bluuebunny/crossref_metadata_2025 huggingface dataset:
Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease
Authors: Mackinnon S , Papadopoulos EB , Carabasi MH , Reich L , Collins NH , Boulad F , Castro-Malaspina H , Childs BH , Gillio AP , Kernan NA
Published: October 2019

Infusions of large numbers (> 10(8)/kg) of donor leukocytes can induce remissions in patients with chronic myeloid leukemia (CML) who relapse after marrow transplantation. We wanted to determine if substantially lower numbers of donor leukocytes could induce remissions and, if so, whether this would reduce the 90% incidence of graft-versus-host disease (GVHD) associated with this therapy. Twenty-two patients with relapsed CML were studied: 2 in molecular relapse, 6 in cytogenetic relapse, 10 in chronic phase, and 4 in accelerated phase. Each patient received escalating doses of donor leukocytes at 4- to 33-week intervals. Leukocyte doses were calculated as T cells per kilogram of recipient weight. There were 8 dose levels between 1 x 10(5) and 5 x 10(8). Lineage-specific chimerism and residual leukemia detection were assessed using sensitive polymerase chain reaction (PCR) methodologies. Nineteen of the 22 patients achieved remission. Remissions were achieved at the following T-cell doses: 1 x 10(7) (n = 8), 5 x 10(7) (n = 4), 1 x 10(8) (n = 3), and 5 x 10(8) (n = 4). To date, 15 of the 17 evaluable patients have become BCR-ABL negative by PCR. The incidence of GVHD was correlated with the dose of T cells administered. Only 1 of the 8 patients who achieved remission at a T-cell dose of 1 x 10(7)/kg developed GVHD, whereas this complication developed in 8 of the 11 responders who received a T-cell dose of > or = 5 x 10(7)/kg. Three patients died in remission, 1 secondary to marrow aplasia, 1 of respiratory failure and 1 of complications of chronic GVHD. Sixteen patients who were mixed T-cell chimeras before treatment became full donor T-cell chimeras at the time of remission. Donor leukocytes with a T-cell content as low as 1 x 10(7)/kg can result in complete donor chimerism together with a potent graft-versus-leukemia (GVL) effect. The dose of donor leukocytes or T cells used may be important in determining both the GVL response and the incidence of GVHD. In many patients, this potent GVL effect can occur in the absence of clinical GVHD.
Characterization and purification of a primitive hematopoietic cell type in adult mouse marrow capable of lymphomyeloid differentiation in long-term marrow "switch" cultures
Authors: Lemieux ME , Rebel VI , Lansdorp PM , Eaves CJ
Published: October 2019

In this report, we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-ICML) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro. The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and then for an additional week under conditions permissive for B-lymphopoiesis. All of the clonogenic pre-B progenitors (colony-forming unit [CFU] pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for > or = 7 days was found to irreversibly terminate CFU-pre-B production and, in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in > 70% of cultures 1 week after the switch), the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells, regardless of the purity of the input population. Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-ICML in normal adult mouse marrow to be 1 per 5 x 10(5) cells with an enrichment of approximately 500-fold in the Sca-1+ Lin-WGA+ fraction, as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC. LTC-ICML also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC, thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S. The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro, without the need for their prior purification, should facilitate studies of totipotent hematopoietic stem cell regulation.
Cell-free transmission of Fv-4 resistance gene product controlling Friend leukemia virus-induced leukemogenesis: a unique mechanism for interference with viral infection
Authors: Kitagawa M , Aizawa S , Kamisaku H , Ikeda H , Hirokawa K , Sado T
Published: October 2019

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We previously demonstrated that mixed radiation bone marrow chimeras containing Fv-4r-bearing BALB/c-Fv- 4Wr (C4W) bone marrow and Fv-4r-bearing C3H/He (C3H) bone marrow grafted into C3H recipient mice (C4W+C3H-->C3H) were resistant to Friend leukemia virus (FLV)-induced leukemogenesis, even when they contained as high as 70% C3H-derived cells. This indicates that FLV- sensitive C3H-derived cells are rendered refractory to infection and/or transformation with FLV when they coexist in mice with Fv-4r-bearing cells. To investigate the mechanism of Fv-4 resistance to FLV-induced leukemogenesis, we first examined the expression of Fv-4r env antigen in the peripheral blood mononuclear cells (PBMC) of these chimeras. The Fv-4r env antigen was present not only on C4W-derived cells, but also on Fv-4r-bearing C3H-derived cells in C4W+C3H-->C3H mixed bone marrow chimeras. The Fv-4r env antigen that binds to the cells surface of C3H cells was found in sera from normal C4W mice, C4W-->C3H chimeras, and C4W+C3H-->C3H mixed chimeras. The serum Fv-4r env antigen binds to ecotropic MuLV receptors, shown by specific binding to transfectant mink cells expressing ecotropic MuLV receptor, but not to parental mink cells. To determine whether the binding of Fv-4r env antigen to the putative MuLV receptors would block FLV infection, C3H thymocytes or spleen cells that had been preincubated with C4W serum were mixed with FLV and the subsequent production of MuLV specific antigens was examined. C3H thymocytes or spleen cells treated with C4W serum became refractory to binding by FLV. These results provide evidence that the Fv-4r env antigen is released from C4W-derived cells in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with FLV. The implication of these findings for gene therapy of retrovirus-induced disease such as acquired immune deficiency syndrome (AIDS) is discussed.
Reverse dot-blot detection of the African-American beta-thalassemia mutations
Authors: Sutcharitchan P , Saiki R , Huisman TH , Kutlar A , McKie V , Erlich H , Embury SH
Published: October 2019

