectable. Abnormal immune response and low levels of antibody to p66 and p17 are early indications of rapid clinical progression.
Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures
Authors: Wilson EL , Rifkin DB , Kelly F , Hannocks MJ , Gabrilove JL
Published: October 2019

We previously showed that basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow (BM) stromal cells and significantly delays their senescence. In the present study, we demonstrated that low concentrations of bFGF (0.2 to 2 ng/mL) enhance myelopoiesis in long-term human BM culture. Addition of bFGF to long- term BM cultures resulted in an increase in (a) the number of nonadherent cells (sixfold), particularly those of the neutrophil granulocyte series; (b) the number of nonadherent granulocyte colony- stimulating factor (G-CSF)- and granulocyte-macrophage colony- stimulating factor (GM-CSF)-responsive progenitor cells; (c) the number of adherent foci of hematopoietic cells (10-fold); and (d) the number of progenitor cells in the adherent stromal cell layer. These effects were not noted with higher concentrations of bFGF (20 ng/mL). Thus, low concentrations of bFGF effectively augment myelopoiesis in human long- term BM cultures, and bFGF may therefore be a regulator of the hematopoietic system in vitro and in vivo.
B-lineage colonies from normal, human bone marrow are initiated by B cells and their progenitors
Authors: McGinnes K , Letarte M , Paige CJ
Published: October 2019

We have recently described a reproducible method whereby colonies containing cells that secrete immunoglobulin (Ig) can be grown from normal, human, adult bone marrow samples. The present report characterizes the cells that initiate these colonies. It is shown that all clonogenic cells express the CD19 surface antigen, as removal of these cells before plating in the B-cell colony assay abolished the subsequent growth of plaque-forming, B-lineage colonies. Cells from both the CD10+ and CD20+ B-lineage subpopulations initiated the growth of B-cell colonies, as removal of either subset resulted in a 50% reduction in the number of resulting B-cell colonies. The removal of activated B cells (CD23+), plasma cells (PCA-1+), or myeloid cells (CD13+) did not lead to a significant depletion in B-cell colony formation. Pre-B cells that were not yet committed to Ig light chain expression were also able to differentiate and proliferate into Ig- secreting colonies under the culture conditions used. Colonies initiated by these light chain uncommitted cells were distinguished using a replicate protein immunoblotting technique, which detects the simultaneous secretion of Ig kappa and Ig lambda from single colonies. These experiments provide evidence that the CD10 antigen is expressed on B-lineage cells before Ig light chain commitment, whereas CD20 is not. In conclusion, this B-cell colony assay provides a system for studying the differentiation of bone marrow-derived B cells and their precursors into Ig-secreting cells.
Tumor necrosis factor regulates the expression of granulocyte- macrophage colony-stimulating factor and interleukin-3 receptors on human acute myeloid leukemia cells
Authors: Elbaz O , Budel LM , Hoogerbrugge H , Touw IP , Delwel R , Mahmoud LA , Lowenberg B
Published: October 2019

Tumor necrosis factor (TNF) acts as a potent enhancer of granulocyte- macrophage colony-stimulating factor (GM-CSF)- and interleukin-3 (IL-3)- induced human acute myeloid leukemia (AML) growth in vitro. We have analyzed the effects of TNF alpha on the expression of GM-CSF and IL-3 receptors on AML cells. Incubation of blasts from seven patients with AML in serum-free medium with TNF (10(3) U/mL) and subsequent binding studies using 125I-GM-CSF and 125I-IL-3 show that TNF increases the specific binding of GM-CSF (30% to 280%) and IL-3 (40% to 600%) in all cases. From Scatchard plot analysis it appears that TNF upregulates (1) low-affinity GM-CSF binding sites, (2) common high-affinity IL-3/GM-CSF binding sites, and (3) unique (non-GM-CSF binding) IL-3 binding sites. The effect of TNF is dose dependent and is half maximal at a concentration of 100 U/mL, and becomes evident at 18 hours of incubation with TNF at 37 degrees C, but not at 0 degree C. The GM-CSF dose-response curve of AML-colony-forming units plateaus at a higher level in the presence of TNF, which indicates that additional numbers of cells become responsive to GM-CSF. Incubation of AML blasts with the phorbol ester 12–0-tetradecanoylphorbol-13-acetate or formyl-Met-Leu- Phe (protein kinase C activators) does not influence GM-CSF receptor expression, suggesting that receptor upregulation by TNF is not mediated through activation of protein kinase C. On the other hand, the protein synthesis inhibitor cycloheximide abrogates receptor upregulation induced by TNF. In contrast to these findings in AML, TNF does not upregulate GM-CSF receptor numbers on blood granulocytes or monocytes. We conclude that TNF exerts positive effects on growth factor receptor expression of hematopoietic cells.
The role of glycosylation in the biosynthesis and acquisition of ligand- binding activity of the folate-binding protein in cultured KB cells
Authors: Luhrs CA
Published: October 2019

