bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between patterns of engraftment in peripheral blood and immune reconstitution after allogeneic bone marrow transplantation for (severe) combined immunodeficiency
Authors: van Leeuwen JE , van Tol MJ , Joosten AM , Schellekens PT , van den Bergh RL , Waaijer JL , Oudeman-Gruber NJ , van der Weijden-Ragas CP , Roos MT , Gerritsen EJ
Published: October 2019

We report the outcome of allogeneic bone marrow transplantation (BMT) as treatment for severe combined immunodeficiency disease (SCID) in 31 patients grafted from 1968 until 1992. The patients received a graft from an HLA-identical related (n = 10), an HLA-haplo-identical related (n = 19), or a closely HLA-matched unrelated (n = 2) donor that resulted in the long-term survival of 6 of 10, 9 of 19, and 0 of 2 children, respectively. Major complications included failure of engraftment and early death caused by respiratory failure. The chimerism pattern and immunologic reconstitution were evaluated in 15 children who survived more than 1 year with sustained engraftment. The pattern of engraftment was investigated within flow-sorted peripheral blood (PB) T- and B-lymphoid, natural killer (NK), and myelomonocytic cell populations using the amplification of variable number of tandem repeats by the polymerase chain reaction. The immunologic reconstitution was assessed by various in vitro and in vivo parameters. Although the number of PB T cells and the in vitro T-cell proliferative response was in the lower region of normal in the majority of cases and even subnormal in some, in all cases donor T-cell engraftment and reconstitution of T-cell immunity was observed. Residual host-type T cells (1% to 5%) were detected in eight cases at multiple occasions. All children showed normal serum IgM and IgG subclass levels and produced specific IgG antibodies after vaccination, irrespective of donor B-cell engraftment. However, three HLA haplo-identical graft recipients with host-type B lymphoid and myeloid cells have a persistent selective IgA deficiency. NK cells were either of donor, host, or mixed origin. Donor NK cell engraftment restored defective in vitro NK cell function of the recipient. We conclude that determination of lineage-specific engraftment patterns provides valuable information for the understanding of the immunologic reconstitution after allogeneic BMT for SCID.
Organization of the gene for human platelet/endothelial cell adhesion molecule-1 shows alternatively spliced isoforms and a functionally complex cytoplasmic domain
Authors: Kirschbaum NE , Gumina RJ , Newman PJ
Published: October 2019

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell-cell adhesion molecule that is expressed on circulating platelets, on leukocytes, and at the intercellular junctions of vascular endothelial cells and mediates the interactions of these cells during the process of transendothelial cell migration. The cDNA for PECAM-1 encodes an open reading frame of 738 amino acids (aa) that is organized into a 27- aa signal peptide, a 574-aa extracellular domain composed of 6 Ig homology units, and a relatively long cytoplasmic tail of 118 aa containing multiple sites for posttranslational modification and postreceptor signal transduction. To provide a molecular basis for the precise evaluation of the structure and function of this transmembrane glycoprotein, we have determined the organization of the human PECAM-1 gene. The PECAM-1 gene, which has been localized to human chromosome 17, is a single-copy gene of approximately 65 kb in length and is broken into 16 exons by introns ranging in size from 86 to greater than 12,000 bp in length. Typical of other members of the Ig superfamily, each of the extracellular Ig homology domains is encoded by a separate exon, consistent with PECAM-1 having arisen by gene duplication and exon shuffling of ancestral Ig superfamily genes. However, the cytoplasmic domain was found to be surprisingly complex, being encoded by seven short exons that may represent discrete functional entities. Alternative splicing of the cytoplasmic tail appears to generate multiple PECAM-1 isoforms that may regulate phosphorylation, cytoskeletal association, and affinity modulation of the mature protein. Finally, a processed pseudogene having 76% identity with PECAM- 1 cDNA was identified and localized to human chromosome 3. These findings should have important implications for structure/function analysis of PECAM-1 and its role in vascular adhesive interactions.
Expression and regulation of tumor necrosis factor, interleukin-2, and hematopoietic growth factor receptors in B-cell chronic lymphocytic leukemia
Authors: Trentin L , Zambello R , Agostini C , Enthammer C , Cerutti A , Adami F , Zamboni S , Semenzato G
Published: October 2019

Leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) express tumor necrosis factor (TNF) and interleukin-2 (IL-2) receptors, but only a low proliferative response can be elicited in vitro by TNF alpha and IL-2. To investigate the functional properties of IL-2 and TNF alpha on leukemic B cells, we evaluated (1) the regulation of expression of TNF receptors (TNF-R) and IL-2 receptors on leukemic B cells after culture with TNF alpha and IL-2; (2) the effect of the combination of TNF alpha and IL-2 in a proliferative in vitro assay; and (3) the expression and regulation by these cytokines of receptors for hematopoietic factors, including IL-3, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage colony- stimulating factor (GM-CSF). Flow cytometry analysis showed that freshly isolated leukemic cells from B-CLL patients bear the 75-kD TNF- R and the 55-kD IL-2R; TNF alpha was able to upregulate the 55-kD IL-2R but not the 75-kD TNF-R. On the other hand, IL-2 was not able to modify the expression of the above-mentioned receptors. Although each cytokine alone was unable to induce a relevant proliferation of leukemic cells, a synergistic proliferative effect was detected when these cytokines were used in combination. Leukemic B cells from B-CLL patients bear receptors for hematopoietic factors (IL-3, G-CSF, and GM-CSF) that were upregulated in vitro by IL-2 via the 55-kD IL-2R. On the contrary, TNF alpha was unable to affect the expression of the above-mentioned receptors. These results indicate (1) that IL-2 and TNF receptors are related to each other on leukemic cells in B-CLL and (2) that the IL-2R is involved in the regulation of other structures, ie, CSF receptors, thus pointing to another functional role of this receptor complex and the related cytokine in leukemic cells.
Marrow transplantation for patients in accelerated phase of chronic myeloid leukemia
Authors: Clift RA , Buckner CD , Thomas ED , Bryant E , Anasetti C , Bensinger WI , Bowden R , Deeg HJ , Doney KC , Fisher LD
Published: October 2019

The records were reviewed of 58 patients receiving transplants in Seattle with unmanipulated marrow from HLA-identical siblings during the accelerated phase (AP) of chronic myeloid leukemia. Variables examined for association with survival and relapse included the interval from diagnosis to transplant, the reasons for categorization as AP, age, regimen, and cytomegalovirus serology. Four patients relapsed. The 4-year probabilities of survival, relapse-free survival, nonrelapse mortality, and relapse were 0.49, 0.43, 0.51, and 0.12, respectively. After completion of the stepwise multivariate analysis, age less than 38 years and categorization as AP solely on the basis of chromosomal abnormalities emerged as being independently significantly associated with improved survival. The 4-year probability of survival for the 16 patients categorized as AP because of chromosomal abnormalities and receiving transplant less than 1 year from diagnosis was 0.74. The low probability of relapse in these patients suggests that more aggressive preparative regimens are not indicated for patients receiving transplants in AP because of the increased risk of transplant-related mortality.
Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates
Authors: Andrews RG , Briddell RA , Knitter GH , Rowley SD , Appelbaum FR , McNiece IK
Published: October 2019

We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG- CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of progenitor cells that can be collected by leukapheresis than does rhG-CSF alone, that blood cells mobilized by low-dose rhSCF plus rhG-CSF contain marrow repopulating cells, and finally that using a single 2-hour leukapheresis to collect cells, the blood cells mobilized by low-dose rhSCF plus rhG-CSF engraft lethally irradiated recipients more rapidly than do blood cells mobilized by rhG- CSF alone.
A mutation located at the 5' splice junction sequence of intron 3 in the p67phox gene causes the lack of p67phox mRNA in a patient with chronic granulomatous disease
Authors: Tanugi-Cholley LC , Issartel JP , Lunardi J , Freycon F , Morel F , Vignais PV
Published: October 2019

Chronic granulomatous disease (CGD) is due to a functional defect of the O2(-)-generating NADPH oxidase of neutrophils. Mutations resulting in CGD have been shown to occur in only four genes, thus identifying the main components of the oxidase complex, namely the two subunits of a membrane-bound cytochrome b and two cytosolic factors of activation of 67 kD (p67phox) and 47 kD (p47phox). The present study deals with the biochemical and genetic analysis of the defect in a patient suffering from a p67phox-deficient form of CGD. The p67phox deficiency was ascertained by immunochemistry and the ability of recombinant p67phox to restore NADPH oxidase activity using a cell-free system of oxidase activation. The cellular extracts from the proband contained no p67phox protein and no p67phox mRNA when assayed by Western and Northern blot analysis. However, reverse transcription of mRNA and subsequent cDNA amplification by polymerase chain reaction using specific p67phox primers showed that trace amounts of a p67phox mRNA deleted for exon 3 were synthesized in the patient immortalized B lymphocytes. Sequence analysis of the genomic DNA showed a T-to-C transition at position +2 of intron 3. This point mutation in the consensus 5′ splice site of the intron 3 was probably responsible for lack of accumulation of mRNA and also for the skipping of exon 3 detected in the few mRNA molecules that escaped cellular degradation.
Long-term treatment of canine cyclic hematopoiesis with recombinant canine stem cell factor
Authors: Dale DC , Rodger E , Cebon J , Ramesh N , Hammond WP , Zsebo KM
Published: October 2019

