bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a monoclonal IgMK (IgMGAS) anti-Gd cold agglutinin (CA). Its coexistence with a monoclonal IgG3K (IgGGAS) without CA activity that might be clonally related to IgMGAS
Authors: De la Fuente MA , Egile C , Pereira A , Juan M , Vivanco F , Roelcke D , Dighiero G , Gallart T
Published: October 2019

A monoclonal IgMK (IgMGAS) cold agglutinin (CA) of the infrequent anti- Gd specificity, found in a patient with Waldenstrom macroglobulinemia, has been characterized. IgMGAS uses a VH gene homologous (93.7%) to the reported VH251 germ-line, one of the two functional genes of the VH5 family, with differences in both framework regions and complementary determining regions (CDR). The VL gene is homologous to the reported 15AVK1 germ-line gene, recently described in an anti-i CA, with differences mostly clustered in CDR. In the patient's serum, IgMGAS coexisted with a monoclonal IgG3K (IgGGAS) that lacked CA activity but expressed the private idiotopes found on IgMGAS. Both Ig lacked reactivity with antibodies detecting VH1 or VHIII or VKIII subgroup regions or the VH4–21 gene product that is expressed by anti-I/i CAs. The K chains from both Igs showed the same isoelectrical mobility. Moreover, the k chains from both serum Igs showed the same N-terminal amino acid sequence. This sequence was identical to that predicted by the nucleotide sequence of VK1GAS gene segment, including one discrepancy at position 15 (Ile for Val) with respect to the consensus VK1 subgroup regions. Although these data do not exclude a possible independent clonal origin, they are consistent with the notion that IgGGAS might be clonally related to IgMGAS.
Identification of Philadelphia-negative granulocyte-macrophage colony- forming units generated by stroma-adherent cells from chronic myelogenous leukemia patients
Authors: Carlo-Stella C , Mangoni L , Piovani G , Garau D , Almici C , Rizzoli V
Published: October 2019

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean (+/- SEM) percentage of Ph- metaphases was 3% +/- 1%. Mononuclear marrow cells from CML patients (n = 18) were incubated with mafosfamide (100 micrograms/mL) or control medium, seeded onto marrow stromal layers and allowed to adhere (2 hours, 37 degrees C). After a short-term (3-day) liquid culture, the cells were harvested, incorporated in methyl-cellulose, and individual colonies were analyzed by single colony karyotyping. The mean (+/- SEM) percentage of Ph- colonies generated from the stroma- adherent fraction was 35% +/- 6%. As compared with marrow colony- forming unit granulocyte-macrophage plated before any manipulation, the mean (+/- SEM) percentage of Ph- clones was significantly increased by stroma adherence (35% +/- 6% v 15% +/- 4%, P < or = .005) and mafosfamide (100 micrograms/mL) incubation of marrow cells before stroma adherence (58% +/- 9% v 35% +/- 6%, P < or = .005). An additive effect was observed by combining mafosfamide treatment and stroma adherence. Single-colony transfer experiments showed that 50% +/- 4% stroma-adherent and 70% +/- 4% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. To further characterize the stroma-adherent fraction, experiments were performed in which CD34+ marrow cells were used. The mean (+/- SEM) output of progenitors generated by 10,000 CD34+, stroma-adherent cells was 888 +/- 188 and 570 +/- 258 for untreated and mafosfamide-treated cells, respectively. Individual colonies were analyzed by single-colony karyotyping and fluorescent in situ hybridization using a biotinylated cosmid DNA probe that hybridize to abl oncogene. The CD34+, stroma-adherent fraction contained 38% +/- 14% (untreated) and 56% +/- 18% (mafosfamide-treated) (P < or = .025) Ph- progenitors. In conclusion, the present data show the possibility to select Ph- clones that (1) have a maintained capability of stroma adherence, (2) are mafosfamide resistant, (3) are derived from the CD34+ fraction, and (4) have high-replating potential.
Interleukin-4 inhibits indoleamine 2,3-dioxygenase expression in human monocytes
Authors: Musso T , Gusella GL , Brooks A , Longo DL , Varesio L
Published: October 2019

Indoleamine 2,3-dioxygenase (IDO), a flavin-dependent enzyme that catalyzes the conversion of tryptophan to kynurenine, is induced in peripheral blood mononuclear cells by interferon-gamma (IFN gamma). Interleukin-4 (IL-4) is a cytokine that modulates the functional properties of monocytes/macrophages, and we investigated the effects of IL-4 on IDO. We showed that IL-4 inhibited the induction of IDO mRNA and IDO activity by IFN gamma in human monocytes. The inhibitory effect was evident with as little as 2 U/mL of IL-4. These results provide the first evidence that a cytokine can provide a negative signal for IDO expression and that IL-4 can influence the catabolism of tryptophan.
Further characterization of cis-acting regulatory sequences in the genomic locus of the murine erythropoietin receptor: evidence for stage- specific regulation
Authors: Youssoufian H
Published: October 2019