DNA-based diagnosis of the beta thalassemias provides accuracy to newborn screening genetic counseling, and prenatal diagnosis. However, the use of polymerase chain reaction (PCR)-based methods is challenged by the great number of different-beta-thalassemia mutations that exist even within defined ethnic groups. In this regard, the reverse dot-blot method offers a means of screening for several mutations with a single hybridization reaction. We have applied the reverse dot-blot method to the detection of the beta-thalassemia mutations of African-Americans. We used two biotin-labeled primer pairs in a duplex reaction to amplify and label two beta-globin target DNA fragments that encompass all known African-American beta-thalassemia mutations. The PCR products were denatured and hybridized to polyT-tailed, membrane-fixed, allele- specific probe pairs for the hemoglobin (Hb) S, Hb C, and 14 beta- thalassemia mutations and their corresponding wild-type sequences. Seven common mutations plus Hb S and Hb C were included on one diagnostic strip, and seven less common beta-thalassemia mutations were included on another strip. Carefully controlled, high stringency hybridization allowed accurate distinction of these alleles. Reverse dot-blot diagnosis of the less common beta-thalassemia mutations precludes the need for alternative, more technically challenging methods. This method provides a rapid, accurate method for diagnosis of beta thalassemia among African-Americans and other ethnic groups in which beta thalassemia occurs.
The significance of HLA-DRB1 matching on clinical outcome after HLA-A, B, DR identical unrelated donor marrow transplantation
Authors: Petersdorf EW , Longton GM , Anasetti C , Martin PJ , Mickelson EM , Smith AG , Hansen JA
Published: October 2019

Despite matching for serologically defined HLA-A, B, DR antigens, acute graft-versus-host disease (GVHD) is a major complication contributing to increased morbidity and mortality in patients who undergo marrow transplantation from unrelated donors. The extent to which unrecognized mismatching for alleles that encode DR1-DR18 contribute to the increased risk of acute GVHD and overall survival is unknown. We analyzed 364 patients and their HLA-A, B, DR serologically matched donors to determine whether molecular typing of DRB1 alleles can allow more accurate donor/recipient matching and thereby improve clinical outcome after marrow transplantation. DRB1 alleles were typed by sequence-specific oligonucleotide probe hybridization methods. Selected alleles were confirmed by DNA sequencing. Of the 364 pairs, 305 were matched and 59 were mismatched for DRB1. The probability of moderate to severe acute GVHD was .48 for the matched and .70 for the mismatched patients. Compared with mismatched patients, the estimated relative risk (RR) of GVHD for matched patients was .58 (95% confidence interval [CI], .40 to .85). DRB1 matching decreased the risk of transplant- related mortality (RR, .66; 95% CI, .44 to .97) and was associated with decreased overall mortality (RR, .71; 95% CI, .51 to 1.0). Therefore, matching DRB1 alleles of the donor and recipient decreases the risk of acute GVHD and improves survival after unrelated marrow transplantation. These results indicate that prospective matching of patients and donors for DRB1 alleles is warranted.
Long-term effects of hepatitis C virus infection in allogeneic bone marrow transplant recipients
Authors: Ljungman P , Johansson N , Aschan J , Glaumann H , Lonnqvist B , Ringden O , Sparrelid E , Sonnerborg A , Winiarski J , Gahrton G
Published: October 2019

A total of 161 patients transplanted between 1978 and 1991 and who had survived at least 2 years after allogeneic bone marrow transplantation (BMT) were studied. Of 161 surviving patients, 28 (17.4%) were positive for hepatitis C virus (HCV) either by serology or polymerase chain reaction (PCR). Twenty-five patients were positive for HCV RNA by PCR, and 26 of the 28 patients had HCV antibodies detected by enzyme-linked immunosorbent assay (ELISA). The median follow-up time of HCV-positive patients was 6.1 years (range, 2.8 to 14.0 years). There was no difference in the frequency or degree of liver dysfunction between patients who were PCR-positive or -negative before BMT. Six patients developed severe liver dysfunction after BMT, and five of these patients did so after discontinuation or tapering of immunosuppression. No patient has developed liver failure. Serum transaminases were abnormal at the time of last follow up in 19 of 28 (68%) patients. Fifteen patients have had liver biopsies. No biopsy showed development of cirrhosis. We conclude that HCV is not a major contributing factor to morbidity and mortality during the first 5 to 10 years after allogeneic BMT.
Human allogeneic stem cell maintenance and differentiation in a long- term multilineage SCID-hu graft
Authors: Fraser CC , Kaneshima H , Hansteen G , Kilpatrick M , Hoffman R , Chen BP
Published: October 2019