The biosynthesis, processing, and ligand-binding function of the membrane-associated and soluble forms of the folate-binding protein (FBP) in KB cells, a cultured human cell line, were studied using pulse- chase labeling with [35S] methionine. The intermediary and mature forms of the protein were isolated by immunoprecipitation and affinity chromatography and analyzed by sodium dodecyl sulfate electrophoresis and autoradiography. The earliest species identified had an Mr of 32 Kd and disappeared over 5 hours concomitant with the appearance of a 38-Kd cellular FBP. As the 38-Kd species disappeared, a 40-Kd form appeared in the medium. When tunicamycin was added to the culture medium to inhibit core glycosylation, a 26-Kd aglycosylated species and minor 28- Kd and 30-Kd forms appeared. Endoglycosidase H, which cleaves high mannose but not complex oligosaccharides, reduced the 32-Kd species to 26-Kd but the enzyme had no effect on the 38-Kd form, indicating that this species is complex glycosylated. Monensin, which blocks complex glycosylation, also inhibited synthesis of the 38-Kd species. Although both the 32-Kd and 38-Kd forms had ligand-binding sites (as demonstrated by binding to a folate-Sepharose matrix), the 26-Kd aglycosylated species, labeled in the presence of tunicamycin, lacked similar binding sites because it did not bind to the affinity matrix. In contrast, the aglycosylated 26-Kd form, which was obtained by treatment of the 32-Kd species with endoglycosidase H, did bind to the folate affinity matrix, indicating that it retained ligand-binding function. Thus, the high mannose oligosaccharide moiety is not required for the folate-binding property of the FBP, but its addition to the polypeptide chain precedes a later step that is necessary for the mature protein to have ligand-binding function.
Human interleukin-3 mRNA accumulation is controlled at both the transcriptional and posttranscriptional level
Authors: Ryan GR , Milton SE , Lopez AF , Bardy PG , Vadas MA , Shannon MF
Published: October 2019

Interleukin-3 (IL-3) is a hematopoietic growth factor that regulates the differentiation of multilineage and committed progenitor cells and the functions of some mature blood cells. The expression of human IL-3 appears to be restricted to stimulated T lymphocytes. We have investigated the kinetics and mechanisms involved in the induction of IL-3 expression in the human T lymphocytic tumor cell line Jurkat. We show that accumulation of IL-3 mRNA is controlled at both the transcriptional and posttranscriptional level. Transcription of the IL- 3 gene in these cells appears to be constitutive but no IL-3 mRNA was detected in unstimulated cells, indicating that in resting cells IL-3 mRNA is highly unstable. Treatment with phytohemagglutinin (PHA) induced a small and transient increase in the IL-3 gene transcription rate and led to the production of detectable levels of IL-3 mRNA and protein. Optimal induction of IL-3 expression required a second stimulus. Costimulation of Jurkat cells with both phorbol myristate acetate and PHA caused both a transient increase in IL-3 gene transcription, which is dependent on new protein synthesis, and also a transient increase in mRNA stability.
1,25-Dihydroxyvitamin D3 receptor RNA: expression in hematopoietic cells
Authors: Kizaki M , Norman AW , Bishop JE , Lin CW , Karmakar A , Koeffler HP
Published: October 2019

