bluuebunny/crossref_metadata_2025 huggingface dataset:
High-resolution analysis of the human gamma-globin gene promoter in K562 erythroleukemia cell chromatin
Authors: Gimble JM , Max EE , Ley TJ
Published: October 2019

We performed high-resolution mapping studies of the DNAse I- hypersensitive sites located just 5′ to the human G gamma- and A gamma- globin genes of K562 erythroleukemia cells, in which these genes are constitutively expressed at low levels. This analysis revealed that the hypersensitive site extends from approximately -210 +/- 5 to -25 +/- 5 base pairs (bp) upstream from the transcription initiation site. Within this region, a GC-rich region located between the proximal CCAAT box and the TATA box is particularly accessible to nuclease digestion; however, the 5′ end of the hypersensitive site is less accessible to nucleases. The pattern of DNAse I cleavage does not change on either strand with hemin induction of K562 cells, which increases the rate of gamma-globin gene transcription about threefold. The region within the hypersensitive site includes all the consensus promoter elements of the gamma-globin genes as well as an octamer sequence located between -182 and -175, and a region associated with a variety of mutations that may cause hereditary persistence of fetal hemoglobin (HPFH).
Interferon alpha-2b as therapy for Ph'-positive chronic myelogenous leukemia: a study of 82 patients treated with intermittent or daily administration
Authors: Alimena G , Morra E , Lazzarino M , Liberati AM , Montefusco E , Inverardi D , Bernasconi P , Mancini M , Donti E , Grignani F
Published: October 2019

The authors treated a total of 82 patients with Ph′-positive chronic myelogenous leukemia (CML) with recombinant interferon alpha-2b (IFN alpha-2b). Sixty-five patients in chronic phase (CP), 28 of whom were untreated and 37 pretreated, and nine patients in accelerated phase (AP), were started on IFN three times a week. Patients in CP were randomized to receive 2 or 5 X 10(6) IU/m2, while patients in AP were all given the dose of 5 X 10(6) IU/m2, in addition to concomitant chemotherapy. Patients in CP who were unresponsive to the lower dose were crossed to the higher dose. Of 63 evaluable patients in CP, 43 (68%) responded, 29 (46%) achieved complete hematologic response (CHR), and 14 (22%) achieved partial hematologic response (PHR). The response rate appeared to be significantly influenced by the IFN dose in pretreated patients. Of the nine patients in AP, two attained PHR and one CHR. More recently, eight previously untreated CP cases were submitted to daily IFN administration at doses from 2 to 5 X 10(6) IU/m2. This daily schedule was also applied to patients who had obtained, with the intermittent treatment, a PHR persisting unmodified for six months (nine patients) or an unstable CHR (five patients). Seven of the eight previously untreated patients, and five of the nine PHR patients crossed to daily IFN reached CHR. In the total series of previously untreated patients, the response rate proved to be significantly influenced by the initial risk status. Cytogenetic improvement was seen in 37 of 53 responders (70%) treated for more than 3 months, the median of Ph′-positive cells declining from 100% to 65% (range 0% to 95%). Complete suppression of Ph′ chromosome was observed in one case. The cytogenetic response was persistent for over 6 months in 21 patients, but the lowest value of Ph′ positivity was usually unstable. At a median follow-up of 56 weeks, 23 of 36 (64%) CHR patients remain in continued disease control with IFN. A blastic transformation (BT) occurred in seven of 21 unresponsive patients and in one of the 36 CHR patients. The authors' data confirm that IFN alpha- 2b, especially at daily doses, is effective in inducing clinical and cytogenetic response in a good proportion of patients with CML in the benign phase. Longer follow-ups will define the exact influence of this agent on the natural course of the disease.
The human T-cell V gamma gene locus: cloning of new segments and study of V gamma rearrangements in neoplastic T and B cells
Authors: Chen Z , Font MP , Loiseau P , Bories JC , Degos L , Lefranc MP , Sigaux F
Published: October 2019