The ability to determine the functional capacity of putative human hematopoietic stem cell (HSC) populations requires in vivo assays in which long-term multilineage differentiation can be assessed. We hypothesized that if human fetal bone was transplanted adjacent to a fetal thymus fragment in severe combined immunodeficient (SCID) mice, a conjoint organ might form in which HSC in the human bone marrow (BM) would mimic human multilineage differentiation into progenitor cells, B cells, and myeloid cells; undergo self-renewal; and migrate to and differentiate into T cells within the thymic microenvironment. To test this possibility, SCID mice were transplanted subcutaneously with HLA class I mismatched fetal bone, thymus, and spleen fragments (SCID-hu BTS). We found that the BM of SCID-hu BTS grafts maintained B cells, myeloid cells, CD34+ cells for at least 36 weeks posttransplant. Assayable hematopoietic progenitors colony-forming units-granulocyte- macrophage were present in 100% (66/66) of grafts over a period of 28 weeks. Cells with a HSC phenotype (CD34+Thy-1+Lin-) were maintained for 20 weeks in SCID-hu BTS grafts. These CD34+Thy-1+Lin- cells had potent secondary multilineage reconstituting potential when isolated and injected into a secondary HLA mismatched SCID-hu bone assay and analyzed 8 weeks later. In addition, early progenitors within the BM of SCID-hu BTS grafts were capable of migrating to the human thymus and undergoing differentiation through immature CD4+CD8+ double-positive T cells and produce mature T cells with a CD4+CD8- or CD8+CD4- phenotype that could be detected for at least 36 weeks. Phenotypically defined human fetal liver (FL) and umbilical cord blood (UCB) hematopoietic stem cell populations were injected into irradiated SCID-hu BTS grafts to assess their multilineage repopulating capacity and to assess the ability of the BTS system to provide an environment where multiple lineages might differentiate from a common stem cell pool. Injection of irradiated grafts with FL HSC or UCB HSC cells resulted in donor- derived B cells, myeloid cells, immature and mature T cells, and CD34+ cells in individual grafts when analyzed 8 weeks postreconstitution, further showing the multipotential nature of these stem cell populations. In addition, a strong correlation was observed between maintenance of host graft-derived CD8+ cells and failure of donor stem cell engraftment.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect of aggressive daunomycin therapy on survival in acute promyelocytic leukemia
Authors: Head D , Kopecky KJ , Weick J , Files JC , Ryan D , Foucar K , Montiel M , Bickers J , Fishleder A , Miller M
Published: October 2019

The Southwest Oncology Group analyzed outcome with cytotoxic chemotherapy for previously untreated acute myeloblastic leukemia (AML) from 1982 through 1986. Results with acute promyelocytic leukemia (APL) prompted comparison with patients from 1986 through 1991 and analysis of factors contributing to APL results. Patient and disease characteristics and treatment outcome were compared for all evaluable patients, with more detailed analysis of factors affecting APL treatment outcome. From 1982 through 1986, median survival and disease- free survival in 45 APL patients were 106 months and greater than 105 months, respectively, versus 6 and 14 months for 417 other AML patients. Such differences were not seen from 1986 through 1991. In the 141 APL patients from 1982 through 1991, after adjusting for significant patient and disease characteristics, higher daunomycin (DNR) doses during induction were significantly associated with higher complete remission rates (P < .0001), longer survival (P < .0001), and longer DFS (P < .0001). Cytosine arabinoside (Ara-C) induction dose, the inclusion of other chemotherapy agents in induction, postremission therapy (consolidation, maintenance, or bone marrow transplantation) other than DNR, APL subtype, and patient age did not appear to significantly affect outcome of APL, except for a significant detrimental effect of high-dose Ara-C in consolidation (P = .0042). Morphologic AML subtypes other than APL did not affect outcome. We conclude that high-dose DNR selectively increases survival in APL. This good survival is important for evaluation of combined all-trans retinoic acid (ATRA)/chemotherapy protocols and for planning future combinations of chemotherapy and ATRA. These results illustrate the need to individualize chemotherapy for subtypes of AML. Therapeutic response of APL is independent of age. Except for APL, morphologic subclassification of AML contributed little prognostic information.
Somatic mutations of PIG-A in Thai patients with paroxysmal nocturnal hemoglobinuria
Authors: Pramoonjago P , Wanachiwanawin W , Chinprasertsak S , Pattanapanayasat K , Takeda J , Kinoshita T
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by clonal blood cells that are deficient in the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins. In the affected cells, the X-chromosomal gene PIG-A, which participates in biosynthesis of the GPI anchor, is somatically mutated. Analyses of Japanese, British, and American patients with PNH have shown somatic mutations of PIG-A in all of them, indicating that PIG-A is responsible for PNH in most, if not all, patients in those countries. Twenty-nine of the reported somatic mutations are small, mostly involving 1 or 2 bases, except for one with a 4-kb deletion. Here we describe an analysis of PIG-A in neutrophils from 14 patients from Thailand where PNH is thought to be more common. We found small somatic PIG-A mutations in all patients. These consisted of six single base deletions, one each of 2-, 3-, 5- and 10-base deletions, two single base insertions and two base substitutions. Thus, the small somatic mutation in the PIG-A gene is also responsible for PNH in Thailand. However, base substitutions were rarer (2 of 14) than in Japan (8 of 16), and deletions of multiple bases were more common, suggesting various causes of mutation.
Regulation of colony-stimulating factor 1-induced proliferation by heterotrimeric Gi2 proteins
Authors: Corre I , Hermouet S
Published: October 2019