Grey collie dogs have cyclic fluctuations in their blood cell counts caused by a regulatory defect of hematopoietic stem cells. To examine the role of stem cell factor (SCF) or its receptor in this disorder, we investigated the stimulatory effects of recombinant canine SCF (rc-SCF) on in vitro marrow cultures, cloned and sequenced the grey collie SCF gene, and treated three grey collies with rc-SCF, either alone or in combination with recombinant canine granulocyte colony-stimulating factor (rcG-CSF). Colony-forming unit granulocyte-macrophage formation from grey collie or normal dog marrow showed similar dose-response curves for rc-SCF. Cloning and sequencing the SCF gene for two grey collies showed no evidence of mutations in the coding region of the SCF gene. Treatment with rc-SCF (10 to 100 micrograms/kg/d) did not induce neutrophilia except at the highest dose (100 micrograms/kg/d), but daily rc-SCF abrogated the neutropenic periods in doses of 20 micrograms/kg/d or greater. Combination of rc-G-CSF (0.5 to 1.0 microgram/kg/d) with rc-SCF treatment (20 to 50 micrograms/kg/d) suggested a synergistic effect, ie, the neutrophil levels on combined therapy were higher than the sum of the levels when these two cytokines were given separately. Long-term treatment of these dogs with rc-SCF in doses of 10 to 30 micrograms/kg/d was generally well tolerated, suggesting that SCF may be useful as a therapy for some chronic hypoproliferative disorders of hematopoiesis.
Selective treatment of SCID mice bearing methotrexate-transport- resistant human acute lymphoblastic leukemia tumors with trimetrexate and leucovorin protection
Authors: Lacerda JF , Goker E , Kheradpour A , Dennig D , Elisseyeff Y , Jagiello C , O'Reilly RJ , Bertino JR
Published: October 2019

Impaired transport of methotrexate (MTX) is a common resistance mechanism of tumor cells to this drug. Trimetrexate (TMTX), a second-generation folate antagonist, is still active against MTX-transport-resistant cells because it enters cells by passive diffusion and does not use the reduced folate transport system for cell entry. Therefore, although leucovorin (LV) protects MTX-sensitive cells from TMTX toxicity, MTX-transport defective cells are poorly rescued by LV. Severe combined immunodeficiency mice bearing MTX-transport-resistant CCRF-CEM acute lymphoblastic leukemia tumors were treated with TMTX alone or with the combination of TMTX and LV, with tumor regressions in both groups (P < .001) and without significant toxicity. These results indicate that TMTX with LV protection may be a useful therapeutic regimen for patients with MTX-transport-defective acute lymphoblastic leukemia. Furthermore, resistance to TMTX plus LV may result in reversion to MTX sensitivity.
Human lymphotoxin has at least equal antitumor activity in comparison to human tumor necrosis factor but is less toxic in mice
Authors: Qin Z , van Tits LJ , Buurman WA , Blankenstein T
Published: October 2019

Because of the severe toxicity of systemically applied tumor necrosis factor (TNF) in cancer patients, considerable efforts have been made to construct mutant TNF molecules, which retain antitumor activity, but display less toxicity. We compared tumor suppression in relation to the toxic effects of human TNF and human lymphotoxin (LT) in mice. The genes for these two cytokines were expressed in Chinese hamster ovary (CHO) cells. Intraperitoneal injection of parental and gene modified CHO cell lines producing similar amounts of biologically active TNF or LT, respectively, into nude mice showed that CHO-TNF cells killed the mice more rapidly than parental cells, but that CHO-LT tumor bearing mice lived significantly longer than mice injected with parental cells. Injection of the cells subcutaneously into severe combined immunodeficiency (SCID) mice allowed direct comparison of tumor suppression and toxic effects of the two cytokines. Both TNF and LT produced by the tumor effectively suppressed tumor growth by an indirect mechanism, LT being at least as effective as TNF. However, mice bearing CHO-TNF cells either died rapidly or developed cachexia, as shown by weight loss. In contrast, mice injected with CHO-LT cells never rapidly died and became cachectic much later than CHO-TNF cell injected animals, though serum levels of LT were higher than those of TNF. Analysis of soluble forms of TNF receptors (TNF-R1 and TNF-R2) in sera of tumor bearing mice showed that soluble TNF-R1 was downregulated in both CHO-TNF and CHO-LT, in comparison with CHO-neo cell injected mice and to normal SCID mice. The soluble form of TNF-R2 was induced by CHO cell lines. In CHO-TNF cell injected SCID mice, serum levels were significantly increased, whereas in mice injected with CHO-LT cells, serum levels of soluble TNF-R2 were decreased. Together, our results show a higher therapeutic index of LT compared with TNF.
Decreased expression of the deleted in colorectal carcinoma gene in non- Hodgkin's lymphoma
Authors: Younes A , Zhao S , Jendiroba D , Kleine HD , Cabanillas F , Andreeff M
Published: October 2019

The putative tumor suppressor gene deleted in colorectal carcinoma (DCC), located on human chromosome band 18q21, is deleted or inactivated in many solid tumors. Its role in the pathogenesis of non- Hodgkin's lymphoma (NHL) has not been studied. Recently, inactivation of this gene was reported in cases of leukemia with monosomy 18. As monosomy 18 is frequently observed in low-grade NHL, we investigated the incidence of altered DCC gene expression in patients with NHL, and correlated it with the number of copies of chromosome 18. Fifteen unselected cases of NHL were studied for evidence of DCC gene expression by reverse transcriptase-polymerase chain reaction. The results were correlated with Southern blot analysis of the DCC gene and with the number of copies of chromosome 18 determined by fluorescent in situ hybridization (FISH). The controls were tissues from normal colon mucosa and normal tonsils. Eight of 15 (53%) NHL cases lacked DCC mRNA, and one expressed substantially less than normal. Southern blot analysis showed normal configuration of the DCC gene in all samples. Two copies of chromosome 18 were found in 9 of 11 samples studied by FISH: one case had a subpopulation of cells with monosomy 18 and one had trisomy 18. All controls expressed DCC. We conclude that DCC gene expression is frequently absent or decreased in NHL and may be involved in the pathogenesis of NHL. Monosomy 18 was not required for DCC inactivation.
Double-stranded RNA induces sickle erythrocyte adherence to endothelium: a potential role for viral infection in vaso-occlusive pain episodes in sickle cell anemia
Authors: Smolinski PA , Offermann MK , Eckman JR , Wick TM
Published: October 2019