The authors have analyzed the involvement of V gamma and J gamma segments in TRG gamma rearrangement from a series of 40 acute lymphoblastic leukemia (ALL), including 25 T- and 15 B-lineage cases, in which TRG gamma are rearranged. Sixty-five rearranged alleles were studied. The authors first describe the cloning and sequencing of two variable segments, V gamma 11 and psi V gamma 12, which rearrange in T- and B-neoplastic cells. To date three subgroups of translatable V gamma segments have been described. The authors show that V gamma 11 is the unique member of a new fourth V gamma subgroup that also rearranges in normal polyclonal T cells and that psi V gamma 12 is located at 5- kilobase (kb) downstream to V gamma 11. As shown by DNA sequence analysis, V gamma 11 shares a 60% homology with V gamma 10 (third subgroup) and a 50% homology with V gamma 9 (second subgroup) but no appreciable homology with the V gamma segments from the first family. In contrast to psi V gamma 12, V gamma 11 is translatable. In this paper the authors have also attempted to determine which V gamma segments were rearranged in the ALL cases by hybridization with a J gamma probe and genomic probes specific of the four subgroups. In the 54 instances in which the rearrangement was consistent with J gamma 1 or J gamma 2 involvement, the authors have identified the corresponding V gamma segments and have not found any other rearrangements suggestive of the existence of further V regions. The V gamma segments, belonging to the first subgroup, were the most frequently used (41 alleles). V gamma 9, V gamma 10, V gamma 11, and psi V gamma 12 were found rearranged in cases 3, 4, 5, and 1, respectively. No cases using the pseudo psi V gamma 1, psi V gamma 5, and psi V gamma 6 segments were found. Pseudo V gamma segments were not found rearranged in T cells, while V gamma 2 and V gamma 4, segments are frequently used. In contrast to the V gamma I gene rearrangement, the involvement of the V gamma II, V gamma III, and V gamma IV subgroups was most frequently observed in T-ALL with stage II differentiation (CD7+, CD4+, and/or CD8+, CD3-), than in those with stage I (CD7+, CD4-, CD8-, CD3-), than in those with stage I (CD7+, CD4-, CD8-, CD3-) and stage III (CD7+, CD4+/-CD8+/-CD3+).(ABSTRACT TRUNCATED AT 400 WORDS)
Serum levels of CD8 antigen in childhood lymphoid malignancies: a possible indicator of increased suppressor cell activity in poor-risk patients
Authors: Pui CH , Ip SH , Dodge RK , Carrabis S , Brown M , Crist WM , Berard CW , Kung P , Dahl GV , Murphy SB
Published: October 2019

Serum concentrations of CD8 antigen were measured at diagnosis with an enzyme-linked immunoassay in children with acute lymphoblastic leukemia (n = 344) or non-Hodgkin's lymphoma (n = 65). All patients had detectable levels of the serum antigen, which in its soluble nonreduced form appeared to be a 52-Kd homodimer as compared with the 66-Kd surface membrane component on most thymocytes and on a subset of functionally distinct T cells (suppressor/cytotoxic). Increased serum levels of CD8 in leukemia patients were significantly related to recognized high-risk prognostic features: high leukocyte count, large liver and spleen size, high serum lactic dehydrogenase level, T-cell immunophenotype, presence of a mediastinal mass, pseudodiploid karyotype, DNA index less than 1.16, and chromosomal translocation. Children with serum CD8 levels greater than or equal to 450 U/mL were more likely to fail treatment than were those with lower levels (P = .002), even in the group with non-T-cell leukemia (P = .003). In a multivariate analysis, serum CD8 antigen contributed independent prognostic information beyond that conveyed by age, leukocyte count, and race (P = .02). High serum CD8 antigen levels also correlated with advanced stages of disease in children with non-Hodgkin's lymphoma or B- cell leukemia. Children with higher serum CD8 antigen levels (greater than or equal to 700 U/mL) had a poorer treatment outcome (P = .003), even after results were adjusted for disease stage and serum lactic dehydrogenase level (P = .05). Measurement of serum levels of CD8 antigen not only has important prognostic value in childhood lymphoid malignancies but also could be useful in assessing the immunoregulatory role of T cells in patients with cancer.
2-Chlorodeoxyadenosine: an effective new agent for the treatment of chronic lymphocytic leukemia
Authors: Piro LD , Carrera CJ , Beutler E , Carson DA
Published: October 2019

2-Chlorodeoxyadenosine, a new lymphocyte-selective, anti-neoplastic drug was administered to 18 patients with advanced chronic lymphocytic leukemia of B-cell origin. All patients were resistant to conventional treatment. A total of 44 courses of 2-chlorodeoxyadenosine were completed with minimal toxicity. An overall response rate of 55% was achieved with four of 18 patients demonstrating partial response and six of 18 patients experiencing clinical improvement. Only minor bone marrow suppression occurred during administration of the drug, indicating a high degree of lymphocyte selectivity. Reduction of lymphocyte infiltration in bone marrow occurred in treated patients including one patient who experienced normalization of the bone marrow. Three of four patients with concurrent autoimmune hemolytic anemia experienced resolution of hemolysis, as indicated by elimination of transfusion requirement, fall in reticulocyte count, elevation of hemoglobin, and ability to taper prednisone without recurrence of hemolysis. Duration of responses ranged from 2 to 15 months without maintenance therapy.
The putative factor IX gene promoter in hemophilia B Leyden
Authors: Reitsma PH , Bertina RM , Ploos van Amstel JK , Riemens A , Briet E
Published: October 2019

Hemophilia B Leyden is characterized by low levels of factor IX antigen and activity before the age of 15, whereas after puberty factor IX levels rise at a rate of about 5% per year. A single base substitution (-A----T) at position -20 was identified in the putative promoter of the gene cloned from a patient with hemophilia B Leyden. This nucleotide change was confirmed in a second patient from the same pedigree and was also found in a patient from a second Dutch pedigree with the same hemophilic phenotype. The results indicate that the two Dutch kindreds are related and point to the functional significance of the -20 position for the expression of the human factor IX gene.
Treatment with monocyte-derived partially purified GM-CSF but not G-CSF aborts the development of transplanted chloroleukemia in rats
Authors: Jimenez JJ , Yunis AA
Published: October 2019