Receptors for hematopoietic cytokines possess intrinsic tyrosine- kinases or are associated with tyrosine-kinases; interactions between metabolic pathways activated by tyrosine-kinases and heterotrimeric G proteins are suspected, but not yet proven. To investigate whether alteration of G protein function affects signal transduction of hematopoietic cytokines, we expressed mutant Gi2 proteins in BAC 1.2F5 cells, a murine macrophage cell line that is dependent from monocyte- macrophage colony-stimulating factor (CSF 1) for its proliferation. Mutations made in alpha subunits constitutively activate (alpha i2- Q205L) or inactivate (alpha i2-G204A) Gi2 heterotrimers. We show that expression of alpha i2-Q205L in BAC 1.2F5 cells does not induce independence from CSF 1, but reduces the cells' requirement in CSF 1, shortens the length of the G1 phase and the cell doubling time in response to CSF 1, and protects cells from death by apoptosis induced by CSF 1 withdrawal, exposure to H2O2 or heat shock, but not mitoxantrone. More importantly, expression of alpha i2-G204A, a dominant negative mutant, inhibits BAC 1.2F5 cell proliferation in response to CSF 1, increases the length of the G1 phase and the cell doubling time, and accelerates apoptotic cell death after withdrawal of CSF 1, exposure to H2O2 or heat shock. We conclude that the metabolic pathways regulated by Gi2 proteins and CSF 1 tyrosine-kinase receptors converge on a common effector necessary for the regulation of macrophage survival and proliferation.
Acute lymphoblastic leukemias with deletion of 11q23 or a novel inversion (11)(p13q23) lack MLL gene rearrangements and have favorable clinical features
Authors: Raimondi SC , Frestedt JL , Pui CH , Downing JR , Head DR , Kersey JH , Behm FG
Published: October 2019

Balanced translocations affecting the 11q23 region are among the most frequent chromosomal abnormalities in childhood acute lymphoblastic leukemia (ALL), comprising 5% to 6%. These cases consistently have a rearranged MLL gene and are associated with high-risk presenting features, hyperleukocytosis and younger age, and a poor treatment outcome. To assess the clinical and biologic significance of 11q23- associated structural chromosomal abnormalities other than translocations, we studied 17 cases of childhood ALL [14 with del(11)(q23) and 3 with inv(11)(p12q23)] that were identified among 785 cases with successful chromosome analysis. In contrast to reported cases with 11q23 and MLL gene rearrangement, our series was characterized by relatively low leukocyte counts (median, 15.1 x 10(9)/L), expression of CD10 antigen but not myeloid-associated CD15 and CDw65 antigens, a relatively high frequency of T-cell immunophenotypes, and a generally favorable prognosis. All 13 cases with interpretable molecular analysis lacked MLL gene rearrangements. We suggest that most cases with deletions or inversions affecting the 11q23 region represent clinically and biologically different entities as compared with those defined by 11q23 translocation.
Stem cell factor contributes to intestinal mucosal mast cell hyperplasia in rats infected with Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor treatment decreases parasite egg production during N brasiliensis infection
Authors: Newlands GF , Miller HR , MacKellar A , Galli SJ
Published: October 2019

We assessed the effects of the c-kit ligand, stem cell factor (SCF), in the jejunal mucosal mast cell hyperplasia that occurs during infection with the intestinal nematodes, Nippostrongylus brasiliensis or Trichinella spiralis in rats. Compared with vehicle-treated rats, rats treated with SCF (25 micrograms/kg/d, intravenous [i.v.] for 14 days) during N brasiliensis infection exhibited significantly higher levels of the rat mucosal mast cell (MMC)-associated protease, rat mast cell protease II (RMCP II) in the jejunum and serum on day 8 of infection, but not on days 10 or 15 of infection. By contrast, in comparison to rats treated with normal sheep IgG, rats treated with a polyclonal sheep antirat SCF antibody exhibited markedly decreased numbers of jejunal MMCs, levels of jejunal RMCP II, and serum concentrations of RMCP II during infection with either nematode, particularly at the earlier intervals of infection (< or = day 10). Taken together, these findings indicate that SCF importantly contributes to MMC hyperplasia and/or survival during N brasiliensis or T spiralis infection in rats, but that levels of endogenous SCF are adequate to sustain near maximal MMC hyperplasia during infection with these nematodes. Notably, treatment of rats with SCF somewhat increased, and treatment with anti- SCF significantly decreased, parasite egg production during N brasiliensis infection. This finding raises the interesting possibility that certain activities of intestinal MMCs may contribute to parasite fecundity during infection with this nematode.
The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions is not responsible for Ca(2+)-induced loss of phospholipid asymmetry in the human erythrocyte: a study in Scott syndrome, a disorder of calcium- induced phospholipid scrambling
Authors: Bevers EM , Wiedmer T , Comfurius P , Zhao J , Smeets EF , Schlegel RA , Schroit AJ , Weiss HJ , Williamson P , Zwaal RF
Published: October 2019

