bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic instability of microsatellite repeats and its association with the evolution of chronic myelogenous leukemia [see comments]
Authors: Wada C , Shionoya S , Fujino Y , Tokuhiro H , Akahoshi T , Uchida T , Ohtani H
Published: October 2019

Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor-suppressor genes. Investigation of genomic instability of microsatellites has indicated a new mechanism for human carcinogenesis in hereditary nonpolyposis colorectal cancer and sporadic cancer and this instability has been shown to be related to inherited predisposition to cancer. This study was conducted to determine whether such microsatellite instability is associated with the evolution of chronic myelogenous leukemia (CML) to the blast crisis. Nineteen CML patients clinically progressing from the chronic phase to accelerated phase or blast crisis and 20 other patients in the CML chronic phase were studied. By polymerase chain reaction assay, DNAs for genomic instability in five separate microsatellites in chromosome arms 5q (Mfd27), 17p (Mfd41), 18q (DCC), 3p (CI3–9), and 8p (LPL) were examined. Differences in unrelated microsatellites of chronic and blastic phase DNAs in 14 of 19 patients (73.7%) were demonstrated. Somatic instability in five microsatellites, Mfd27, Mfd41, DCC, CI3–9, and LPL, was detected in 2 of 19 (10.5%), 8 of 19 (42.1%), 11 of 19 (57.9%), 4 of 17 (23.5%), and 4 of 17 (23.5%) cases. In 10 of 19 cases (52.6%), genetic instability in at least two of five microsatellites was observed and was categorized as replication error (RER+) phenotype. CML evolution cases with myeloid, lymphoid, and mixed phenotypes and the blast crisis and accelerated phase showed somatic instability in a number of microsatellites. No alterations in leukemic cells at the chronic phase could be detected in any microsatellites. These data indicate instability of microsatellites (RER+) but not familial predisposition to possibly be a late genetic event in the evolution of CML to blast crisis. In the microsatellite of the DCC gene, complicated alterations in band patterns caused by instability as well as loss of heterozygosity (LOH) were observed in 13 of 19 cases (68.4%): instability in 9 cases, instability plus LOH in 2 cases, and only LOH in 2 cases. These highly frequent alterations in microsatellites, including instability and LOH, suggesting that secondary events due possibly to loss of fidelity in replication and repair machinery may be significantly associated with CML evolution.
The gamma subunit of the interleukin-2 receptor is expressed in human monocytes and modulated by interleukin-2, interferon gamma, and transforming growth factor beta 1
Authors: Bosco MC , Espinoza-Delgado I , Schwabe M , Russell SM , Leonard WJ , Longo DL , Varesio L
Published: October 2019

The interleukin-2 receptor gamma (IL-2R gamma) chain is a newly recognized component of the IL-2R of lymphoid cells that is required for their response to IL-2. We investigated the expression of IL-2R gamma protein in human monocytes by Western blot analysis using an antiserum specific for IL-2R gamma. We found that IL-2R gamma subunit is constitutively expressed in human monocytes and upregulated by the monocyte-activating factors IL-2 and interferon gamma (IFN gamma). Furthermore, we show that transforming growth factor beta 1 (TGF beta 1) downmodulates, in a dose-dependent manner, basal and IL-2-induced, but not IFN gamma-induced, IL-2R gamma chain expression, and this effect may be responsible for TGF beta 1 suppressive activity on IL-2- activated monocytes. Overall, these results show that the expression of the IL-2R gamma subunit in human monocytes is tightly regulated by the cytokine network, suggesting a critical role played by this protein on monocyte activation.
Importance of effective central nervous system therapy in isolated bone marrow relapse of childhood acute lymphoblastic leukemia. BFM (Berlin- Frankfurt-Munster) Relapse Study Group
Authors: Buhrer C , Hartmann R , Fengler R , Schober S , Arlt I , Loewke M , Henze G
Published: October 2019