Expression of the murine erythropoietin receptor (EpoR) gene was investigated in progenitor cell lines representing distinct stages of hematopoietic differentiation. In murine erythroid cell lines, the EpoR mRNA level was fivefold higher in the more mature murine erythroleukemia (MEL) cells than in CB-5 cells and very low in granulocyte/macrophage-like FDC-P1 cells. GATA-1 mRNA was present in equivalent levels in both erythroid cell lines, but at a low level in FDC-P1 cells. To account for the elevated levels of EpoR mRNA, the activity of the promoter and expression of DNase I hypersensitive sites were assessed as markers of transcriptional activity in various cell lines. Among a series of 5′ flanking restriction fragments linked to a reporter gene, a 83-bp fragment that includes binding sites for the transcription factors GATA-1 and Sp-1 gave low levels of erythroid- specific activity, and a 256-bp fragment that includes, in addition, two sites for the putative CACCC-binding protein gave the highest level of erythroid-specific transcription. DNase I footprinting showed binding of a constitutive factor to the proximal CACCC-binding site, and deletion or mutation of this site significantly reduced the overall expression while maintaining tissue-specificity. Three DNase I hypersensitive sites were detected in the 5′ flanking region of the EpoR gene, two of which were unique to MEL cells. These sites were situated over the promoter region and approximately 0.5 kb and 2.4 kb upstream of the transcriptional initiation sites. A 0.8-kb restriction fragment spanning the distal site caused approximately a four-fold rise in transcription from the endogenous or a heterologous promoter in MEL cells independent of its orientation and up to 1.5-fold rise in CB-5 cells, but it was inactive in COS-1 cells that were cotransfected with an expression plasmid encoding GATA-1. These results show that (1) basal activity as well as tissue specificity of the EpoR promoter can be accounted for by its interaction with GATA-1, and (2) upstream sites regulate the strength of the promoter. Expression of the distal DNase I hypersensitive site and the corresponding enhancer activity in MEL cells suggests a role for this element in stage-specific transcriptional control.
Retrovirus-mediated gene transduction into canine peripheral blood repopulating cells
Authors: Kiem HP , Darovsky B , von Kalle C , Goehle S , Stewart D , Graham T , Hackman R , Appelbaum FR , Deeg HJ , Miller AD
Published: October 2019

Genetically marked peripheral blood progenitor cells were used to investigate their contribution to long-term hematopoietic reconstitution after autologous marrow and peripheral blood cell transplantation. After autologous marrow harvest and cryopreservation, canine peripheral blood progenitor cells were mobilized in three dogs by treatment with recombinant canine stem cell factor for 8 days. Peripheral blood mononuclear cells were collected and enriched for major histocompatibility complex (MHC) class II antigen-positive cells by avidin-biotin immunoadsorption, thereby enriching for repopulating cells. Subsequently, the cells were cocultivated for 24 hours on irradiated vector-producing packaging cells (PA317/LN), followed by an 11-day incubation in a vector containing long-term marrow culture system. On the day of transplantation, the animals were irradiated with 9.2 Gy total body irradiation (TBI), and transduced peripheral blood cells and untransduced cryopreserved marrow cells were infused within 2 hours of TBI. All three dogs engrafted. Two dogs are long-term survivors showing intermittently G418-resistant marrow-derived colony- forming unit granulocyte-macrophage colonies at a median of 1% and 2%, respectively (range, 1% to 10%), for now up to 48 weeks after transplantation. Neo-specific sequences were detected by polymerase chain reaction in peripheral blood granulocytes for now up to 65 weeks and in peripheral blood lymphocytes for up to 75 weeks after transplantation. Peripheral blood samples of the dogs were free of helper virus and no side effects from the transduction were observed. One of the three dogs died from chronic canine distemper sclerosing encephalitis on day 84, whereas the other two dogs are alive at 15 and 17 months. Our data show successful retroviral transduction of canine peripheral blood repopulating cells. Long-term persistence of marked myeloid and lymphoid cells after transplantation suggests that peripheral blood contains repopulating cells that contribute to long- term hematopoietic reconstitution after otherwise lethal TBI.
Expression and functionality of the trkA proto-oncogene product/NGF receptor in undifferentiated hematopoietic cells
Authors: Chevalier S , Praloran V , Smith C , MacGrogan D , Ip NY , Yancopoulos GD , Brachet P , Pouplard A , Gascan H
Published: October 2019

The expression of the low-affinity NGF receptor (p75) and the trkA proto-oncogene product was analyzed in a series of human hematopoietic cell lines at protein and RNA levels. We did not detect any form of NGF receptor in cell lines displaying a myelomonocytic phenotype (HL60 and U937). In contrast, cells displaying a more immature erythroleukemic phenotype (TF1 and K562) expressed TrkA in the absence of detectable p75. Scatchard analysis showed a single high-affinity site for NGF (kd = 10(-10) mol/L), with a copy number ranging from 300 to 3,000 sites per cell depending on the studied cell line. In addition, NGF induced autophosphorylation of TrkA and could substitute for granulocyte- monocyte colony-stimulating factor to trigger the proliferation of the TF1 cell line, with a half-maximal signal observed at 50 pmol/L, indicating that p75 is not required for DNA synthesis in this cell line. The physiologic relevance of NGF in early hematopoiesis was confirmed by showing that 12% to 15% of progenitor blood cells from mice treated with 5-fluorouracil expressed TrkA and that these cells could be induced to proliferate and differentiate in response to NGF in association with macrophage colony-stimulating factor. Our study demonstrates for the first time that trkA proto-oncogene expression and activation is not restricted to the nervous system, but is also an important element in early hematopoiesis.
Plasma P-selectin is increased in thrombotic consumptive platelet disorders
Authors: Chong BH , Murray B , Berndt MC , Dunlop LC , Brighton T , Chesterman CN
Published: October 2019