Elevation of cytoplasmic Ca2+ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca(2+)- induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP2 content and the metabolic-resistant pool of PIP2 that remains after incubation with Ca2+ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP2 in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP2 gave rise to a Ca(2+)-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP2-loaded cells were also found to undergo progressive Ca(2+)-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP2 did not abolish their impaired lipid scrambling, even in the presence of a Ca(2+)-ionophore. Finally, artificial lipid vesicles containing no PIP2 or 1 mole percent of PIP2 were indistinguishable with respect to transbilayer movement of NBD- phosphatidylcholine in the presence of Ca2+. Our findings suggest that Ca(2+)-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP2, and imply that such lipid movement is regulated by other cellular processes.
Deferiprone (L1) chelates pathologic iron deposits from membranes of intact thalassemic and sickle red blood cells both in vitro and in vivo
Authors: Shalev O , Repka T , Goldfarb A , Grinberg L , Abrahamov A , Olivieri NF , Rachmilewitz EA , Hebbel RP
Published: October 2019

Red blood cell (RBC) membranes from patients with the thalassemic and sickle hemoglobinopathies carry abnormal deposits of iron presumed to mediate a variety of oxidative-induced membrane dysfunctions. We hypothesized that the oral iron chelator deferiprone (L1), which has an enhanced capacity to permeate cell membranes, might be useful in chelating these pathologic iron deposits from intact RBCs. We tested this hypothesis in vitro by incubating L1 with RBCs from 15 patients with thalassemia intermedia and 6 patients with sickle cell anemia. We found that removal of RBC membrane free iron by L1 increased both as a function of time of incubation and L1 concentration. Thus, increasing the time of incubation of thalassemic RBCs with 0.5 mmol/L L1 from 0.5 to 6 hours, enhanced removal of their membrane free iron from 18% +/- 9% to 96% +/- 4%. Dose-response studies showed that incubating thalassemic RBC for 2 hours with L1 concentrations ranging from 0.125 to 0.5 mmol/L resulted in removal of membrane free iron from 28% +/- 15% to 68% +/- 11%. Parallel studies with sickle RBCs showed a similar pattern in time and dose responses. Deferoxamine (DFO), on the other hand, was ineffective in chelating membrane free iron from either thalassemic or sickle RBCs regardless of dose (maximum, 0.333 mmol/L) or time of incubation (maximum, 24 hours). In vivo efficacy of L1 was shown in six thalassemic patients whose RBC membrane free iron decreased by 50% +/- 29% following a 2-week course of L1 at a daily dose of 25 mg/kg. As the dose of L1 was increased to 50 mg/kg/d (n = 5), and then to 75 mg/kg/d (n = 4), 67% +/- 14% and 79% +/- 11%, respectively, of their RBC membrane free iron was removed. L1 therapy-- both in vitro and in vivo--also significantly attenuated the malondialdehyde response of thalassemic RBC membranes to in vitro stimulation with peroxide. Remarkably, the heme content of RBC membranes from L1-treated thalassemic patients decreased by 28% +/- 10% during the 3-month study period. These results indicate that L1 can remove pathologic deposits of chelatable iron from thalassemic and sickle RBC membranes, a therapeutic potential not shared by DFO. Furthermore, membrane defects possibly mediated by catalytic iron, such as lipid peroxidation and hemichrome formation, may also be alleviated, at least in part, by L1.
The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene
Authors: Chaplin T , Bernard O , Beverloo HB , Saha V , Hagemeijer A , Berger R , Young BD
Published: October 2019

The gene on chromosome 10 at band p12 (AF10), involved in the t(10;11) translocation in acute myeloid leukemia, has been identified and shown to contain conserved zinc finger and leucine zipper domains. These regions are highly homologous to the equivalent regions on AF17, the gene involved in the t(11;17) translocations. A series of adult, childhood, and infant leukemias with either simple or complex versions of the t(10;11) has been examined by Southern analysis and shown to involve rearrangement to the HRX locus. Reverse transcriptase- polymerase chain reaction from either bone marrow or peripheral blood cells showed that HRX sequence was fused to AF10 sequence in all 8 cases and subsequent sequence analysis showed an in-frame fusion between the HRX and AF10 sequence. A consistent feature of these fusions was the juxtaposition of the leucine dimerization motif of AF10 onto the NH2-terminal region of HRX. The published data suggest that a similar conclusion can be drawn about the t(11;17) translocation, implying a critical role for this motif in the chimaeric HRX protein.
JAK3 protein tyrosine kinase mediates interleukin-7-induced activation of phosphatidylinositol-3' kinase
Authors: Sharfe N , Dadi HK , Roifman CM
Published: October 2019