We have previously demonstrated that cultured rat chloroleukemia cells, MIA C51, will terminally differentiate to macrophages when treated with rat lung-conditioned medium in vitro and in vivo. In the present study we fractionated rat monocyte-conditioned medium by ultrafiltration according to molecular size. The fraction with molecular weight (mol wt) 30 to 50 Kd containing partially purified granulocyte-macrophage colony-stimulating factor (GM-CSF) activity caused the differentiation of C51 cells to macrophages in vitro and in diffusion chambers in vivo. Treatment of young rats with this fraction aborted the development of chloroleukemia from transplanted C51 cells. In contrast, the fraction with mol wt 10 to 30 Kd containing virtually all the G-CSF activity exhibited no differentiation activity either in vitro or in vivo. It is concluded that in this rat myelogenous leukemia model partially purified GM-CSF but not G-CSF contains the effector molecule(s) causing terminal differentiation of C51 cells and tumor cell rejection.
Release of C8 binding protein (C8bp) from the cell membrane by phosphatidylinositol-specific phospholipase C
Authors: Hansch GM , Weller PF , Nicholson-Weller A
Published: October 2019

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol- containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.
Effect of alpha-interferon therapy on bone marrow fibrosis in hairy cell leukemia
Authors: Laughlin M , Islam A , Barcos M , Meade P , Ozer H , Gavigan M , Henderson E , Han T
Published: October 2019

Iliac crest trephine biopsy specimens from 16 patients treated with recombinant alpha 2-interferon (alpha-IFN) for hairy cell leukemia (HCL) were examined for reticulin and collagen content. These data were compared with the hairy cell index (HCl), the proportion of hairy cells to the overall cellularity of the bone marrow. Specimens were studied immediately before alpha-IFN therapy, at 6-month intervals during, and in six patients 6 months after cessation of therapy. All patients presented with increased bone marrow fibrosis ranging from focally increased reticulin to a diffuse increase in both reticulin and collagen content. This fibrosis was observed to decrease during alpha- IFN therapy inasmuch as the hairy cell population was diminished in the bone marrow in 13 patients. Regression analysis of HCl v bone marrow fibrosis showed a positive correlation (r = .73, P less than .02). Six patients demonstrated a reduction in bone marrow reticulin and collagen to normal levels during alpha-IFN therapy. Two of six patients demonstrated increased bone marrow fibrosis and HCl 6 months after cessation of alpha-IFN therapy. Three of 16 patients exhibited no decrease in bone marrow reticulin content during therapy despite a decreased bone marrow hairy cell population.
Prevention of HLA immunization with leukocyte-poor packed red cells and platelet concentrates obtained by filtration
Authors: Andreu G , Dewailly J , Leberre C , Quarre MC , Bidet ML , Tardivel R , Devers L , Lam Y , Soreau E , Boccaccio C
Published: October 2019

HLA immunization is a common complication of transfusion therapy in 30% to 60% of oncohematologic patients. Evidence shows that leukocytes present in cellular blood products are the main component involved in the occurrence of HLA immunization, and several studies showed that leukocyte-poor blood products are less able to induce it. However, leukocyte-poor platelet concentrates obtained by conventional techniques, ie, centrifugation, frequently have a high level of remaining leukocytes. Cotton wool filter Imugard IG 500 can be used to obtain leukocyte-poor cellular blood products. The technique is easy to perform, even in an emergency, and can be used with either packed RBCs or platelet concentrates. Means of 97%, 92%, and 76% elimination of leukocytes are obtained for packed RBCs, pooled standard platelet concentrates, and single-donor platelet concentrates, respectively. Patients were randomized to receive either standard (control group) or filtered (leukocyte-poor group) blood products. Of 112 randomized patients, 69 were evaluable, 35 in the control group and 34 in the leukocyte-poor group. Both groups are comparable according to age, diagnosis, sex ratio, previous transfusions, and pregnancies. There is a significant difference in regard to the HLA immunization rate (31.4% in the control v 11.7% in the leukocyte-poor group, P less than .05) and frequency of refractoriness to platelet transfusions (46.6% v 11.7%, P less than .05). We conclude that this filtration technique can be an efficient means to reduce the HLA immunization rate in polytransfused oncohematologic patients.
Bone involvement in young patients with non-Hodgkin's lymphoma: efficacy of chemotherapy without local radiotherapy
Authors: Haddy TB , Keenan AM , Jaffe ES , Magrath IT
Published: October 2019

Of 95 young non-Hodgkin's lymphoma patients entered consecutively on the National Cancer Institute (NCI) Protocol 7704, 26 (27.4%) had involvement of one or more bones. The mean age of these 26 patients was 16.6 years, and the male to female ratio was 3.3:1. Tumor histology included undifferentiated Burkitt's lymphoma in 12, undifferentiated non-Burkitt's lymphoma in two, undifferentiated, unspecified lymphoma in one, diffuse large cell lymphoma in three, and lymphoblastic lymphoma in eight patients. Most had extensive disease; two patients had isolated bone lesions, one had lesions of two bones without involvement of other tissues, and 23 had either multiple bone lesions or single bone lesions with involvement of other tissues. Eight of the 26 patients had bone marrow involvement. Of a subgroup of 12 patients with jaw disease, 11 had undifferentiated lymphoma and one had diffuse large cell lymphoma. Only one had primary a jaw tumor, with two quadrants of the jaw involved. All 26 patients were treated with chemotherapy; only two received radiotherapy initially for bone lesions. Predicted survival of the 26 patients at 5 years is 53.2%. The 12 patients who remain disease free have a mean survival of 62.1 months (range, 22 to 100 months). Our results call into question the role of radiotherapy in the treatment of bone lesions in non-Hodgkin's lymphoma.
Identification of a second transforming gene, rasn, in a human multiple myeloma line with a rearranged c-myc allele
Authors: Ernst TJ , Gazdar A , Ritz J , Shipp MA
Published: October 2019