Presymptomatic central nervous system (CNS) treatment in children with a late isolated first bone marrow (BM) relapse of acute lymphoblastic leukemia (ALL) was based on intermediate-dose systemic and intrathecal (IT) methotrexate (MTX) in the multicenter trial, ALL-REZ BFM 85. Because this was associated with an excess of overt second CNS relapses, cranial radiotherapy (cRT) plus prolonged triple IT therapy with MTX, cytarabine, and prednisone was instituted during the course of the subsequent trial, ALL-REZ BFM 87. Results of children with or without cRT, but otherwise identical chemotherapy, are presented here. Between April 1985 and March 1990, 93 children with their first late isolated BM relapse of ALL were entered on protocols ALL-REZ BFM 85M and ALL-REZ BFM 87. An intensive 6-month phase of multiagent chemotherapy that included 8 courses of systemic MTX (1 g/m2) plus IT MTX was followed by 2 years of conventional maintenance therapy with daily 6-thioguanine and biweekly MTX. Children with bone marrow transplantation excluded, 73 were in complete remission at the end of intensive polychemotherapy, 40 of whom received fractionated cRT plus triple IT therapy during the following 6 months; 11 did not receive cRT but prolonged triple IT; 22 received neither cRT nor prolonged triple IT. Except for a higher percentage of children who had received cRT in front-line protocols (29 of 33 v 20 of 40), the patient groups without or with salvage cRT were comparable. Of 33 children without salvage cRT, 26 relapsed, compared with 21 of 40 who had received cRT (P < .05). The difference was solely attributable to second relapses with CNS involvement (10 of 33 v 1 of 40; P < .01). Estimated 6-year event- free survival rates were .18 for children without cRT and .46 for children with cRT (P < .01). In patients without cRT, no impact of prolonged IT therapy could be shown. The data suggest that second CNS prophylaxis with cRT and prolonged triple IT therapy in children with late isolated BM relapse of ALL is effective in preventing CNS relapses, in reducing the overall relapse rate, and in increasing the overall survival rate.
Demonstration that Thy(lo) subsets of mouse bone marrow that express high levels of lineage markers are not significant hematopoietic progenitors
Authors: Morrison SJ , Lagasse E , Weissman IL
Published: October 2019

We have been unable to reproduce experiments suggesting the existence of three lineage-restricted progenitor populations from mouse bone marrow. Thy1.1loMac-1+B220+ cells were reported to give rise to greatly expanded numbers of myeloid and lymphoid cells, while Thy1.1loMac- 1+B220- and Thy1.1loMac-1-B220+ cells were reported to be highly proliferative myeloid and B-lineage-restricted progenitors, respectively. Both Mac-1+ cell types appear to be much less frequent than previously reported, and we observed no activity consistent with their characterization as committed progenitors of expanded numbers of cells. The original identification of these populations may have resulted from a failure to distinguish bonafide signals from autofluorescent background and nonspecific staining. The progenitor activities originally associated with these populations may have been due to hematopoietic stem cell contamination. This study shows that low levels of Mac-1 are expressed on cells with multipotent progenitor activity. Thy1.1loB220+Mac-1- cells can be purified from bone marrow, but in these experiments they do not give rise to detectable levels of progeny on injection into lethally irradiated mice. Thy1.1loB220+Mac-1- cells appear to be pro-B cells without significant proliferation potential in vivo. The finding that the described populations do not have the reported progenitor activities leaves the pathways of stem cell differentiation open to further study.
C-kit gene is expressed by skin mast cells in embryos but not in puppies of Wsh/Wsh mice: age-dependent abolishment of c-kit gene expression
Authors: Yamazaki M , Tsujimura T , Morii E , Isozaki K , Onoue H , Nomura S , Kitamura Y
Published: October 2019