Vaso-occlusive pain episodes in sickle cell anemia are hypothesized to be precipitated by adherence of sickle erythrocytes to vascular endothelium in the microcirculation. Febrile episodes, thought to be viral in etiology, are frequently associated with vaso-occlusion; however, a direct link between viral infection and vascular occlusion has not yet been established. Many pathogenic viruses contain double-stranded RNA or replicate through double-stranded RNA intermediates. Double-stranded RNA has been shown to induce vascular cell adhesion molecule-1 (VCAM-1) protein expression on endothelial cells. Recently, a new adhesion pathway has been described between VCAM-1 expressed on cytokine stimulated endothelium and the alpha 4 beta 1 integrin complex expressed on sickle reticulocytes. Based on these observations, the hypothesis was developed that viral infection, through double-stranded RNA intermediates, increases endothelial VCAM-1 expression leading to sickle erythrocyte adhesion to endothelium via an alpha 4 beta 1-VCAM-1- -dependent mechanism. In support of this hypothesis, endothelial cells exposed to the synthetic double-stranded RNA poly(I:C) or the RNA virus parainfluenza 1 (Sendai virus) express increased levels of VCAM-1 and support increased sickle erythrocyte adherence under continuous flow at 1.0 dyne/cm2 shear stress as compared with unstimulated endothelium. Blocking antibodies directed against either VCAM-1 on the endothelium or alpha 4 beta 1 on sickle erythrocytes inhibit nearly all of the increased sickle cell adherence caused by poly(I:C) or Sendai virus. These results support the hypothesis that viruses, through double-stranded RNA elements, can induce sickle erythrocyte adherence to endothelium through alpha 4 beta 1-VCAM-1--mediated adhesion and provide a potential link between viral infection and microvascular occlusion precipitating sickle cell pain episodes.
Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells
Authors: Oria R , Sanchez L , Houston T , Hentze MW , Liew FY , Brock JH
Published: October 2019

Nitric oxide (NO) is known to increase the affinity of the intracellular iron-regulatory protein (IRP) for iron-response elements (IREs) in transferrin receptor and ferritin mRNAs, suggesting that it may act as a regulator of cellular iron metabolism. In this study, exogenous NO produced by adding the NO-generator S-nitroso-N-acetyl penicillamine gave a dose-dependent upregulation of transferrin receptor expression by K562 erythroleukemia cells and increased levels of transferrin receptor mRNA. NO did not affect the affinity of transferrin binding by the transferrin receptor. NO alone did not alter intracellular ferritin levels, but it did abrogate the inhibitory effect of the iron chelator desferrioxamine and potentiated the stimulatory effect of additional iron. NO also caused some increase in ferritin mRNA levels, which might mask any IRP-/IRE-mediated inhibitory effect of NO on ferritin translation. Although NO did not affect net iron uptake, it increased release of iron from K562 cells pulsed previously with 59Fe, and subcellular fractionation showed that it also increased the proportion of intracellular iron bound to ferritin. These findings provide direct evidence that NO can affect cellular iron metabolism and suggest that NO produced in vivo by activated bone marrow macrophages might affect erythropoiesis.
Tyrosine phosphorylation and activation of JAK family tyrosine kinases by interleukin-9 in MO7E cells
Authors: Yin T , Yang L , Yang YC
Published: October 2019

Interleukin-9 (IL-9) is a T-cell-derived multifunctional cytokine that can stimulate the proliferation of a human megakaryocytic leukemia cell line, MO7E. Previous studies suggested that protein tyrosine phosphorylation may be involved in IL-9 signaling pathways. However, tyrosine kinases activated by IL-9 have not been identified. In this report we show that IL-9 induces tyrosine phosphorylation and activation of the JAK family tyrosine kinases including JAK1, JAK3, and Tyk2. The kinetic studies indicate that tyrosine phosphorylation and activation of JAK kinases induced by IL-9 occurred within 1 minute, peaked by 5 to 10 minutes, and persisted at least for 45 minutes. Furthermore, we show that signal transducers and activators of transcription (Stat) 91 or related protein and an 88-kD Stat 91-associated protein are rapidly tyrosine phosphorylated following IL-9 treatment. Gel shift assays confirm that nuclear extracts from MO7E cells stimulated with IL-9 specifically interact with a DNA element termed gamma activated site. These results suggest that actions of IL-9 may, in part, be mediated through JAK kinase-Stat signaling cascades.
Hypercoagulability in centenarians: the paradox of successful aging
Authors: Mari D , Mannucci PM , Coppola R , Bottasso B , Bauer KA , Rosenberg RD
Published: October 2019