Multiple myeloma is a disease characterized by a long, slowly progressive phase and a final, more aggressive one. Little is known about the mechanism of transformation of myeloma cells, although the clinical characteristics of the disease suggest a multi-step process. Recently, a myeloma cell line, NCI-H929, was isolated from a patient with aggressive preterminal disease and found to have a rearranged myc allele. This myeloma cell line has been further characterized in a focus formation assay to determine whether its unusual growth characteristics were associated with a second activated transforming gene. We now report that the NCI-H929 myeloma cell line has an activated rasn allele in addition to a rearranged myc allele. This is the first identification of an activated transforming gene in a multiple myeloma cell line; furthermore, the characterization of two independently activated oncogenes in this B cell malignancy has implications for both the pathogenesis and evolution of the disease.
Variability of the molecular defects corresponding to the presence of a Philadelphia chromosome in human hematologic malignancies
Authors: Saglio G , Guerrasio A , Tassinari A , Ponzetto C , Zaccaria A , Testoni P , Celso B , Rege Cambrin G , Serra A , Pegoraro L
Published: October 2019

By analyzing a total of 107 patients affected by chronic myelogenous leukemia (CML; chronic and blast crisis) or lymphoid and myeloid Philadelphia chromosome (Ph′) positive acute leukemias, we have investigated the relationship between the molecular defect on the Ph′ chromosome and the associated hematologic phenotype. As expected, approximately half of the Ph′ positive acute leukemias showed a breakpoint on chromosome 22 falling outside the “breakpoint cluster region” (bcr) known to be involved in CML. Surprisingly, seven of 80 CML cases in chronic phase also showed rearrangements falling outside the bcr region. In two of these cases the breakpoint on chromosome 22 was mapped between 9 and 12 kb upstream to the bcr region. In another case, the breakpoint was located approximately 16 kb downstream to bcr. In the remaining four cases, the precise position of the rearrangement could not be localized with the available bcr probes. DNAs from patients with CML blast crises showed classical bcr rearrangements. No molecular changes were observed during the progression of the disease in six patients whose DNA from both a chronic and acute phase was available. Our results seem to indicate a greater degree of variability of chromosome 22 breakpoints in CML than previously observed, and the lack of additional rearrangements on the Ph′ chromosome in CML blast crises with respect to chronic phase.
A novel leukemia cell line, MR-87, with positive Philadelphia chromosome and negative breakpoint cluster region rearrangement coexpressing myeloid and early B-cell markers
Authors: Okamura J , Yamada S , Ishii E , Hara T , Takahira H , Nishimura J , Yumura K , Kawa- Ha K , Takase K , Enomoto Y
Published: October 2019

We developed a Philadelphia chromosome (Ph) positive cell line, designated MR-87, from a 4-year-old boy with Ph+-acute leukemia. MR-87 cells grew in single cell suspensions with a doubling time of 120 to 144 hours. Both MR-87 and original leukemia cells were positive for myeloperoxidase (MPO) and myeloid antigen CD13. These cells exhibited the early B-cell phenotype, ie, terminal deoxynucleotidyl transferase+, Ia+, CD19+, and CD10+. Rearrangement of the immunoglobulin heavy chain was confirmed in both. Approximately 80% of MR-87 cells coexpressed CD13 and lymphoid antigens CD10 or CD19, as confirmed by a two-color analysis. Simultaneous expression of MPO and CD19 on a single MR-87 cell was demonstrated at ultrastructural level. Thus, MR-87 is a Ph+ leukemia cell line exhibiting a hybrid phenotype. The breakpoint cluster region (bcr) was not rearranged in the MR-87 cells and subsequent analysis using antisera revealed that these cells expressed a novel protein, P190c-abl, which was immunoprecipitated with anti-abl and anti-phosphotyrosine antibodies. The MR-87 line will be most useful for investigating the biology and pathogenesis of Ph+ bcr- acute leukemia.
Immunoblot analysis shows changes in factor VIII inhibitor chain specificity in factor VIII inhibitor patients over time
Authors: Fulcher CA , Lechner K , de Graaf Mahoney S
Published: October 2019