The interleukin-7 (IL-7) receptor is expressed throughout T-cell differentiation and, although lacking a tyrosine kinase domain, mediates tyrosine phosphorylation in T cells. We have identified IL-7- induced activation of three cyoplasmic tyrosine kinases in T cells, Jak1, Jak3, and the src-like kinase p56lck. Many members of the cytokine receptor superfamily activate the Jak protein tyrosine kinase family, with resultant phosphorylation of the Stat transcriptional activator factors. We describe here a novel function of the Jak kinases, because Jak kinase activity is not only required for Stat activation but also for P13 kinase response to IL-7 in human T cells. We show that IL-7 receptor-mediated Jak activation can occur independently of p56lck activity. IL-7-induced P13 kinase activation, mediated by tyrosine phosphorylation of the P13 kinase p85 subunit, is essential to the IL-7 proliferative signal and also occurs in the absence of src family kinase activity. Jak3 is found associated with the p85 subunit of P13 kinase in an IL-7-responsive manner in T cells and appears to regulate IL-7-induced P13 kinase activation by mediating tyrosine phosphorylation of the p85 subunit. Specific inhibition of IL- 7-induced Jak kinase activity ablates p85 tyrosine phosphorylation, subsequent P13 kinase activation, and, ultimately, proliferation. The ability to regulate P13 kinase activity indicates a more generalized role for the Jak family than activation of gene transcription via the Stat family in cytokine receptor signal transduction.
The value of high-dose systemic chemotherapy and intrathecal therapy for central nervous system prophylaxis in different risk groups of adult acute lymphoblastic leukemia
Authors: Cortes J , O'Brien SM , Pierce S , Keating MJ , Freireich EJ , Kantarjian HM
Published: October 2019

Although central nervous system (CNS) leukemic relapse is frequent in adult acute lymphocytic leukemia (ALL), the need for prophylaxis in different risk groups for CNS relapse, the value of high-dose systemic and intrathecal (IT) chemotherapy, and the timing of prophylaxis are not well defined. This analysis was conducted to investigate these questions and to assess the value of a risk-oriented CNS prophylaxis approach. We analyzed the incidence of CNS leukemia after initiation of therapy in patients treated on 4 consecutive trials for adult ALL including different CNS prophylactic modalities. The treatment groups included (1) the program preceeding the vincristine-Adriamycin- dexamethasone (VAD) regimen, with no CNS prophylaxis; (2) the VAD regimen with prophylaxis using high-dose systemic chemotherapy; (3) the modified VAD program with high-dose systemic chemotherapy to all patients and IT chemotherapy for high-risk patients after achieving complete remission; and (4) the hyperCVAD program with early high-dose systemic and IT chemotherapy starting during induction to all patients, with more IT injections (16IT) administered to the high-risk group for CNS relapse compared with the low-risk group (4IT). A total of 391 patients were included, 73 of whom were treated with preVAD, 112 with VAD, 114 with modified VAD, and 92 with hyperCVAD. The overall CNS relapse rates were 31%, 18%, 17%, and 3%, respectively for the 4 groups (P < .001). For the high-risk group for CNS relapse, they were 42%, 26%, 20%, and 2%, respectively (P < .001). The differences in CNS relapse rates in the low-risk group were not statistically significant. At 3 years, the overall CNS leukemia event-free rates were 48%, 76%, and 98%, respectively (P < .001). In the high-risk group, the CNS event- free rates were 38%, 66%, 75%, and 98%, respectively (P < .001); however, there was no difference in the low-risk group. We conclude that (1) high-dose systemic chemotherapy is a useful prophylactic measure; (2) early IT chemotherapy is necessary to reduce the incidence of CNS leukemia overall and in the high-risk group; and (3) a risk- oriented approach is appropriate to tailor the intensity of CNS prophylaxis.
In vivo biologic effects of PIXY321, a synthetic hybrid protein of recombinant human granulocyte-macrophage colony-stimulating factor and interleukin-3 in cancer patients with normal hematopoiesis: a phase I study
Authors: Vadhan-Raj S , Broxmeyer HE , Andreeff M , Bandres JC , Buescher ES , Benjamin RS , Papadopoulos NE , Burgess A , Patel S , Plager C
Published: October 2019

PIXY321 is a novel fusion protein of recombinant human granulocyte- macrophage colony-stimulating factor and interleukin-3 that exhibits biologic effects of both its parent cytokines in vitro and in preclinical studies. To evaluate the clinical safety and hematopoietic effects of this hybrid cytokine, PIXY321 was administered by subcutaneous injection twice daily at doses of 25 to 1,000 micrograms/m2/day over 14 days to 24 patients with sarcoma before chemotherapy as part of a phase I trial. The treatment was associated with significant increases in white blood cell, neutrophil, platelet, and reticulocyte counts (all P < .001). The increase in neutrophil count was dose-related and was seen during treatment with the cytokine, whereas the increase in platelet count was gradual and peaked after the cessation of the cytokine treatment and was not clearly dose related. PIXY321 treatment also increased bone marrow (BM) cellularity and the percentage of BM cells in S phase (P < .001). In addition, there was a significant increase in the number of CD34+ cells and committed and multipotential progenitors in the peripheral blood. The ex vivo expansion capacity of peripheral blood and BM progenitor cells was preserved after the in vivo treatment with PIXY321. The treatment was well tolerated, with the most common side-effect being injection site reactions. The results of this study show the biologic and clinical activity of a genetically engineered fusion molecule of two hematopoietic cytokines in humans with normal hematopoietic function.
Bone marrow and splenic granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta mRNA levels in irradiated mice
Authors: Chang CM , Limanni A , Baker WH , Dobson ME , Kalinich JF , Jackson W , Patchen ML
Published: October 2019