With advancing age, an increasing number of healthy individuals have laboratory signs of heightened coagulation enzyme activity. Such biochemical hypercoagulability might be the basis of either the increased thrombotic tendency occurring with age or a harmless manifestation of this process. To see whether these alterations are also present in the very elderly who had aged successfully, 25 healthy centenarians were studied and results of coagulation and fibrinolysis measurements were compared with those obtained in two control groups of healthy adults, 25 ranging in age from 18 to 50 years and 25 from 51 to 69 years. Older controls had, in general, slightly higher values of several coagulation and fibrinolysis measurements than younger controls. Centenarians had striking signs of heightened coagulation enzyme activity, as assessed directly by measuring activated factor VII in plasma (P < .01, compared with either control group) or indirectly by measuring the plasma levels of the activation peptides of prothrombin, factor IX, factor X, and thrombin-antithrombin complexes (all P < .001). Heightened coagulation enzyme activity was accompanied by signs of enhanced formation of fibrin (high fibrinopeptide A, P < .001) and secondary hyperfibrinolysis (high D-dimer and plasmin-antiplasmin complex, P < .001). Plasma concentrations of fibrinogen and factor VIII were higher than in controls, whereas other coagulation factors were not elevated. In conclusion, this study shows the very elderly do not escape the state of hypercoagulability associated with aging, but that this phenomenon is compatible with health and longevity. Hence, high plasma levels of the coagulation activation markers in older populations do not necessarily mirror a high risk of arterial or venous thrombosis.
Purification and characterization of a C5a-inactivating enzyme from human peritoneal fluid
Authors: Ayesh SK , Azar Y , Barghouti II , Ruedi JM , Babior BM , Matzner Y
Published: October 2019

Earlier work has suggested that familial Mediterranean fever, an inherited disorder characterized by sporadic episodes of inflammation involving the pleural and peritoneal cavities and the joints, is caused by the lack of a C5a inactivator normally found in serosal fluid. We have purified this inactivator from ascites fluid and obtained a protein of molecular weight 53 to 56 kD with a specific activity 10,000- fold greater than the crude material. On Western blot, an inhibitory antibody recognized a single antigenic species at the same molecular weight. The enzyme had no activity against denatured bovine serum albumin. With recombinant C5a as substrate, the Km and Vm were 3.4 mumol/L and 52 nmol C5a/min/mg protein, respectively.
Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells
Authors: Shibuya A , Nagayoshi K , Nakamura K , Nakauchi H
Published: October 2019

We have established a cell culture system without stromal cells that allows the CD34+ hematopoietic progenitor cells (HPC) to differentiate into natural killer (NK) cells. CD34+Lin (CD3, CD16, CD56)-cells were purified using fluorescence-activated cell sorting from normal adult bone marrow (BM) and cultured for 28 days in medium supplemented with interleukin-2 (IL-2) and stem cell factor (SCF). NK (CD3-CD16-CD56+) cells were generated in a dose-dependent manner in response to SCF. NK cells originated from CD34+CD33+Lin- cells, but they were barely detectable in cultures of CD34+CD33-Lin- cells. However, on addition of IL-3, an induced differentiation of NK cells from CD34+CD33-Lin- cells was observed, although at a lower frequency. Supplementing of the cell cultures with SCF alone or both SCF and IL-3 for the first 7 days followed by IL-2 for the next 21 days is essential for production of NK cells from CD34+CD33+Lin- cells and from CD34+CD33-Lin- cells, respectively. These data provide direct evidence that NK cells arise from CD34+HPC and show the minimum lymphokine requirement for their differentiation.
Prognostic value of cellular proliferation and histologic grade in follicular lymphoma
Authors: Martin AR , Weisenburger DD , Chan WC , Ruby EI , Anderson JR , Vose JM , Bierman PJ , Bast MA , Daley DT , Armitage JO
Published: October 2019

The clinical usefulness of histologic grading in follicular lymphoma (FL) is controversial and is further compromised by the subjective nature and poor reproducibility of most systems in current use. Therefore, we decided to objectively evaluate the importance of cellular proliferation in FL, along with the current grading systems. We studied 106 patients with FL who were uniformly staged and aggressively treated. A proliferative index (PI) was determined quantitatively using an automated image analyzer and a new Ki-67 antibody that stains archival paraffin tissues. The cases were also subclassified according to the Berard, Rappaport, Luke-Collins, and Jaffe methods, and survival analysis was performed. Patients with a low PI (< 40%) had a significantly longer overall survival (OS) than those with a high PI (> or = 40%), but the PI did not predict failure-free survival (FFS). The mean PI correlated well with the subgroups in each of the various classifications. All four of the classification methods were predictive of OS, but only the Berard method appeared to predict FFS and suggest that a proportion of patients with FL may be curable. In multivariate analysis, histologic classification was the only independent predictor of OS (Berard method: relative risk, 3.1) and the International Prognostic Index was the only independent predictor of FFS (relative risk, 2.3). We conclude that the Berard method for grading of FL is clinically useful and, along with the International Prognostic Index, should be included in future clinical studies of FL. The measurement of cellular proliferation does not appear to add additional useful information in FL.
Leukocyte integrin CD11b promoter directs expression in lymphocytes and granulocytes in transgenic mice
Authors: Back A , East K , Hickstein D
Published: October 2019