P-selectin is a 140-kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was approximately 3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody. We have measured plasma P- selectin and beta-thromboglobulin (beta TG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) patients with idiopathic thrombocytopenic purpura (ITP); and (3) healthy controls. Patients with DIC, HIT, and TTP/HUS, but not ITP, had significantly elevated plasma P-selectin and beta TG levels when compared with their age-matched healthy controls. The increased plasma P-selectin and beta TG in patients with thrombotic disorders were likely to be the result of in vivo platelet and endothelial cell damage or activation. We also found that avoidance of veno-occlusion and other tedious measures customarily taken during blood collection and sample preparation to prevent in vitro platelet activation did not affect plasma P-selectin assay results. In addition, plasma P-selectin levels were not influenced by the presence of renal failure or heparin administration. These results indicate that plasma P- selectin may be a useful new marker for thrombotic diseases.
Leu 697-->Val mutation in mature von Willebrand factor is responsible for type IIB von Willebrand disease
Authors: Hilbert L , Gaucher C , de Romeuf C , Horellou MH , Vink T , Mazurier C
Published: October 2019

Type IIB von Willebrand disease is characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers from plasma and enhanced platelet agglutination of platelet-rich-plasma in the presence of low concentrations of ristocetin. We identified, in two related patients, a C-->G transversion resulting in the substitution of Valine for Leucine at position 697 of the mature subunit of vWF. We reproduced this mutation in vWF cDNA and expressed the recombinant protein in Cos-7 cells. The subunit composition and multimeric structure of mutated protein (rvWFLeu697Val) were similar to the wild- type recombinant (WTrvWF). Ristocetin-induced binding of rvWFLeu697Val to platelets was markedly increased in the presence of low doses of ristocetin and slightly increased with botrocetin as compared with that for WTrvWF, whereas collagen binding was not affected by the mutation. These data show that the Leu 697-->Val substitution is not a rare polymorphism but is responsible for the subtype IIB characteristic abnormalities identified in the two affected patients; however, it is not located in the area of vWF (amino acid 540 to amino acid 578) where most of the other type IIB mutations have already been reported.
Murine recombinant interleukin-3 induces recovery of T and B cells in irradiated mice
Authors: Frasca D , Leter G , Doria G
Published: October 2019

Injection of murine recombinant interleukin-3 (IL-3) into (C57BL/10 x DBA/2)F1 mice, sublethally irradiated with 300 cGy and killed 14 days later, induced in the thymus recovery of the cell number and mitotic responsiveness to concanavalin A (Con A), as well as an increase in number of double-negative CD4-CD8-, double-positive CD4+ CD8+, and single-positive CD4+CD8- and CD4-CD8+ cells. Also in the spleen, the cell count and mitotic responsiveness to Con A and lipopolysaccharide were increased to normal levels by IL-3 treatment. If the assays were performed 21 or 28 days after irradiation, IL-3 treatment was able to restore thymus and spleen cell counts as well as T- and B-cell mitotic responsiveness, even when mice were exposed to 400 or 500 cGy, respectively. These results altogether indicate that IL-3 induces differentiation and growth of thymocytes and recovery of T- and B-cell functions in mice exposed to sublethal irradiation.
Differential attachment of human neoplastic B cells to purified extracellular matrix molecules
Authors: Segat D , Pucillo C , Marotta G , Perris R , Colombatti A
Published: October 2019

Recirculation of normal and neoplastic lymphocytes occurs via binding to the endothelial luminar surface, followed by extravasation and the subsequent interaction of the cells with components of the underlying basement membrane and stromal extracellular matrix (ECM). To identify matrix constituents that could be involved in the tissue dissemination of neoplastic B cells, we have examined the ability of three lymphoma B- cell lines and one Philadelphia chromosome (Ph1)-positive cell line established from the lymphoid transformation of a chronic myeloid leukemia (CML) to adhere to a range of purified ECM molecules. Immunophenotyping with a panel of markers suggested that the lines derived from cells that had undergone transformation at distinct stages of B-cell maturation. The four cell lines displayed a differential ability to adhere to the ECM molecules tested. BV-173, Ci-1, and Sc-1 cells attached to various degrees to fibronectin (FN). Ri-1, Ci-1, and Sc-1 cells attached to human laminin (LN) variants, whereas only Ci-1 and Sc-1 cells showed some affinity for collagen (Col) type VI. All four cell lines interacted with fibrillar Col I, but only BV-173 and Ri- 1 cells attached to fibrillar Col III. The subendothelial Col VIII only was active as a substratum for BV-173 cells. In all cases, cells bound to fibrillar collagens when they were assembled into polymeric fibrils, and were incapable of adhering to monomeric and denatured collagen. In contrast to cell adhesion to FN and LN, which showed a plateau at high substrate concentrations, adhesion to fibrillar Col I reached a peak at intermediary concentrations and decreased thereafter, suggesting that cells respond to a definite macromolecular arrangement of collagenous fibrils. Adhesion to individual ECM molecules was not directly correlated with the apparent maturation state of the cells, nor with the relative density of known ECM receptors. Taken together, these results suggest that interaction of neoplastic B cells with selected matrix components may influence their dispersion throughout tissues. We further suggest that the use of quantitative cell adhesion assays in vitro may provide means of defining the behavioral traits of neoplastic B cells in vivo.
Prognostic importance of mutations in the ras proto-oncogenes in de novo acute myeloid leukemia
Authors: Neubauer A , Dodge RK , George SL , Davey FR , Silver RT , Schiffer CA , Mayer RJ , Ball ED , Wurster-Hill D , Bloomfield CD
Published: October 2019