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients; these effects are probably mediated through the 1,25(OH)2D3 receptor. Little is known of expression of 1,25(OH)2D3 receptor RNA in hematopoietic cells. We examined the expression and modulation of expression of 1,25(OH)2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells. Constitutive expression of 1,25(OH)2D3 receptor RNA was detected in various kinds of hematopoietic cells, including macrophages and activated T lymphocytes, as well as in cell lines KG-1 (myeloblasts), HL-60 (promyelocytes), ML-3 (myelomonoblasts), U937, THP-1 (monoblasts), K562 (erythroblasts), and S-LB1 (HTLV-1-transfected T lymphocytes). Receptor transcripts were 4.6 kilobases (kb), and no variant sizes were observed. All cell lines examined in this group also expressed 1,25(OH)2D3 receptors. Most B lymphocyte lines expressed negligible levels of 1,25(OH)2D3 receptor RNA and protein; however; analysis of a lymphoid/myeloid somatic hybrid suggested that suppression of expression of 1,25(OH)2D3 receptor RNA in B lymphocytes may be a dominant characteristic. HL-60 cells were cultured with 10(-7) mol/L 1,25(OH)2D3 for 24 to 72 hours, and levels of expression of 1,25(OH)2D3 receptor and its RNA were examined. Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand. Levels of occupied 1,25(OH)2D3 receptor protein increased in these HL-60 cells; but the total number of 1,25(OH)2D3 receptors decreased about 50% at 24 hours and returned toward normal at 72 hours. Steady-state levels of 1,25(OH)2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages. Nondividing macrophages from normal individuals also expressed 1,25(OH)2D3 receptor RNA. In contrast, nondividing peripheral blood lymphocytes from normal individuals did not express 1,25(OH)2D3 receptor RNA; with stimulation of proliferation of these cells, accumulation of 1,25(OH)2D3 receptor RNA increased markedly. Half-life (t1/2) of 1,25(OH)2D3 receptor RNA in T lymphocytes was short (1 hour) as determined by measuring decay of the message after addition of actinomycin D. Consistent with this short t1/2, accumulation of 1,25(OH)2D3 receptor RNA increased in cells as their protein synthesis was inhibited. Further studies are required to understand the physiologic role of 1,25(OH)2D3 receptors in myeloid cells and proliferating T lymphocytes.
Protection from arabinofuranosylcytosine and n-mustard-induced myelotoxicity using hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys monomer and dimer
Authors: Paukovits WR , Moser MH , Binder KA , Paukovits JB
Published: October 2019

We have previously shown that the synthetic peptide pGlu-Glu-Asp-Cys- Lys (pEEDCK monomer) inhibits the cytostatic drug-induced proliferation of hematopoietic stem cells CFU-S. Keeping CFU-S quiescent by pEEDCK treatment renders them insensitive to cycle-specific cytostatic drugs and leads to reduced toxicity. Here we show that pEEDCK application during repeated (twice) administration of clinically relevant (nonlethal) 1-beta-D-arabinofuranosylcytosine (Ara-C) doses reduced the percentage of CFU-S in S-phase from 60%-70% to 25%-30% and led to a sustained stem cell number in the bone marrow (BM), whereas unprotected mice had lost about 75% of their CFU-S population. Owing to its cysteine content, the pEEDCK monomer is easily oxidized. The resulting dimer (pEEDCK)2 is a potent stimulator of hematopoiesis. As we show, it can be used for postchemotherapy acceleration of hematologic recovery, similar to the use of recombinant hematopoietic growth factors. A single injection of 30 micrograms/kg pEEDCK monomer to mice 2 hours before the second Ara-C injection retarded onset of neutropenia (by 2 to 3 days) and improved recovery after depression. The quantitative degree of neutropenia was not changed. Postchemotherapy (Ara-C administered twice, followed by N-mustard) infusion of the stimulatory (pEEDCK)2 dimer (1.4 micrograms/kg/d) produced a 4.6-fold increase of progenitor levels (6.7 CFU-GM/1,000 BM cells v 1.45 CFU-GM/1,000 in normal mice) 2 days after the end of the cytostatic treatment when CFU- GM were not detectable in unprotected mice. This increase was followed after several days by strongly elevated granulocyte counts, which remained high for approximately 1 week. Up to 75% of the peripheral leukocytes were mature polymorphonuclear leukocytes (PMN) during this phase. Ara-C (twice) and monomer treatment as above followed by dimer infusion resulted in the complete protection of hematopoiesis. Mice treated with the protective pEEDCK monomer plus stimulatory dimer did not develop the leukocyte depression noted in unprotected animals. The inhibitory monomer appears to keep the stem cell population numerically and qualitatively intact, thus providing optimum target cell conditions for the subsequent stimulator (dimer) treatment. Our results show that the hemoregulatory peptide monomer and dimer can be used for improving the hematologic status of mice treated with clinically relevant doses of cytostatic drugs (antimetabolite and alkylating, alone and in combination). Combining both peptides can prevent occurrence of neutropenia completely. Both peptides can be obtained easily by chemical synthesis and are also active on human cells. They are thus highly promising candidates for application as multilevel hemoprotectors in cancer chemotherapy.
Molecular characterization of beta-thalassemia intermedia in patients of Italian descent and identification of three novel beta-thalassemia mutations
Authors: Murru S , Loudianos G , Deiana M , Camaschella C , Sciarratta GV , Agosti S , Parodi MI , Cerruti P , Cao A , Pirastu M
Published: October 2019