We have used immunoblotting of purified factor VIII (FVIII) to determine whether or not changes in FVIII chain specificity occur during the course of an inhibitor. Serial plasma samples from 15 inhibitor patients (13 hemophilic and two spontaneous) were analyzed. Nine of the 15 antibodies, all with epitopes on the 44-kilodalton (Kd) thrombin fragment of the 92-Kd FVIII heavy chain and/or the 72-Kd thrombin fragment of the 80-Kd FVIII light chain, showed no change in FVIII chain specificity. However, six of the inhibitors analyzed showed changes in FVIII fragment specificity. Four inhibitors (three hemophilic and one spontaneous) reactive with 72-Kd thrombin fragment also became reactive with the 44-Kd thrombin fragment after an anamnestic response to FVIII infusion. Another inhibitor with epitopes on both the 54-Kd and 44-Kd thrombin fragments lost most of its reactivity with the 44-Kd fragment but retained its reactivity with the 54-Kd fragment following a FVIII infusion. The inhibitor later regained its 44-Kd-fragment reactivity but lost its 54-Kd-fragment reactivity following treatment with FEIBA, FVIII inhibitor bypassing activity. The last inhibitor studied had an antibody to either the 44-Kd fragment or to both the 44-Kd and 72-Kd fragments during anamnestic responses to FVIII. These data indicate that a FVIII inhibitor patient can potentially produce antibody to multiple areas on the FVIII molecule and that this must be taken into account in the design of specific therapeutic products.
Production of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor by human mononuclear cells stimulated with granulocyte- macrophage colony-stimulating factor [see comments]
Authors: Sisson SD , Dinarello CA
Published: October 2019

Recent studies have examined the synergistic effects of granulocyte- macrophage colony-stimulating factor (GM-CSF) and hematopoietin-1 (now identified as Interleukin-1, IL-1) on bone marrow colony formation. In the present report, human peripheral blood mononuclear cells (MNCs) were stimulated in vitro with recombinant human GM-CSF (rGM-CSF) and production of IL-1 alpha, IL-1 beta, and tumor necrosis factor (TNF) was measured by specific radioimmunoassays. In the MNCs of 20 individuals, rGM-CSF's ability to induce the three cytokines was variable. Nearly all donors responded to low-dose rGM-CSF (0.02 to 2 ng/mL) with production of TNF, whereas some individuals did not produce IL-1 alpha or IL-1 beta. The MNCs from some subjects stimulated with high-dose rGM-CSF (10 to 80 ng/mL) produced as much cytokine as in response to 10 ng/mL endotoxin. Localization (ie, extracellular or cell- associated cytokine) was specific for the cytokine rather than the stimulus. Indomethacin increased the amount of cytokine produced in response to rGM-CSF for IL-1 beta and TNF but not for IL-1 alpha. In addition, interferon-gamma (INF-gamma) upregulated the amount of TNF induced by rGM-CSF in all donors examined, with variable effect on IL-1 alpha and IL-1 beta. Suboptimal levels of endotoxin incubated with rGM- CSF did not alter the amount of IL-1 produced as compared with cells stimulated with rGM-CSF alone, whereas TNF production showed either no change or a slight decrease in production. These data suggest that GM- CSF may play an important role in the host defense response by stimulating production of these cytokines.
Evaluation of clinical competence in hematology training programs
Authors: Schrier SL , Benson JA Jr
Published: October 2019

The American Board of Internal Medicine (ABIM) has called on directors of hematology training programs to establish systems to evaluate, document, and substantiate those components of overall clinical competence considered essential for certification in the subspecialty. Many of these can be assessed only by repeated direct observations. In particular, proficiency is now required in the preparation of blood smears, bone marrow aspiration and biopsy, administration of chemotherapy, management of indwelling vascular access, lumbar puncture with chemotherapy, bleeding time, phlebotomy, and exchange transfusion. The goal of this expanded evaluation program is to ensure that the public and the profession can identify, through certification, physicians with demonstrated excellence in hematology.
Defective T suppressor-inducer cell function in human immune deficiency virus-seropositive hemophilia patients
Authors: Sjamsoedin-Visser LJ , Heijnen CJ , Zegers BJ , Stoop JW
Published: October 2019

In human immune deficiency virus (HIV)-seropositive hemophilia patients, a low number of CD4 + lymphocytes is found, as well as a low CD4+/CD8+ ratio. In previous studies, it has been shown that antigen- specific T-helper cell (CD4+) function was present and no excessive antigen-specific T-suppressor cell (CD8+) function could be demonstrated. In this report, we studied another activity of CD4+ cells, namely the capacity to induce T-suppressor cell activity. The results clearly show a selective dysfunction of CD4+ suppressor-inducer (Tsi) cell function. Since these HIV-seropositive hemophilia patients showed the presence of activated B cells in the peripheral circulation refractory to antigen-specific T-helper cell signals and secreting specific antibodies spontaneously, we raised the hypothesis that the activated B cells in the patients activate the Tsi cells in vivo. This constant activation leads to a functional exhaustion of the Tsi cell pool.
Regulation of hematopoiesis-IV: The role of interleukin-3 and bryostatin 1 in the growth of erythropoietic progenitors from normal and anemic W/Wv mice
Authors: Leonard JP , May WS , Ihle JN , Pettit GR , Sharkis SJ
Published: October 2019