The human leukocyte integrin subunit CD11b is expressed predominantly on myelomonocytic cells. To identify CD11b promoter sequences important for myelomonocytic gene expression and to assess the utility of the CD11b promoter for expressing heterologous genes in vivo, we generated transgenic mice with a human CD4 reporter gene driven by CD11b promoter constructs composed of 1.5, 0.3, or 0.1 kb of DNA sequence 5′ to the transcription start site. Using flow cytometry to detect the human CD4 reporter on murine leukocytes, two of three 1.5-kb CD11b promoter founder lines showed surface expression of the human CD4 transgene in granulocytes and lymphocytes. The transgene expression observed in lymphocytes was inappropriate relative to the normal pattern of CD11b expression. Of the eight 0.3-kb or 0.1-kb founder lines, only one 0.1- kb founder line showed transgene expression. The overall pattern of transgene expression among the 11 founder lines does not parallel expression of the endogenous CD11b gene. These studies indicate that additional CD11b regulatory elements will be required to express a reporter gene in vivo in a lineage-specific pattern that mimics the endogenous CD11b gene.
The pro region of human neutrophil defensin contains a motif that is essential for normal subcellular sorting
Authors: Liu L , Ganz T
Published: October 2019

Human defensins (human neutrophil peptides) HNP 1–3 are 29–30 amino acid antibiotic and cytotoxic peptides highly abundant in the cytoplasmic granules of polymorphonuclear leukocytes. The peptides are produced from 94 amino acid (aa) prepropeptides by proteolytic cleavage of the signal sequence and stepwise removal of the 44–45 aa anionic propiece. To study the role of the propiece, we constructed five in- frame deletions in preproHNP-1 cDNA between the signal peptidase site and the amino-terminus of the mature defensin region (aa 21–64). The wild type HNP-1 cDNA and the deletion mutants were ligated into the pBabe-Neo retroviral vector, expressed in GP+E86 packaging derivative of NIH 3T3 cells, then transduced into the 32D cl3 granulocytic cell line. For each construction and both cell lines, we measured the accumulation of the various defensin forms in cells and media by 24- hour labeling or pulse-chase with 35S-cysteine- and immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Deletions in the amino-terminal two-fifths of the propiece, delta 21–28 and delta 21–38, had only minor effects on defensin biosynthesis in both cell lines and did not interfere with the accumulation of mature defensin in the granules of 32D cl3 cells. Deletions in the carboxyterminal three-fifths of the propiece (delta 21– 51 and delta 21–64) diminished net defensin synthesis, blocked constitutive secretion of prodefensin in both cell lines, and interfered with defensin accumulation in cytoplasmic granules of 32D cl3 cells. These effects were reproduced by the smaller deletion delta 40–51, which contains highly conserved secondary structure. The propiece segment 40–51 appears to be essential for the subcellular trafficking and sorting of HNP-1 defensin.
Differential CD26-mediated activation of the CD3 and CD2 pathways after CD6-depleted allogeneic bone marrow transplantation
Authors: Kameoka J , Sato T , Torimoto Y , Sugita K , Soiffer RJ , Schlossman SF , Ritz J , Morimoto C
Published: October 2019

Patients who have undergone allogeneic bone marrow transplantation (allo-BMT) are susceptible to a variety of opportunistic infectious complications in the months to years after engraftment. Impaired in vitro T-cell functions have been documented in these patients, and these T-cell dysfunctions contribute to the prolonged immune deficiency after allo-BMT. In the present study, we examined the expression of CD26 as well as the reconstitution of CD26-mediated T-cell costimulation via the CD3 and CD2 pathways at various times in patients aged greater than 18 years after CD6-positive, T-cell depleted allo- BMT. We found that the percentage of CD26- and CD3-positive cells, as well as the levels of expression of both antigens, was lower than in normal controls during the first 4 months after CD6-depleted allo-BMT. Subsequently, the amount of lymphocytes expressing CD3 and CD26 and the quantitative surface expression of CD3 and CD26 were not significantly different in patients and normal controls. Functional studies showed that CD26-mediated T-cell proliferation via the CD3 pathway was considerably improved and almost reached normal levels by 1 year, whereas recovery of CD26-mediated T-cell proliferation via the CD2 pathway was delayed for at least 2 years after CD6-depleted allo-BMT. As CD26 involvement in the regulation of human thymocyte activation is restricted preferentially to the CD3 pathway--unlike its involvement with both CD3 and CD2 pathways of peripheral T cells--our results suggest that the different effects of CD26-mediated costimulation via the CD3 and CD2 pathways after CD6-depleted allo-BMT may be a reflection of peripheral T-cell immaturity in those individuals, similar to that seen in mature medullary thymocytes or cord T lymphocytes.
FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver
Authors: Muench MO , Roncarolo MG , Menon S , Xu Y , Kastelein R , Zurawski S , Hannum CH , Culpepper J , Lee F , Namikawa R
Published: October 2019