In this study, we have defined by dot-blot analysis with allelic specific oligonucleotide probes or direct sequencing on amplified DNA the beta-thalassemia mutations in a large group of patients (23) of Italian descent with thalassemia intermedia. These patients had one parent with either the silent beta-thalassemia carrier phenotype or borderline-normal hemoglobin A2 (HbA2) levels (2.5% to 3.5%). Nearly all were genetic compounds for a severe beta-thalassemia mutation and a beta-thalassemia mutation associated with high residual output of beta- globin chains (beta + intervening sequence [IVS]-I-nt6, beta -87, beta - 101), indicating that inheritance of a mild beta-thalassemia allele, even in a single dose, is the most common molecular mechanism producing thalassemia intermedia in the Italian population. In three cases, in whom we failed to define by dot-blot analysis the mutations, we sequenced the beta + globin gene and found three novel beta-thalassemia mutations, which are certainly very rare because they have been hitherto detected solely in a single patient. These mutations consist of: (1) a T-A substitution at position 2 of IVS-I, in a patient compound heterozygote for this mutation and the -87 promoter mutation; (2) a G-C substitution at position 844 of IVS-II, in a patient heterozygous for this mutation who showed normal sequences at the in trans beta-globin gene (The reason for the presence of clinical manifestations in a beta-thalassemia heterozygote has not been defined.); and (3) a deletion of one nucleotide (-T) at codon 126, resulting in a frameshift and readthrough of the 5′ untranslated region and most likely producing an elongated Hb molecule of 156 amino acid residues, in a patient heterozygous for this mutation with normal beta- globin gene sequences at the other locus.
Molecular defect of a phosphoglycerate kinase variant (PGK-Matsue) associated with hemolytic anemia: Leu----Pro substitution caused by T/A- ---C/G transition in exon 3
Authors: Maeda M , Yoshida A
Published: October 2019

We have identified the mutation in a phosphoglycerate kinase variant (PGK-Matsue) associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders. The mRNA coding for PGK was reverse transcribed and amplified by the polymerase chain reaction. Nucleotide sequencing of the variant cDNA showed a point mutation, a T/A----C/G transition in exon 3 of the variant gene. No other mutation was found in all coding regions of PGK-Matsue. The nucleotide change created an additional NciI cleavage site in the variant gene; thus, the NciI fragment types detected by Southern blot hybridization differ in the variant DNA and normal DNA. The mutation should cause Leu----Pro substitution at the 88th position from the NH2- terminal Ser of PGK. Because the Leu----Pro substitution is expected to induce serious perturbation and instability in the protein structure, the severe enzyme deficiency is mainly caused by more rapid in vivo denaturation and degradation of the variant enzyme.