We and others have established a role for T lymphocytes and their products in the regulation of erythropoiesis. Interleukin-3 (IL-3) is a multipotential lymphokine with burst-promoting activity that is produced by activated T lymphocytes. In the anemic, stem cell-defective W/Wv mouse we have described the absence of a functionally active thymocyte population that in normal animals enhances erythroid progenitor growth and stem cell self-renewal. In studies reported here we find that W/Wv mouse marrow responds to exogenous IL-3 by increased erythroid progenitor cell growth. The BFU-E and CFU-E from anemic donors are more sensitive to IL-3 than are those in +/+ marrow. We have recently observed a stimulatory effect of bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate) on normal erythropoiesis in human bone marrow progenitor assays. To test the effects of this molecule on murine normal and anemic W/Wv cells we grew these cells in the presence of increasing doses of bryostatin 1. Bryostatin mimics the stimulatory action of IL-3 on W/Wv bone marrow. Polyclonal antibody directed against murine IL-3 blocks the stimulatory effect of bryostatin on erythropoiesis. Otherwise inactive thymocytes from W/Wv mice in coculture with W/Wv bone marrow showed stimulation of erythropoiesis in the presence of bryostatin. We believe that bryostatin may in part act by stimulating T lymphocytes to release physiologic concentrations of lymphokines.
Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant
Authors: Porter JB , Gyparaki M , Burke LC , Huehns ER , Sarpong P , Saez V , Hider RC
Published: October 2019

A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4- ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2- ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.
Phorbol ester induces expression and function of interleukin-2 receptors on a human leukemic cell line with a T-cell precursor phenotype
Authors: Kees UR
Published: October 2019

We show here that a human leukemic cell line, PER-117, bearing the markers of a T-cell precursor phenotype, can be induced to express receptors for interleukin-2 (IL-2). These IL-2 receptors could be demonstrated to mediate a physiologic response to the lymphokine for which the high-affinity form of the IL-2 receptor appears to be essential. The phenotype of PER-117 cells corresponds to the earliest identifiable stage of T-cell differentiation, which is defined by the lack of the T3-T-cell receptor complex and the presence of the 40 Kd protein recognized by monoclonal antibodies of the CD7 group. Further evidence for the clonality and T-cell lineage of this cell line was obtained by analysis of rearrangements of genes for the T-cell receptor (TCR) beta chain and for the immunoglobulin heavy-chain (IgJH) genes. PER-117 cells could be shown to have rearranged TCR beta genes but no rearrangement of the IgJH genes. Cell line PER-117 provides a model to investigate the requirements for induction of IL-2 receptors in a cell expressing the first T-cell-specific marker and may help to elucidate the role of IL-2 during thymic differentiation and in the uncontrolled proliferation of T-cell leukemias.
Effects of recombinant human interleukin-3 on CD34-enriched normal hematopoietic progenitors and on myeloblastic leukemia cells
Authors: Saeland S , Caux C , Favre C , Aubry JP , Mannoni P , Pebusque MJ , Gentilhomme O , Otsuka T , Yokota T , Arai N
Published: October 2019

Induction of proliferation and differentiation in response to recombinant human interleukin-3 (hIL-3) was studied in liquid and semisolid cultures of umbilical cord blood and bone marrow cells that were fractionated by “panning” with anti-My10 antibody according to expression of CD34 antigen. Cells from enriched fractions (70% to 90% CD34+) were found to proliferate strongly in response to hIL-3. Phenotypic analysis and morphologic characterization of the proliferating cells demonstrated a rapid decrease in CD34+ cells and an exponential increase in the number of cells belonging to the neutrophilic, eosinophilic, monocyte/macrophage, and thrombocytic lineages. When combined with recombinant human erythropoietin, burst colonies and cells expressing glycophorin-A were detected, thereby demonstrating the effects of hIL-3 on erythroid progenitors. Further, the development of mixed-erythroid colonies indicated that multipotential cells within CD34-enriched fractions responded to hIL-3. In addition, we examined the effect of hIL-3 on the proliferation of primary acute myeloblastic leukemia cells in liquid culture. We found that hIL-3 was able to induce cell proliferation in a proportion of the cases tested. Heterogeneity of the responses to hIL-3 was in part related to French-American-British classification but could not be correlated with CD34 antigen expression by the leukemic cells. These results indicate that, although the effects of hIL-3 on proliferation and differentiation of cells obtained from normal hematopoietic specimens were primarily borne by CD34+ cells, expression of the CD34 molecule per se is an insufficient condition to determine a growth response to this lymphokine.
Interleukin-1 alpha enhances the in vitro survival of purified murine granulocyte-macrophage progenitor cells in the absence of colony- stimulating factors
Authors: Williams DE , Broxmeyer HE
Published: October 2019