The Wsh is a mutant allele at the W (c-kit) locus of mice, but no significant abnormalities are found at the coding region of the Wsh allele. Since cultured mast cells derived from the spleen of Wsh/Wsh mice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c- kit mRNA expression in the skin of Wsh/Wsh mice of various ages. The number of mast cells in the skin of Wsh/Wsh embryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kit mRNA was detectable in the skin of 18-day pc Wsh/Wsh embryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wsh mice. The number of c-kit protein- containing cells was significantly greater in the skin of 18-day pc Wsh/Wsh embryos than in the skin of 5-day-old Wsh/Wsh mice. The abolishment of c-kit mRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kit mRNA was detectable by in situ hybridization in skin mast cells of 5- day-old Wsh/Wsh mice. Taken together, the expression of c-kit mRNA was abolished first, then the content of c-kit protein dropped to undetectable levels, and then the disappearance of Wsh/Wsh mast cells themselves followed.
Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor
Authors: Christophe O , Ribba AS , Baruch D , Obert B , Rouault C , Niinomi K , Pietu G , Meyer D , Girma JP
Published: October 2019

We compared the properties of plasma von Willebrand factor (vWF) from normal individuals and from two patients with type IIA (Glu875Lys) and type IIB (duplication of Met 540) von Willebrand disease (vWD) with the corresponding fully multimerized recombinant proteins. We included cryosupernatant from normal human plasma and type IIA plasma (Cys509Arg). Functions of vWF were analyzed by binding assays to platelets in the presence of ristocetin or botrocetin. Parameters of binding (number of binding sites per vWF subunit, and dissociation constant Kd) were quantitatively estimated from the binding isotherms of 125I-botrocetin or glycocalicin to vWF, independently of the size of the multimers. We found that ristocetin- or botrocetin-induced binding to platelets was correlated in all cases with the size of vWF multimers. In the absence of inducer, only type IIB rvWF Met-Met540 spontaneously bound to platelets. No significant difference of binding of purified botrocetin to vWF was found between normal and patients' plasma, or between wild-type rvWF (rvWF-WT) and rvWF-Lys875. In contrast, affinity of botrocetin for type IIB rvWF Met-Met540 was decreased. Botrocetin-induced binding of glycocalicin to vWF from all plasma and cryosupernatant was similar. Compared with rvWF-WT, binding of glycocalicin to rvWF-Lys875 was normal. In contrast, the affinity for type IIB rvWF Met-Met540 was 10-fold greater. Thus, our data suggest that, in the patients tested, the abnormal IIA phenotype results from the lack of large-sized multimers and is independent of the point mutations. In contrast, the type IIB mutation is directly involved by providing a conformation to the vWF subunits that allows the high molecular weight multimers to spontaneously interact with platelet glycoprotein Ib.
The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible
Authors: Michelson AD , Benoit SE , Kroll MH , Li JM , Rohrer MJ , Kestin AS , Barnard MR
Published: October 2019

Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time- dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb- IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation- induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.
Mechanisms regulating the mRNA levels of interleukin-5 and two other coordinately expressed lymphokines in the murine T lymphoma EL4.23
Authors: Naora H , Young IG
Published: October 2019

The mechanisms that regulate the mRNA levels of interleukin-5 (IL-5) were compared with those regulating the mRNA levels of two other coordinately expressed lymphokines in the murine T lymphoma EL4.23. Our results indicate that IL-5 mRNA levels are independently regulated from those of IL-2 and granulocyte-macrophage colony-stimulating factor (GM- CSF) mRNAs. The induction of IL-5 mRNA by phorbol 12-myristate 13- acetate (PMA) stimulation was found to be cyclosporin A-resistant, in contrast to the induction of IL-2 and GM-CSF mRNAs. Although the three lymphokine mRNAs were not detected in unstimulated cells by Northern blot analysis, the GM-CSF gene was found by nuclear run-off analysis to be constitutively transcribed. However, the IL-2 and IL-5 genes were transcriptionally inactive in the absence of PMA stimulation. The induction of IL-5 mRNA by PMA stimulation primarily involved increased transcriptional activity. In contrast, GM-CSF mRNA induction predominantly involved enhanced mRNA stability. Both transcriptional and mRNA stabilization mechanisms appeared to regulate IL-2 mRNA induction. The activation of IL-2 and IL-5 gene transcription was dependent on de novo protein synthesis. Cellular treatment with cycloheximide enhanced IL-2 gene transcription once activation was initiated, implicating the involvement of a labile repressor(s). Furthermore, IL-5 mRNA was more stable than IL-2 and GM-CSF mRNAs. These latter two species were stabilized by cycloheximide, suggesting that a labile mechanism may regulate their degradation.
Regulation of 92-kD gelatinase release in HL-60 leukemia cells: tumor necrosis factor-alpha as an autocrine stimulus for basal- and phorbol ester-induced secretion
Authors: Ries C , Kolb H , Petrides PE
Published: October 2019