The effects of a myeloablative sublethal 775 cGy 60C gamma radiation exposure on endogenous bone marrow (BM) and splenic granulocyte- macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta (TGF-beta) mRNA levels were assayed in B6D2F1 female mice. BM and spleen were harvested from normal mice and irradiated mice on days 2, 4, 7, 10, and 14 after exposure. Cytokine mRNA levels were determined using reverse transcription-polymerase chain reaction. After irradiation, GM-CSF mRNA levels were significantly increased in the BM from days 2 to 10 and in the spleen from days 4 to 10. However, when BM and splenic GM-CSF protein levels were measured using Western dot blot, no increased protein levels were detected. Serum GM-CSF levels were likewise unchanged. Radiation exposure did not affect BM or splenic TGF- beta mRNA levels and this cytokine is known to be produced by cell populations similar to those that produce GM-CSF. These data suggest that radiation injury to hemopoietic tissues results in differential effects on GM-CSF and TGF-beta mRNA levels and that, in the case of GM- CSF, increased mRNA levels are not matched by increased protein production.
50-kD integrin-associated protein does not detectably influence several functions of glycoprotein IIb-IIIa complex in human platelets
Authors: Fujimoto T , Fujimura K , Noda M , Takafuta T , Shimomura T , Kuramoto A
Published: October 2019

A 50-kD integrin-associated protein (IAP) has been reported to be associated with beta 3 integrins and to modulate their function, especially vitronectin receptor in human erythroleukemia (HEL) cells and leukocyte response integrin in neutrophils. We studied the involvement of IAP in the function of platelet beta 3 integrin, glycoprotein (GP) IIb-IIIa complex. IAP was a widely distributed protein and was also expressed in the cells that do not have beta 3 integrin. Platelets from a patient with thrombasthenia, which lack GPIIb and IIIa, expressed IAP as well as normal platelets. Neither platelet aggregation nor intracellular Ca2+ elevation after stimulation was influenced by the anti-IAP antibody, B6H12, which was reported to be inhibitory for other beta 3 integrins. The expression level of GPIIb- IIIa complex was not influenced by coexpression of human IAP in the transfected Chinese hamster ovary (CHO) cells. IAP did not facilitate the binding of soluble fibrinogen to the CHO cells expressing GPIIb- IIIa complex. Furthermore, cell adhesion onto the immobilized fibrinogen via GPIIb-IIIa complex was not inhibited by B6H12 in HEL cells and was not altered by coexpression of human IAP in CHO cells. We concluded that expression of IAP is regulated independently with that of GPIIb-IIIa complex and that IAP does not influence the function of GPIIb-IIIa complex.
Calcium-dependent homotypic adhesion through leukocyte function- associated antigen-1/intracellular adhesion molecule-1 induces interleukin-1 and parathyroid hormone-related protein production on adult T-cell leukemia cells in vitro
Authors: Wake A , Tanaka Y , Nakatsuka K , Misago M , Oda S , Morimoto I , Eto S
Published: October 2019

Adult T-cell leukemia (ATL) is a human T-cell leukemia virus type I (HTLV-I)-infected lymphoproliferative disorder that shows a characteristic nodular infiltration into various tissues, hypercalcemia, and subsequent rapid increase of peripheral ATL cell number. ATL cells and HTLV-I-infected T-cell lines also make cluster formation rapidly after the non-stimulative culture. However, the mechanism of the acute proliferation of ATL cells remains to be understood. We report the following novel features of homotypic adhesion via leukocyte function-associated antigen-1 (LFA- 1)/intracellular adhesion molecule-1 (ICAM-1) pathway that suggest a role for it in cytokine production and rapid proliferation of ATL cells: (1) ATL cells show clustering in a calcium dependent manner, even at the higher concentration; (2) ATL cells consistently and highly express ICAM-1 and an active form of LFA-1, whereas integrin expression, except for LFA-1, is rather lower compared with that of normal CD4+ T cells; (3) ATL cells make conjugate formation within 6 minutes and clustering within 48 hours, both of which are inhibited by the addition of monoclonal antibodies (MoAbs) against LFA-1 and ICAM-1; (4) spontaneous mRNA transcription and protein secretion of both interleukin-1 and parathyroid hormone-related protein are observed consistently in ATL cells, and these productions are inhibited by anti- LFA-1 and anti-ICAM-1 MoAbs but are markedly increased by cross-linking of LFA-1 and ICAM-1 by the immobilized specific MoAbs; and (5) proliferative responses of ATL cells are also inhibited by these MoAbs. We propose that ATL cells proliferate in sequential events: the homotypic and calcium-dependent adhesion through LFA-1/ICAM-1, the signal transduction through these adhesion molecules, the production of cytokines, and the proliferation.
Hypermetaphase fluorescence in situ hybridization for quantitative monitoring of Philadelphia chromosome-positive cells in patients with chronic myelogenous leukemia during treatment
Authors: Seong DC , Kantarjian HM , Ro JY , Talpaz M , Xu J , Robinson JR , Deisseroth AB , Champlin RE , Siciliano MJ
Published: October 2019