Interleukin-1 (IL-1), recently identified as hemopoietin-1, affects hematopoiesis by presumably direct and indirect mechanisms. IL-1 stimulates primitive hematopoietic stem cells to express receptors for the granulocyte-macrophage colony-stimulating factors (GM-CSFs) and stimulates CSF release from accessory cells. We used highly purified murine granulocyte-macrophage progenitor cells (CFU-GM, up to 92% cloning efficiency) to assess the effects of IL-1 alpha on the proliferation, differentiation, and survival of these cells. The results demonstrated that IL-1 alpha does not directly influence the proliferation or differentiation of purified CFU-GM in the presence of plateau concentrations of purified natural or recombinant CSFs, and IL- 1 alpha lacks intrinsic CSF activity of its own. CSF deprivation studies showed that IL-1 alpha rapidly (within one hour) promoted CFU- GM survival in the absence of CSF. This enhanced survival stimulated by IL-1 alpha was observed with CFU-GM responding to purified recombinant GM-CSF, natural M-CSF, recombinant G-CSF, recombinant IL-3, or IL-3 in WEHI-conditioned medium, and no effect on the pattern of CFU-GM differentiation occurred in cultures incubated with IL-1 alpha in the absence of CSF. The IL-1 alpha effects on CFU-GM are probably due to a direct action on progenitor cells because the presence or absence of accessory cells did not alter the results and concentrations of the CSFs that were too low to stimulate the proliferation of CFU-GM could not mimic the IL-1 alpha effect on CFU-GM survival.
Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B
Authors: Thompson AR , Chen SH , Smith KJ
Published: October 2019

In hemophilia B, assays based on a monoclonal antifactor IX specific for the Thr-148 variant of an exonic polymorphism have diagnosed carriers in selected families by either establishing linkage or by indicating the presence or absence of a given normal factor IX. The sensitivity of the immunoassays for detecting heterozygous women was explored by comparing results from immunoassays with solid-phase polyclonal v the monoclonal antifactor IXs. Factor IX with the normal Ala-148 variant gave a flat dilution curve, qualitatively distinct from factor IX with the Thr-148 variant in the monoclonal assay. The two were indistinguishable in the polyclonal assay. Mixtures of equal amounts of the two types gave an intermediate result, about half as reactive in the monoclonal as compared with the polyclonal assay system. Whereas mixtures with 10% Ala-148 and 90% Thr-148 factor IXs could not readily be distinguished from Thr-148 factor IX plasma, as little as 1% of the Thr-148 protein was detected in Ala-148 factor IX plasma. The frequency of the Ala-148 variant varied in individuals with different ethnic backgrounds; it was found in 29% of white, 12% of black, and none of Asian blood donors' factor IX genes in Seattle. Only 4% of samples from South African black men were nonreactive (ie, Ala- 148). The Thr/Ala-148 dimorphism is in strong linkage disequilibrium with Taql restriction fragment length polymorphisms (RFLPs). Three recombinations were noted in normal white genes and one in a normal black factor IX gene (less than 2% of those examined). In 34 white families with at least one woman being a possible carrier, genetically, the immunoassay results were informative in 18. RFLP analyses were informative in eight of the 15 families tested. In five families each, assignment of carrier status was made to a woman by only DNA or only immunoassay results, whereas the other approach was noninformative. The immunoassays provide a rapid, inexpensive screening test and complement DNA analysis in white women who are potential carriers of hemophilia B.
Flow-cytometric detection of terminal deoxynucleotidyl transferase and other intracellular antigens in combination with membrane antigens in acute lymphatic leukemias
Authors: Slaper-Cortenbach IC , Admiraal LG , Kerr JM , van Leeuwen EF , von dem Borne AE , Tetteroo PA
Published: October 2019

Development of a new fixation procedure allowed flow-cytometric analysis of nuclear and other intracellular antigens in acute lymphatic leukemia (ALL). A short fixation of the cells with buffered formaldehyde acetone (BFA) rendered the cell membrane permeable, allowing the monoclonal antibodies (MoAbs) to penetrate the cell. Through this method, a rapid analysis of intracellular antigens, specific for acute lymphatic leukemia [such as terminal deoxynucleotidyl transferase (TdT), immunoglobulin M (IgM) heavy chain, and antigens recognized by the CD22 or CD3 MoAbs) was performed by flow cytometry. The surface antigens remained intact after this fixation procedure, enabling simultaneous detection of membrane and intracellular antigens. The binding of biotinylated antibodies against several B- and T-lymphoid membrane antigens was detected with streptavidin-phycoerythrin (red fluorescence), whereas the intracellular antigens were stained with FITC-labeled polyclonal antibodies, or indirectly with FITC-labeled goat anti-mouse IgG (green fluorescence). Through this combination of markers, minor cell populations can be detected and a rapid and quantitative immunodiagnosis can be performed.
Development of the human coagulation system in the healthy premature infant
Authors: Andrew M , Paes B , Milner R , Johnston M , Mitchell L , Tollefsen DM , Castle V , Powers P
Published: October 2019