Mutations of the N- and K-ras genes are the most frequent genetic aberrations in acute myeloid leukemia (AML) and their detection in preleukemic conditions such as the myelodysplastic syndrome (MDS) suggests a role in the earliest phases of leukemogenesis. Despite these observations, little is known about the clinical importance of ras mutations in AML. We studied the clinical impact of ras mutations in 99 patients with de novo AML. All patients were treated in two prospective multicenter trials. The polymerase chain reaction was used to amplify areas surrounding the codons 12, 13, and 61 of the three ras genes N-, K-, and H-ras from DNA from bone marrow cells, ras mutations were detected by an algorithm based on allele-specific oligonucleotide hybridization. Eighteen of 99 (18%) patients harbored mutations in either N- or K-ras. All of the observed mutations occurred in N-ras (N = 10) and K-ras (N = 5) or concurrently in both N- and K-ras (N = 3). There were no significant differences between ras-negative and ras- positive patients according to age, sex, blood counts, cytogenetic abnormalities, or French-American-British classification. However, univariate analysis suggested a longer survival in ras-positive patients (P = .11). When adjusted for age, which was the most important factor affecting outcome, the presence of a ras mutation emerged as a significant predictor for improved survival (P = .03) and along with lower bone marrow blast counts (P = .02) and better cytogenetic category (P = .01). However, the presence of an aberrant ras allele was strongly correlated with lower bone marrow blast counts (P = .007). Thus, whether a mutation in the N-ras or K-ras proto-oncogenes directly affects treatment outcome or indirectly through an association with lower leukemic burden remains to be determined. Nevertheless, these findings counter the prevailing bias that oncogene mutations lead to more aggressive behavior in human malignancies.
Bone marrow cells from vitamin B12- and folate-deficient patients misincorporate uracil into DNA
Authors: Wickramasinghe SN , Fida S
Published: October 2019

Bone marrow cells from 15 patients with normal deoxyuridine (dU) suppression test results, 3 healthy subjects, and 11 patients with megaloblastic anemia caused by vitamin B12 or folate deficiency were examined for misincorporation of uracil into DNA. Cells were incubated with [5–3H] uridine for 2 hours and their DNA extracted. The DNA was hydrolyzed to deoxyribonucleosides with DNase 1, phosphodiesterase and alkaline phosphatase, and any dU present was separated from other deoxyribonucleosides by Aminex A6 chromatography. The quantity of dU/mg DNA and the radioactivity in the dU peak/mg DNA were then calculated. The results clearly showed that there was markedly increased uracil misincorporation into the DNA of vitamin B12- or folate-deficient marrow cells. Misincorporation of uracil into DNA may be an important biochemical lesion underlying both the megaloblastic change and the ineffectiveness of hematopoiesis in vitamin B12 and folate deficiency.
Regulation of beta-globin mRNA accumulation by heme in dimethyl sulfoxide (DMSO)-sensitive and DMSO-resistant murine erythroleukemia cells
Authors: Fukuda Y , Fujita H , Garbaczewski L , Sassa S
Published: October 2019

The level of mRNA encoding beta-globin was examined in dimethyl sulfoxide (DMSO)-sensitive (DS), and DMSO-resistant (DR) murine erythroleukemia (MEL) cells. DR cells lack erythroid-specific delta- aminolevulinate (ALA) synthase (AL-AS-E), and fail to undergo erythroid differentiation following treatment with DMSO. Treatment of cells with DMSO markedly increased ALAS-E mRNA in DS cells, while the same treatment downregulated the nonspecific ALA synthase (ALAS-N) mRNA levels in both DS and DR cells. The levels of beta-globin mRNA, heme content, and hemoglobin in DS cells increased, while those in DR cells decreased following treatment with DMSO. Treatment of DR cells with hemin caused an increase in beta-globin mRNA and hemoglobin, and partially restored the DMSO-mediated suppression of beta-globin mRNA and hemoglobin synthesis. DMSO treatment decreased heme oxygenase (HO) mRNA in hemin-treated DS cells, but not in hemin-treated DR cells. These findings indicate that heme is necessary for accumulation of the beta-globin transcript during erythroid differentiation, and that hemin- mediated HO induction becomes markedly downregulated in differentiated erythroid cells, presumably because less free heme is available for HO induction by a greater demand for the synthesis of hemoglobin.
Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification
Authors: Craig JE , Barnetson RA , Prior J , Raven JL , Thein SL
Published: October 2019