Using Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) as a model, our aim has been to develop a molecular cytogenetic method of high resolution analysis for monitoring the frequency of cells with nonrandom chromosome rearrangements in the bone marrow of patients receiving treatment for hematologic malignancies. Long-term exposure (24 hours) of bone marrow cultures to colcemid (0.1 microgram/mL) maximized a high frequency of metaphase collection. Such preparations were subjected to fluorescence in situ hybridization (FISH) using a 5 Mb probe that overlapped the region of the translocation at chromosome 9q34. This detected the Ph translocation in the resultant large number of overly contracted chromosome spreads. The procedure was validated and verified by studying 70 double-blind marrow samples from patients in different stages of Ph+ CML and from patients with Ph- hematologic malignancies (controls). This hypermetaphase FISH (HMF) method clearly identified Ph+ metaphases and allowed the analysis of 500 hypermetaphases per sample in less than 1 hour after FISH. HMF (1) identified statistically significant differences between the frequencies of Ph+ cells in samples that differed by less than 4%; (2) resolved such differences among patient samples that were all judged 100% Ph+ by standard G-band cytogenetics (CG); (3) resulted in the reclassification of response status in 23% of the patients initially classified by CG; (4) recognized Ph+ cells in 16% of patients characterized as having a complete cytogenetic response and in one patient with an original diagnosis of Ph- CML; and in one patient with an original diagnosis of Ph- CML; and (5) was informative where insufficient metaphases were obtainable for analysis by CG. HMF appears to be uniquely suitable for monitoring the status of patients with CML receiving treatment. It should also be applicable for patients with any hematologic diseases where chromosomal alterations are known and appropriate FISH probes are available.
Del (7)(q32) is associated with a subset of small lymphocytic lymphoma with plasmacytoid features
Authors: Offit K , Louie DC , Parsa NZ , Noy A , Chaganti RS
Published: October 2019

Deletions of the long arm of chromosome 7, previously documented in myelodysplasias and myeloid leukemias, have also been noted in lymphoid malignancies. Of 558 karyotypically abnormal specimens of non-Hodgkin's lymphoma (NHL) serially ascertained over an 8-year period, del(7q) was identified in 24 cases, 10 of which were of the small lymphocytic (sm lym) subtype. Del(7q) was the third most common karyotypic abnormality among the cohort of 61 sm lym cases in this ascertainment. Mapping of the deletions identified a region of common deletion affecting 7q32, which was the sole karyotypic abnormality in 2 cases. Eight of the ten sm lym cases were characterized by plasmacytoid features in histologic sections of lymphoma tumors or circulating cells in the peripheral blood. The del(7)(q32) was accompanied by 14q32-associated translocations in 11 of the 14 cases with histologies other than sm lym, compared with 2 of the sm lym cases. Extranodal involvement was more frequent in the del(7)(q32) sm lym NHLs, although median survival was typical of other low-grade lymphomas. These results suggest that loss or inactivation of a putative tumor-supressor gene at 7q32 may play a role the progression of lymphomas as well as constitute an early event in the pathogenesis of lymphoplasmacytoid tumors.
Increase of bcr-abl chimeric mRNA expression in tumor cells of patients with chronic myeloid leukemia precedes disease progression
Authors: Gaiger A , Henn T , Horth E , Geissler K , Mitterbauer G , Maier-Dobersberger T , Greinix H , Mannhalter C , Haas OA , Lechner K
Published: October 2019

The translocation t(9;22) in chronic myeloid leukemia (CML) generates a bcr-abl fusion gene that codes for an aberrant chimeric mRNA. Cell lines established from CML patients in blast crisis show higher expression of this aberrant bcr-abl transcript than cells from patients in chronic phase of the disease. This observation provided the stimulus to investigate whether increased expression of the aberrant bcr-abl fusion transcript is critical to the progression of CML from chronic phase to blast crisis. We have monitored the bcr-abl mRNA expression in 25 patients by serial quantitative polymerase chain reaction analyses during a follow-up period of 12 to 156 months after diagnosis, with a median observation time of 28 months. In all patients who have shown disease progression to accelerated phase (n = 4) or blast crisis (n = 7), an increase in bcr-abl mRNA expression was detected up to 16 months before laboratory or clinical parameters showed phenotypic transformation of the malignant clone. To investigate whether the elevated levels of bcr-abl mRNA reflected an increase in the proportion of leukemic cells in the samples analyzed or primarily enhanced transcriptional activity of the bcr-abl fusion gene, we performed quantitative analyses of the bcr-abl gene at the DNA level and of the Ph chromosome at the cytogenetic level and compared these data with steady-state bcr-abl mRNA levels. We show that increased levels of the bcr-abl transcript did not reflect increased proportions of leukemic cells but elevated steady-state levels of the chimeric mRNA in the malignant cells before disease progression. Therefore, our data strongly suggest that an increase of the chimeric mRNA expression in the leukemic cells precedes the phenotypic transformation of the malignant clone.