The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in short-term cultures of CD34++ CD38+ lineage (Lin)- light-density fetal liver (LDFL) cells and the more primitive CD34++ CD38- Lin- LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CF) and/or low-proliferative potential (LPP)-CFC in cultures of CD34++ CD38+ Lin- and CD34++ CD38- Lin- LDFL- cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34+ Lin- LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34++ CD38- Lin- LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34++ CD38- Lin- LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors.
Genomic structure, chromosomal localization, and conserved alternative splice forms of thrombopoietin
Authors: Gurney AL , Kuang WJ , Xie MH , Malloy BE , Eaton DL , de Sauvage FJ
Published: October 2019

Thrombopoietin (TPO), the ligand for c-mpl, is a novel cytokine comprising an amino terminal domain with homology to erythropoietin and a glycosylated carboxyl terminal domain that does not bear overall homology to other known proteins. We report the cloning of cDNAs encoding the porcine and murine TPO and the characterization of the human TPO gene. The cDNA for an additional splice form (TPO-2) with a four-amino-acid deletion within the erythropoietin-like domain has been isolated and is conserved between humans, pigs, and mice. Species comparison of TPO shows that the amino terminal erythropoietin-like domain is highly conserved, while the carboxyl terminal domain is less conserved. Recombinant murine TPO and human TPO are each able to activate both the murine and human c-mpl receptors, indicating an absence of strict species specificity. Human TPO is encoded by a single gene consisting of six exons and located on chromosome 3q271–28.
All-trans retinoic acid for the treatment of newly diagnosed acute promyelocytic leukemia. Japan Adult Leukemia Study Group [see comments]
Authors: Kanamaru A , Takemoto Y , Tanimoto M , Murakami H , Asou N , Kobayashi T , Kuriyama K , Ohmoto E , Sakamaki H , Tsubaki K
Published: October 2019

We conducted a multicenter trial of treatment with all-trans retinoic acid (ATRA) for newly diagnosed acute promyelocytic leukemia (APL) in the AML-92 study and compared it with our previous study with standard intensive chemotherapy, the AML-89 study, in the view of complete remission (CR) rate, incidence of early death, and event-free survival (EFS). Patients were scheduled to receive oral ATRA 45 mg/m2 daily until CR. If patients had leukocyte counts above 3 x 10(9)/L at the start of therapy, they received daunorubicine (DNR) 40 mg/m2 for 3 days and behenoyl cytosine arabinoside (BHAC) 200 mg/m2 for 5 days in addition to ATRA. During the ATRA therapy, if patients showed myeloblast plus promyelocyte counts higher than 1 x 10(9)/L in the peripheral blood, they received additional DNR and BHAC in the same schedule, as well. A total of 110 patients were entered into the study. Median age was 43 years (range, 16 to 74). Twenty-eight (26%) of 109 patients (one died before the start of therapy) received ATRA alone. Ninety-seven patients (89%) achieved CR; 48 of 49 (98%) aged less than 40 years, 44 of 52 (84%) aged between 40 and 69, and 5 of 8 (63%) aged above 70 achieved CR, respectively; 25 of 28 (89%) with ATRA alone, 46 of 51 (90%) with ATRA plus initial chemotherapy and 26 of 30 (87%) with ATRA plus later chemotherapy attained CR, respectively. Nine (8%) patients died within 28 days after the start of therapy. In contrast, 44 of 62 patients (71%) attained CR, and 13 (21%) died within 28 days in the AML-89 study with the combination of DNR, BHAC, 6-mercaptopurine and prednisolone. Seven developed retinoic acid syndrome and one died of it in the present study. Other toxicities associated with this drug included cheilitis, desquamation, muscle pain, and hypertriglyceridemia. Predicted 23 months EFS for all ATRA-treated patients and disease-free survival (DFS) in the CR cases were 75% and 81%, respectively, in a median follow-up period of 21 months. Compared to the AML-89 study, there was a highly significant difference in remission rate (P = .004), EFS (P = .0007), and also early mortality rate (P = .02). Present results demonstrated that ATRA with or without chemotherapy gives a statistical improvement in CR rate and early mortality rate, as well as superior survival in newly diagnosed APL.
The 175 antigen expressed on myeloid and erythroid cells during differentiation is associated with serine protease activity
Authors: Deane D , Inglis L , Haig D
Published: October 2019

Monoclonal antibody 175 recognizes a cell-surface antigen on more than 80% of nucleated ovine bone marrow cells (BMC). The distribution is unusual, as the majority of differentiated myeloid and erythroid cells express the antigen (175 antigen), whereas mast cells, basophils, and the majority of lymphocytes do not. The level of 175 antigen expression has been shown to increase as BMC differentiate during hematopoiesis. Previous attempts to identify the 175 antigen have been unsuccessful. In this study, the 175 antigen was affinity-purified and shown to contain serine protease activity. Immunoblot analysis following sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of bone marrow cell lysates run under reducing or nonreducing conditions showed two closely adjacent protein bands (a doublet) of 28 to 30 kD molecular weight. N-linked deglycosylation showed that the 30-kD band was a glycosylated form of the 28-kD protein. Both protein bands shared the same N-terminal amino acid sequence over 20 residues, with high homology with serine proteases. Affinity-purified 175 antigen was proteolytic in substrate gels, the activity being inhibited by the 175 monoclonal antibody (Mab) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), but not by metallo, thiol, or acid protease-specific inhibitors. The 175 antigen is therefore part of a growing family of cell-surface proteases associated with hematopoietic cell differentiation.
Detection of the chromosome 16 CBF beta-MYH11 fusion transcript in myelomonocytic leukemias
Authors: Poirel H , Radford-Weiss I , Rack K , Troussard X , Veil A , Valensi F , Picard F , Guesnu M , Leboeuf D , Melle J
Published: October 2019