Matrix metalloproteinase 9 (MMP-9), also known as 92-kD type IV collagenase/gelatinase, is believed to play a critical role in tumor invasion and metastasis. Here, we report that MMP-9 was constitutively released from the human promyelocytic cell line HL-60 as determined by zymographic analysis. Tumor necrosis factor-alpha (TNF-alpha) enhanced the enzyme release threefold to fourfold and the protein kinase C (PKC) activator and differentiation inducer 12-O-tetradecanoylphorbol-13- acetate (TPA) eightfold to ninefold. Gelatinase induction by TNF-alpha and TPA was inhibited by actinomycin D or cycloheximide, indicating that de novo protein synthesis was required. Neutralizing monoclonal antibodies to TNF-alpha (anti-TNF-alpha) decreased the basal MMP-9 release of these cells. In addition, these antibodies also significantly interfered with the TPA-induced enzyme release. Agents that inhibit TNF-alpha expression in HL-60 cells, such as pentoxifylline and dexamethasone, completely abrogated both the constitutive and TPA-evoked MMP-9 release. Diethyldithiocarbamate, which is known to stimulate TNF-alpha production in HL-60 cells, exerted a positive effect on MMP-9 release in untreated cells but was inhibitory in TPA-treated HL-60 cells. The PKC inhibitor staurosporine at low concentrations (100 ng/mL) caused a significant augmentation of MMP-9 release in untreated cultures that was blocked by the addition of anti-TNF-alpha. High concentrations (2 mumol/L) of staurosporine completely abolished the extracellular enzyme activity both in untreated and TPA-stimulated cells. These results suggest, that TNF- alpha is required for basal and PKC-mediated MMP-9 release in HL-60 leukemia cells. Thus, MMP-9 secretion may be regulated by TNF-alpha not only in a paracrine but also in an autocrine fashion. This may potentiate the matrix degradative capacity of immature leukemic cells in the processes of bone marrow egress and the evasion of these cells into peripheral tissue.
Rearrangement of CCND1 (BCL1/PRAD1) 3' untranslated region in mantle- cell lymphomas and t(11q13)-associated leukemias
Authors: Rimokh R , Berger F , Bastard C , Klein B , French M , Archimbaud E , Rouault JP , Santa Lucia B , Duret L , Vuillaume M
Published: October 2019

Rearrangement and overexpression of CCND1 (BCL1/PRAD1), a member of the cyclin G1 gene family, are consistent features of t(11q13)-bearing B- lymphoid tumors (particularly mantle-cell lymphoma [MCL]). Its deregulation is thought to perturb the G1-S transition of the cell cycle and thereby to contribute to tumor development. As suggested by previously published studies, rearrangement of the 3′ untranslated region (3′ UTR) of CCND1 may contribute to its activation in some lymphoid tumors. To define further the prevalence of such rearrangements, we report here the result of the molecular study of 34 MCL and six t(11q13)-associated leukemias using a set of probes specific to the different parts of the CCND1 transcript. We also sequenced the entire cDNA of the overexpressed CCND1 transcripts in a t(11q13)-associated leukemia. DNA from four of these 40 patients showed rearrangement of the 3′ UTR of CCND1 coexisting with major translocation cluster (MTC) rearrangement. Southern blot and sequence analyses showed that, as a result of these rearrangements, the 3′ AU- rich region containing sequences involved in mRNA stability and in translational control is eliminated. Moreover, the finding that the CCND1 mRNA half-life was greater than 3 hours (normal tissues, 0.5 hours) in three t(11q13)-associated cell lines stresses the importance of posttranscriptional derangement in the activation of CCND1. Finally, we did not observe any mutation in the coding frame of the CCND1 cDNA analyzed.
Increased expression of the G gamma and A gamma globin genes associated with a mutation in the A gamma enhancer
Authors: Balta G , Brickner HE , Takegawa S , Kazazian HH , Papayannopoulou T , Forget BG , Atweh GF
Published: October 2019