This study was designed to determine the postnatal development of the human coagulation system in the healthy premature infant. Consecutive mothers of healthy premature infants born at either St Joseph's Hospital or McMaster University Medical Centre in Hamilton were asked for consent. One hundred thirty-seven premature infants (30 to 36 weeks of gestational age) entered the study. The premature infants did not have any major health problems and did not require ventilation or supplemental oxygen. Demographic information and a 20-mL blood sample were obtained in the postnatal period on days 1, 5, 30, 90, and 180. Between 40 and 96 premature infants were studied on each day for each of the following tests: prothrombin time, activated partial thromboplastin time, thrombin clotting time, plasminogen; 13 factor assays [fibrinogen, II, V, VII, VIII, IX, X, XI, XII, XIII, high-mol-wt kininogen (HMWK), prekallikrein (PK), von Willebrand factor (vWF)] and eight inhibitors [antithrombin III (AT-III), heparin cofactor II, alpha 2-antiplasmin, alpha 2-macroglobulin, alpha 1-antitrypsin, C1 esterase inhibitor, protein C (PC), and protein S (PS)]. A combination of biologic and immunologic assays were used. Between 30 to 36 weeks there was a minimal effect of gestational age for levels of AT-III, PC, and factors II and X only; therefore, the entire data set was used to generate reference ranges for these components of the coagulation system for premature infants. Next, the results for the premature infants were compared with those of a previously published study in 118 fullterm infants and with those for adults. An effect of gestational age was shown for plasminogen, fibrinogen, factors II, V, VIII, IX, XI, XII, HMWK, and all eight inhibitors. In general, the postnatal maturation towards adult levels was accelerated in premature infants as compared with the fullterm infants. By 6 months of age, most components of the coagulation system in premature infants had achieved near adult values.
Regulation of streptokinase-human plasmin complex by the plasma proteinase inhibitors alpha 2-antiplasmin and alpha 2-macroglobulin is species specific and temperature dependent
Authors: Gonias SL , Figler NL , Braud LL
Published: October 2019

Streptokinase-plasmin complex (SkPl) was prepared with human plasminogen. Regulation of SkPl and plasmin by the plasma proteinase inhibitors, alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M), was studied as a function of temperature in plasminogen- depleted human plasma, mouse plasma, and solutions of purified proteins. The reaction of plasmin with proteinase inhibitors in human plasma was complete. alpha 2AP was the predominant inhibitor. The fraction of alpha 2M-plasmin recovered was not affected significantly by incubation temperature. In contrast, the reaction of SkPl with human proteinase inhibitors was markedly temperature dependent. The apparent second-order rate constant for the reaction of SkPl with purified alpha 2AP at 37 degrees C (1.5 x 10(2) mol/L-1 s-1) was greater than 150-fold higher than the constant derived at 4 degrees C. In human plasma and in solutions containing mixtures of purified human proteins, alpha 2AP was the principal inhibitor of SkPl. Elevating the temperature enhanced the reaction of SkPl with alpha 2AP and alpha 2M comparably. Equivalent results were obtained when incubations were performed in platelet-rich plasma (PRP) or whole blood. In murine plasma, SkPl reacted readily with the proteinase inhibitors. The principal inhibitor of SkPl was alpha 2M. Maximum reaction between SkPl and murine alpha 2M was observed at 37 degrees C; however, significant reaction also occurred at 4 degrees C. alpha 2 AP was the predominant inhibitor of plasmin in mouse plasma. Reaction of alpha 2AP with SkPl in murine plasma was significant only after the alpha 2M was inactivated with methylamine. These results were not affected by platelets or whole blood cells. We conclude that the thrombolytic efficacy of streptokinase reflects not only the nature of the plasminogen activator complex but also the function of the proteinase inhibitors.
Effects of recombinant human granulocyte-macrophage colony-stimulating factor on intracellular pH in mature granulocytes
Authors: Sullivan R , Griffin JD , Wright J , Melnick DA , Leavitt JL , Fredette JP , Horne JH Jr , Lyman CA , Lazzari KG , Simons ER
Published: October 2019

We studied the effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes using the fluorescent probe BCECF. GM-CSFrh did not directly alter the resting pH of granulocytes isolated from the peripheral blood; however, when the cells were preincubated for 90 minutes with the growth factor and then activated with the chemotactic peptide N-formyl met leu phe (fMLP), they exhibited both an acceleration in the initial rate of acidification and a marked delay in realkalinization. The kinetic changes both in initial acidification and in subsequent realkalinization induced by GM-CSFrh priming were not prevented by protein synthesis inhibitors and were observed in granulocytes harvested from patients with both sex-linked and autosomal recessive chronic granulomatous disease (CGD). By directly quantitating H+ ion secretion, by monitoring the effects of sodium repletion on intracellular pH, and through use of the sodium channel inhibitors amiloride and dimethyl amiloride and the Na+/K+-ATPase inhibitor ouabain, we showed that the altered kinetics of intracellular acidification and alkalinization following fMLP stimulation of GM-CSFrh- primed granulocytes could not be accounted for by changes in transmembrane proton exportation regulated by the Na+/H+ antiport channel. Although the initial acidification following fMLP was abrogated by 2-deoxy-D-glucose in both GM-CSFrh-pretreated and GM-CSFrh- untreated granulocytes, retardation of the subsequent phase of alkalinization was observed in GM-CSFrh-primed cells even after inhibition of both glycolytic and mitochondrial metabolism. Our data indicate that the increased cytosolic acidification following fMLP stimulation in granulocytes “primed” with GM-CSFrh does not result from disordered proton excretion but instead from increased release of intracellular free acid which is only partially coupled to glucose catabolism or to the generation of superoxide anion (O2-).