Karyotypic detection of chromosomal 16 abnormalities classically associated with AML M4Eo can be difficult. Characterization of the two genes involved in the inv(16)(p13q22), CBF beta and MYH11, has allowed the detection of fusion transcripts by reverse-transcriptase polymerase chain reaction (RT-PCR). We have analyzed CBF beta-MYH11 fusion transcripts by RT-PCR in myelomonocytic leukemias, with or without eosinophilia, to determine whether their presence correlates with morphology. Fifty-three cases (11 AML M4Eo; 1 AML M4 with atypical abnormal eosinophils (AML M4 “Eo”); 29 AML M4; 8 AML M5; 3 CMML; and 1 AML M2 with eosinophilia) were analyzed. All 11 typical AML M4Eo were CBF beta-MYH11 positive. The single case of AML M4 with distinctive eosinophil abnormalities was negative by karyotype, RT-PCR and fluorescent in situ hybridization (FISH). Three of 29 (10%) AML M4 without abnormal eosinophils were CBF beta-MYH11 positive, 1 of which did not show any apparent chromosome 16 abnormalities by classical metaphase analysis (2 not tested). Both cases tested also showed MYH11 genomic rearrangement. None of the other leukemias were RT-PCR positive. Follow-up of three patient showed residual positivity in apparent complete remission. These data show that CBF beta-MYH11 fusion transcripts occur not only in the vast majority of typical AML M4Eo, but also in approximately 10% of AML M4 without eosinophilic abnormalities, a much higher incidence than the sporadic reports of chromosome 16 abnormalities in AML M4 would suggest. Taken together with the detection of CBF beta-MYH11 transcripts in the absence of apparent chromosome 16 abnormalities by classical banding techniques, these data show that additional screening by either RT-PCR or FISH should be performed in all AML M4, regardless of morphologic features, to allow accurate evaluation of the prognostic importance of this fusion transcript.
High-dose chemotherapy with or without total body irradiation followed by autologous bone marrow and/or peripheral blood stem cell transplantation for patients with relapsed and refractory Hodgkin's disease: results in 85 patients with analysis of prognostic factors
Authors: Nademanee A , O'Donnell MR , Snyder DS , Schmidt GM , Parker PM , Stein AS , Smith EP , Molina A , Stepan DE , Somlo G
Published: October 2019

Eight-five consecutive patients with relapsed or refractory Hodgkin's disease (HD) underwent high-dose chemotherapy or chemo/radiotherapy followed by autologous bone marrow (ABMT) and/or peripheral blood stem cell (PBSC) transplantation. Two preparative regimens were used. Twenty- two patients (26%) without prior radiation received fractionated total body irradiation (FTBI) 1,200 Gy in combination with high-dose etoposide (VP-16) 60 mg/kg and cyclophosphamide (CTX) 100 mg/kg. Sixty- three patients (74%) with prior radiotherapy received carmustine (BCNU) 450 mg/m2 instead of FTBI. The median age was 32 years (range, 16 to 56). The median number of prior chemotherapy regimens was three (range, 1 to 7). Forty-three patients (51%) received transplants in first relapse or second complete remission (CR), whereas 33 (39%) received transplants after second or subsequent relapse. All relapsed patients, except one, received conventional salvage chemotherapy and/or radiotherapy in an attempt to reduce tumor bulk before transplant. At the time of analysis in April 1994, fifty-seven patients (67%) are alive, including 44 (52%) in continuous CR, with a median follow-up for the surviving patients of 28 months (range, 7 to 66). Thirty patients (35%) relapsed at a median of 9 months (range, 1 to 43). Eleven patients (13%) died of transplant-related complications including veno- occlusive disease of the liver (VOD) in five, acute and late interstitial pneumonitis in three, graft failure in one, cerebral hemorrhage in one, and therapy-induced myelodysplasia (MDS)/acute leukemia in one patient. At a median follow-up of 25 months (range, 0.6 to 66), the cumulative probability of 2-year overall and disease-free survival (DFS) of all 85 patients is 75% (95% confidence interval [CI] 64% to 84%) and 58% (95% CI 47% to 69%), respectively. Three independent prognostic variables were identified by univariate analysis: number of prior chemotherapy regimens, prior radiotherapy, and extranodal disease at ABMT. Multivariate stepwise Cox regression identified the number of prior chemotherapy regimens as the only significant prognostic factor predicting for both relapse and DFS. There were no significant differences in the outcome of the treatment between the two preparative regimens. Our results confirm that high- dose therapy and ABMT is an effective therapy for patients with relapsed or refractory HD. Earlier transplantation is recommended before the development of drug resistance and end organ damage that results from repeated attempts of salvage therapy.