A considerable number of deletions of variable size and position that involve the beta-globin gene complex on chromosome 11 are associated with the clinical entities of hereditary persistence of fetal hemoglobin (HPFH) and delta beta thalassemia. Specific deletions appear to be associated with consistent phenotypes and some are known to be recurrent. To facilitate the molecular diagnosis of uncharacterized patients with HPFH and delta beta thalassemia, oligonucleotide primers have been designed to enzymatically amplify deletion-specific products for nine known deletions, which include those responsible for HPFH-1, HPFH-2, HPFH-3, Spanish (delta beta)zero thalassemia, hemoglobin (Hb) Lepore, Sicilian (delta beta)zero thalassemia, Chinese G gamma(A gamma delta beta)zero thalassemia, Asian-Indian inversion-deletion G gamma(A gamma delta beta)zero thalassemia, and Turkish inversion-deletion (delta beta)zero thalassemia. Using this approach, we have successfully characterized the molecular basis for delta beta thalassemia in 23 individuals from 16 families of diverse ethnic origins. Thirteen individuals from this group were shown to be heterozygous for the 13.4- kb Sicilian deletion, two were heterozygous for the 100-kb Chinese G gamma(A gamma delta beta)zero deletion, four were heterozygous for the Turkish form of inversion-deletion delta beta thalassemia, and three were heterozygous for the Asian-Indian form of inversion-deletion G gamma(A gamma delta beta)zero thalassemia. One Vietnamese subject was heterozygous for a 12.6-kb deletion, which we have fully characterized at the molecular level. Sequence analysis of the breakpoint regions of the Chinese deletion and the Turkish rearrangement indicates that, in each case, the mutation is likely to have arisen from a single origin. This hypothesis is supported by the evident geographical clustering of the various deletions described here.
Immunoglobulin V regions and the B cell
Authors: Stewart AK , Schwartz RS
Published: October 2019

There is now substantial evidence that a small group of V genes predominates in the Ig repertoire of preimmune B cells. This phenomenon of V gene restriction may reflect preferential accessibility of these genes to recombinase, homology-directed V gene rearrangement, promoters and enhances of V gene transcription, or positive and negative selection mediated by the anti-self binding properties of the B cells surface Ig. These mechanisms may operate alone or in combination to influence V gene rearrangement and populations of immature B cells. Although constraints on the pool of rearranged V genes may seem disadvantageous to the immune system, the mechanisms that generate the CDR3s of heavy and light chains ensure extensive diversity in the pre-B- cell population. In mature B cells, somatic mutation of V genes adds further diversity. CDR3 sequences and somatic mutations not only provide potentially useful clonal markers but also help to identify the normal counterparts of malignant B cells.
Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia
Authors: Manabe A , Coustan-Smith E , Kumagai M , Behm FG , Raimondi SC , Pui CH , Campana D
Published: October 2019

We investigated the effects of interleukin-4 (IL-4) on the survival of leukemic and normal B-cell progenitors cultured on bone marrow stroma. IL-4 (at 100 U/mL) was cytotoxic in 16 of 21 cases of B-lineage acute lymphoblastic leukemia, causing reductions in CD19+ cell numbers that ranged from 50% to greater than 99% (median 83.5%) of those in parallel cultures not exposed to the cytokine. All nine cases with the t(9;22)(q34;q11) or the t(4;11)(q21;q23), chromosomal features that are often associated with multidrug resistance and a fatal outcome, were susceptible to IL-4 toxicity. IL-4 cytotoxicity resulted from induction of programmed cell death (apoptosis); there was no evidence of cell killing mediated by T, natural killer, or stromal cells. IL-4 cytotoxicity extended to a proportion of normal B-cell progenitors. After 7 days of culture with IL-4 at 100 U/mL, fewer CD19+, CD34+ normal lymphoblasts (the most immature subset) survived: in five experiments the mean (+/- SEM) reduction in cell recoveries caused by IL-4 was 60.0% +/- 6.0%. By contrast, reductions in recovery of more differentiated bone marrow B cells (CD19+, CD34-, surface Ig+) were low (6.6% +/- 2.2%; P < .001 by t-test). Our findings indicate that IL-4 is cytotoxic for human B-cell precursors and support clinical testing of IL-4 in cases of high-risk lymphoblastic leukemia resistant to conventional therapy.
Effect of recombinant canine granulocyte-macrophage colony-stimulating factor on hematopoietic recovery after otherwise lethal total body irradiation
Authors: Nash RA , Schuening FG , Seidel K , Appelbaum FR , Boone T , Deeg HJ , Graham TC , Hackman R , Sullivan-Pepe M , Storb R
Published: October 2019

Recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) was studied in normal dogs and in dogs receiving otherwise lethal total body irradiation (TBI) without marrow transplant. Five normal dogs receiving 25 micrograms/kg of rcGM-CSF by subcutaneous (SC) injection twice daily (BID) for 14 days showed increases in peripheral blood neutrophil counts of three to five times the baseline. Platelet counts decreased during administration of rcGM-CSF to a mean nadir of 52,800. Ten dogs received 400 cGy TBI at 10 cGy/min from two opposing 60Co sources and no marrow graft. Within 2 hours of TBI, rcGM-CSF was begun at a dose of 50 micrograms/kg SC BID for 5 doses and then continued at 25 micrograms/kg SC BID for 21 days. Only 1 of the 10 dogs receiving rcGM-CSF survived with complete and sustained recovery of hematopoiesis. One of 13 historical control dogs survived after 400 cGy with no hematopoietic growth factor or marrow infusion. Results with rcGM-CSF were compared with previous and concurrent data with G-CSF studied in the same model. Of 10 dogs receiving G-CSF, 8 survived with complete and sustained hematopoietic recovery, a significantly better survival than that seen with rcGM-CSF (P = .006). Neutrophil counts were sustained at higher levels after TBI for the first 18 days in the G-CSF group (P < .016) and the neutrophil nadirs were higher. No differences in neutrophil nadirs were noted between the rcGM-CSF and control groups. Dogs treated with rcGM-CSF experienced a more rapid decline of platelet counts than G-CSF-treated or control dogs over the first 18 days (P < .001). The nadir of the platelet count was higher in the control group than in either the G-CSF or rcGM-CSF group and no significant difference was observed between the G-CSF and rcGM-CSF groups. After otherwise lethal TBI (400 cGy) in dogs, rcGM-CSF was not effective in promoting hematopoietic recovery or improving survival.
Interleukin-11: a multifunctional growth factor derived from the hematopoietic microenvironment
Authors: Du XX , Williams DA
Published: October 2019

IL-11 is a unique growth factor derived from cells making up the HM. Although cloned based on IL-6-like bioactivity, IL-11 and IL-6 have distinct biologic profiles (Table 1). IL-11, like many recently cloned growth factors, has pleiotropic effects on hematopoietic cells presumably depending on the cytokine and cellular environment into which it is introduced. However, some general findings are consistent (Table 2). In addition, IL-11 has significant effects, either primary or secondary, on nonhematopoietic cells, including neurons, small intestine crypt progenitor/stem cells, and preadipocytes. The institution of human trials with IL-11 will provide important information on the pharmacologic effects of IL-11 on human hematopoietic cells in the context of frequently used chemotherapy protocols. The physiologic role(s) of IL-11 are unknown but will become clear (at least in the mouse) with gene targeting experiments underway in several laboratories.
Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction
Authors: Thoma SJ , Lamping CP , Ziegler BL
Published: October 2019

A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity > 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.
An endogenous glycosylphosphatidylinositol-specific phospholipase D releases basic fibroblast growth factor-heparan sulfate proteoglycan complexes from human bone marrow cultures
Authors: Brunner G , Metz CN , Nguyen H , Gabrilove J , Patel SR , Davitz MA , Rifkin DB , Wilson EL
Published: October 2019

Basic fibroblast growth factor (bFGF) is a hematopoietic cytokine that stimulates stromal and stem cell growth. It binds to a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan on human bone marrow (BM) stromal cells. The bFGF- proteoglycan complex is biologically active and is released by addition of exogenous phosphatidylinositol-specific phospholipase C. In this study, we show the presence of an endogenous GPI-specific phospholipase D (GPI-PLD) that releases the bFGF-binding heparan sulfate proteoglycan and the variant surface glycoprotein (a model GPI-anchored protein) from BM cultures. An involvement of proteases in this process is unlikely, because released proteoglycan contained the GPI anchor component, ethanol-amine, and protease inhibitors did not diminish the release. The mechanism of release is likely to involve a GPI-PLD and not a GPI-specific phospholipase C, because the release of variant surface glycoprotein did not reveal an epitope called the cross- reacting determinant that is exposed by phospholipase C-catalyzed GPI anchor cleavage. In addition, phosphatidic acid (which is specifically a product of GPI-PLD-catalyzed anchor cleavage) was generated during the spontaneous release of the GPI-anchored variant surface glycoprotein. We also detected GPI-PLD-specific enzyme activity and mRNA in BM cells. Therefore, we conclude that an endogenous GPI-PLD releases bFGF-heparan sulfate proteoglycan complexes from human BM cultures. This mechanism of GPI anchor cleavage could be relevant for mobilizing biologically active bFGF in BM. An endogenous GPI-PLD could also release other GPI-anchored proteins important for hematopoiesis and other physiologic processes.
Expansion of T cells expressing low CD4 or CD8 levels in B-cell chronic lymphocytic leukemia: correlation with disease status and neoplastic phenotype
Authors: Dianzani U , Omede P , Marmont F , DiFranco D , Fusaro A , Bragardo M , Redoglia V , Giaretta F , Mairone L , Boccadoro M
Published: October 2019