We have previously described a unique type of delta beta-thalassemia in a Chinese family characterized by increased expression of the G gamma and A gamma fetal globin genes in the absence of a large deletion in the beta-globlin gene cluster. Our earlier study of the beta-globin gene on this delta beta-thalassemia chromosome showed a promoter mutation in the TATA box. In this report, we describe the results of our study of the fetal globin domain of this delta beta-thalassemia chromosome. We have cloned a 13-kb DNA fragment that includes the G gamma and the A gamma genes and the 3′ A gamma enhancer element of this delta beta-thalassemia chromosome. DNA sequence analysis of the G gamma and A gamma-globin genes including their promoters did not show any mutations, but analysis of the putative enhancer element downstream from the A gamma-globin gene showed a C to T substitution 2,401 nucleotides downstream from the A gamma cap site. We performed DNA linkage analysis to determine if this mutation is unique to this chromosome or represents a common polymorphism. Our linkage analysis showed that this mutation is not a common polymorphism and that it is also not an intrinsic part of the haplotype of the chromosome on which it was found. We also studied the interaction of nuclear proteins from erythroid and nonerythroid cells with the DNA sequences surrounding this mutation. We have shown by in vitro DNase I footprinting that this mutation falls within a region that is occupied by a novel DNA-binding protein that binds to this site in nuclear extracts from erythroid, but not nonerythroid cells. The binding of this nuclear protein to DNA appears to be dependent on GATA-1 binding to an adjacent GATA-1 site. We have also developed a new functional assay to compare the activity of the normal and mutant A gamma enhancer elements in erythroid cells. Analysis of the activity of the mutant enhancer shows that the mutation completely eliminates all enhancer activity in this assay. These findings suggest that this mutation of the A gamma enhancer on a chromosome that carries a partially inactivated beta-globin gene may be responsible for the increased expression of both gamma-globin genes seen in this condition.
Patient characteristics associated with successful mobilizing and autografting of peripheral blood progenitor cells in malignant lymphoma
Authors: Haas R , Mohle R , Fruhauf S , Goldschmidt H , Witt B , Flentje M , Wannenmacher M , Hunstein W
Published: October 2019

For patients with advanced-stage or poor-prognosis malignant lymphoma, high-dose therapy with peripheral blood progenitor cell (PBPC) support may become a first-line treatment. The duration of severe cytopenia in this setting is inversely related to the number of PBPCs autografted. In a retrospective analysis, we therefore looked for factors influencing the yield of PBPCs in 61 patients (16 with high-grade and 29 with low-/intermediate-grade non-Hodgkin's lymphoma [NHL], and 16 with Hodgkin's disease) who received cytotoxic chemotherapy and filgrastim (R-metHuG-CSF, 300 micrograms/d; median, 4.2 micrograms/kg/d; range, 2.7 to 6.6 micrograms/kg/d; subcutaneously). Sixteen patients had active disease, while 45 were in partial remission (PR) or complete remission (CR) after conventional therapy. A median of three leukaphereses (range, one to 10) resulted in a median of 5.7 x 10(6) CD34+ cells/kg (range, 0.03 to 31.1 x 10(6)). Previous cytotoxic chemotherapy and irradiation adversely affected the yield of CD34+ cells. Each cycle of chemotherapy is associated with an average decrease of 0.2 x 10(6) CD34+ cells/kg per leukapheresis in nonirradiated patients, while large-field radiotherapy reduces the collection efficiency by an average of 1.8 x 10(6)/kg CD34+ cells. The collection efficiency was also significantly lower in patients with Hodgkin's disease. However, except for one, all had been previously irradiated. In contrast, age, sex, disease status, bone marrow involvement during mobilization, and the time since the last chemotherapy or radiotherapy were not significantly related to the collection efficiency. Following high-dose conditioning therapy, 42 patients were autografted with filgrastim-mobilized PBPCs. Hematological recovery (neutrophils > or = 0.5 x 10(9)/L and an unsupported platelet count > or = 20 x 10(9)/L) within 2 weeks was observed in patients autografted with > or = 2.5 x 10(6) CD34+ cells/kg. In seven patients, the quantity of CD34+ cells reinfused was below this threshold. They required a median of 17 days (range, 11 to 34) and 31 days (range, 13 to 141) for neutrophil and platelet recovery, respectively. If autografting with PBPCs in malignant lymphoma with poor prognosis is being considered, mobilization and harvesting should be planned early after initial diagnosis to avoid exhaustion of hematopoiesis by cumulative toxicity.
Kinetics of committed and primitive blood progenitor mobilization after chemotherapy and growth factor treatment and their use in autotransplants
Authors: Sutherland HJ , Eaves CJ , Lansdorp PM , Phillips GL , Hogge DE
Published: October 2019