Peripheral blood (PB) T cells from 56 patients with B-cell chronic lymphocytic leukemia (B-CLL) were analyzed by two- and three-color immunofluorescence (IF) to determine the expansion of distinct T-cell subsets and their relationship with the clinical and biological features of the disease. We detected the expansion of an unusual T-cell subpopulation expressing lower CD4 or CD8 levels (CD4lo, CD8lo) than classic T cells (CD4hi, CD8hi). This subpopulation also expressed low levels of the CD3/TCR alpha/beta complex and was CD19-CD13-CD14-. A phenotypic analysis probing the activation level of CD4lo, CD8lo, CD4hi, and CD8hi cells showed that they comprised increased counts of HLA-DR+, CD11b+, CD45R0+, and CD45RA+ cells. Subset expansion ranged from 2.1- to 13.6-fold. Statistical analysis showed that the size of some of these subsets was correlated to intrinsic features of the tumor. First, CD4loHLA-DR+ cell counts were higher in patients with stage A than those with stages B and C disease. Second, CD8loHLA-DR+ cell counts were higher in patients in stable remission than in those at diagnosis. Third, CD4loHLA-DR+, CD4loCD45R0+, CD4loCD45RA+, and CD4hiCD11b+ cell counts were higher in patients whose tumor cells expressed high levels of surface immunoglobulin (sIg) than in those expressing low levels. The involvement of CD4lo and CD8lo cells in most of these correlations suggests that they may be tumor-reactive cells. Similar cells described in human and murine autoimmune disease have been shown to be autoreactive anergic cells, which may derive from nonclassic pathways of T-cell development.
Bone marrow stromal cell blockade of human leukemic cell differentiation
Authors: Weber MC , Tykocinski ML
Published: October 2019

Bone marrow (BM) stromal cell inhibition of leukemic cell differentiation was studied in cellular coculture experiments. In coculture, a significant percentage of cells from the human myeloid leukemic cell lines HL-60, PLB-985, and K562 adhere to fibroblastic KM- 102 BM stromal cells. A sensitive two-color immunofluorescence assay was developed to monitor stromal cellular effects upon leukemic cell differentiation. After chemical induction with 1 alpha,25- dihydroxyvitamin D3, strongly adherent HL-60 and PLB-985 cells were inhibited from differentiating into more mature monocytic cells, as measured by the monocytic surface marker CD14. In contrast, loosely adherent and nonadherent HL-60 and PLB-985 leukemic cells in the same cocultures, as well as both adherent and nonadherent K562 cells induced with phorbol ester, were not blocked in their capacity to differentiate. Scanning electron microscopy and intercellular dye transfer experiments correlated intimate stromal cell/leukemic cell interaction and intercellular communication with the blockade of leukemic cell differentiation. These studies indicate that there is significant variability among leukemic lines with respect to the nature of their adhesion to stromal cells. Moreover, the data implicate gap- junction formation as a potentially significant event in stromal cell- mediated leukemic cell regulation.
Analysis of Ig and T-cell receptor genes in 40 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis
Authors: Beishuizen A , Verhoeven MA , van Wering ER , Hahlen K , Hooijkaas H , van Dongen JJ
Published: October 2019

The rearrangement patterns of Ig and T-cell receptor (TcR) genes were studied by Southern blot analysis in 30 precursor B-cell acute lymphoblastic leukemias (B-ALLs) and 10 T-ALLs at diagnosis and subsequent relapse. Eight precursor B-ALLs appeared to contain biclonal/oligoclonal Ig heavy-chain (IgH) gene rearrangements at diagnosis. Differences in rearrangement patterns between diagnosis and relapse were found in 67% (20 cases) of precursor B-ALLs (including all eight biclonal/oligoclonal cases) and 50% (five cases) of T-ALLs. In precursor B-ALLs, especially changes in IgH and/or TcR-delta gene rearrangements were found (17 cases), but also changes in TcR-beta, TcR- gamma, Ig kappa, and/or Ig lambda genes (11 cases) occurred. The changes in T-ALLs concerned the TcR-beta, TcR-gamma, TcR-delta, and/or IgH genes. Two precursor B-ALLs showed completely different Ig and TcR gene rearrangement patterns at relapse, suggesting the absence of a clonal relation between the leukemic cells at diagnosis and relapse and the development of a secondary leukemia. The clonal evolution in the other 23 ALL patients was based on continuing rearrangement processes and selection of subclones. The development of changes in Ig and TcR gene rearrangement patterns was related to remission duration, suggesting an increasing chance of continuing rearrangement processes with time. These immunogenotypic changes at relapse occurred in a hierarchical order, with changes in IgH and TcR-delta genes occurring after only 6 months of remission duration, whereas changes in other Ig and TcR genes were generally detectable after 1 to 2 years of remission duration. The heterogeneity reported here in Ig and/or TcR gene rearrangement patterns at diagnosis and relapse might hamper polymerase chain reaction (PCR)-mediated detection of minimal residual disease (MRD) using junctional regions of rearranged Ig or TcR genes as PCR targets. However, our data also indicate that in 75% to 90% of ALLs, at least one major rearranged IgH, TcR-gamma, or TcR-delta band (allele) remained stable at relapse. We conclude that two or more junctional regions of different genes (IgH, TcR-gamma, and/or TcR-delta) should be monitored during follow-up of ALL patients for MRD detection by use of PCR techniques. Especially in biclonal/oligoclonal precursor B-ALL cases, the monitoring should not be restricted to rearranged IgH genes, but TcR-gamma and/or TcR-delta genes should be monitored as well, because of the extensive changes in IgH gene rearrangement patterns in this ALL subgroup.
Mutations of the N-ras gene in juvenile chronic myelogenous leukemia
Authors: Miyauchi J , Asada M , Sasaki M , Tsunematsu Y , Kojima S , Mizutani S
Published: October 2019

Juvenile chronic myelogenous leukemia (JCML), a myeloproliferative disorder of childhood, is distinct from adult-type chronic myelogenous leukemia (CML) and bears resemblance to chronic myelomonocytic leukemia (CMMoL). Since mutations in the N-ras gene have been found at high frequencies in CMMoL, but only rarely in CML, we analyzed mutations activating the N-ras gene in 20 patients with JCML. We used the strategy for analysis of gene mutations based on in vitro DNA amplification by polymerase chain reaction (PCR) followed by single- strand conformation polymorphism (SSCP) analysis and/or direct sequence analysis. Nucleotide sequence analysis showed single nucleotide substitutions involving codons 12, 13, or 61 in six of 20 patients (30%). Four of six patients with mutations were in chronic phase and the other two in blast crisis, indicating no apparent correlation with disease stage. Most of the patients with mutations were in the older age group with poor prognosis, although one patient in the younger age group also harbored the mutation. These data suggest that N-ras gene mutations may be involved in the pathogenesis and/or prognosis of JCML and provide further evidence that JCML is an entity distinct from CML.
Bcl2 inhibits apoptosis associated with terminal differentiation of HL- 60 myeloid leukemia cells
Authors: Naumovski L , Cleary ML
Published: October 2019

The Bcl2 protein inhibits apoptosis (programmed cell death) induced by a variety of noxious stimuli. However, relatively little is known about its effect on apoptosis that occurs after terminal differentiation. Bcl2 protein levels decrease during differentiation of myeloid cells into granulocytes that subsequently undergo apoptosis, but the potential role of Bcl2 in coupling survival and differentiation remains undefined. To ascertain the relationship between decreasing Bcl2 levels and the onset of apoptosis in differentiating myeloid cells, Bcl2 was hyperexpressed in the HL-60 cell line after retroviral gene transfer. After treatment of HL-60/BCL2 cells with all-trans retinoic acid or phorbol myristic acid, Bcl2 levels did not decrease as in normal HL-60 cells but, rather, increased because of activation of the viral promoter. Differentiation of the Bcl2-overexpressing cells was similar to that of normal HL-60 cells, but they showed little evidence for apoptosis and had a prolonged survival. These studies show that the survival-enhancing properties of Bcl2 counteract programmed cell death that accompanies terminal differentiation; however, Bcl2 has no significant effect on differentiation itself, suggesting that apoptosis and differentiation are regulated independently in myeloid cells.
Effects of 1 alpha,25-dihydroxyvitamin D3 on macrophage colony- stimulating factor production and proliferation of human monocytic cells
Authors: Kaneki M , Inoue S , Hosoi T , Mizuno Y , Akedo Y , Ikegami A , Nakamura T , Shiraki M , Ito H , Suzu S
Published: October 2019

1 alpha-25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates the proliferation of human monocytes in vitro. In the present study, we investigated a possible role of macrophage colony-stimulating factor (M- CSF) in 1 alpha,25(OH)2D3-induced proliferation of human circulating monocytes and the effects of 1 alpha,25(OH)2D3 on M-CSF production by human monocytic cells. Both 1 alpha,25(OH)2D3 and recombinant human M- CSF increased 2.5-fold the nucleus number of human circulating monocytes on day 6 of the culture. These effects were inhibited by antihuman M-CSF antibody as well as by anti-c-fms antibody, although these antibodies themselves did not affect the nucleus number when added to control culture. These results indicated that M-CSF is required for 1 alpha,25(OH)2D3-stimulated monocyte proliferation. In addition, 1 alpha,25(OH)2D3 stimulated M-CSF secretion from human circulating monocytes. Secretion and mRNA expression of M-CSF by 12–0- tetradecanoylphorbol-13-acetate (TPA)-treated THP-1 cells (human monocytic leukemia cell line) and TPA-treated HL-60 cells (human promyelocytic leukemia cell line) were also increased by 1 alpha,25(OH)2D3. M-CSF secretion from TPA-treated THP-1 cells was increased by 1 alpha,25(OH)2D3 in a dose-dependent and metabolite- specific manner. The present study demonstrates that 1 alpha,25(OH)2D3 is a potent stimulator for M-CSF production by human monocytic cells and that the proliferative effect of 1 alpha,25(OH)2D3 on human monocytes may be attributed, at least in part, to the stimulated secretion of M-CSF.