Peripheral blood cells (PBCs) collected by leukapheresis after progenitor mobilization with chemotherapy and growth factors have been used successfully to replace marrow autografts in protocols requiring stem-cell support. Moreover, such transplants are often associated with more rapid recovery of blood cell counts than is routinely achieved with bone marrow. While conditions that mobilize colony-forming cells (CFCs) into the circulation are becoming increasingly well characterized, little information is available as to how these or other mobilizing treatments may influence the release of more primitive cells into the peripheral blood. To quantitate the peripheral blood content of such cells, we used the long-term culture-initiating cell (LTC-IC) assay, which detects a cell type that is able to produce progeny CFCs after a minimum of 5 weeks in cultures containing marrow fibroblasts. In this report, we present the findings on 21 patients who were transplanted over a 7-year period at our institution with PBCs alone. PBCs were collected in steady-state (n = 6) or during the recovery phase after high-dose cyclophosphamide (Cy; n = 15, nine with and six without additional growth factor administration). PBCs collected from another 11 patients given granulocyte colony-stimulating factor (G-CSF) were transplanted together with autologous marrow. Time-course studies of nine patients after Cy +/- granulocyte-macrophage CSF (GM-CSF) showed that CD34+ cells, CFCs, and LTC-ICs fell from normal to undetectable levels after Cy, and increased at the time of white blood cell (WBC) recovery: LTC-ICs to a mean of sixfold and CFCs to a mean of 26-fold higher than normal. The mean number of CD34+ cells, CFCs, and LTC-ICs present in the PBC harvest was twofold to 10-fold higher after mobilization than in steady-state collections; however, more than 2-log interpatient variability was observed. After PBC transplantation, the median time to a WBC count more than 10(9)/L was 12 days; polymorphonuclear leukocyte (PMN) count more than 0.5 x 10(9)/L, 15 days; and platelet count more than 20 x 10(9)/L, 17 days, although patients who received fewer than 1.5 x 10(5) CFCs/kg had a more than 50% chance of delayed count recovery (> 28 days). Patients who received Cy + GM-CSF-stimulated PBCs had more rapid and consistent platelet recoveries as compared with other groups receiving Cy mobilized or steady-state PBCs alone, and a rapid WBC recovery after Cy predicted a rapid WBC recovery after transplantation.
The der(11)-encoded MLL/AF-4 fusion transcript is consistently detected in t(4;11)(q21;q23)-containing acute lymphoblastic leukemia
Authors: Downing JR , Head DR , Raimondi SC , Carroll AJ , Curcio-Brint AM , Motroni TA , Hulshof MG , Pullen DJ , Domer PH
Published: October 2019

The t(4;11)(q21;q23) is the most common translocation involving band 11q23 and is found predominantly in acute lymphoblastic leukemias (ALLs) of infants. Recent studies have shown that this translocation involves the MLL gene on chromosome 11 and the AF-4 gene on chromosome 4. Using oligonucleotide primers derived from these genes, we established reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of the fusion transcripts from both the der(11) and der(4) chromosomes. Using these assays we analyzed 23 pediatric cases of t(4;11) containing ALL. RT-PCR analysis for the der(11)-derived MLL/AF-4 fusion transcript resulted in its detection in every case at a sensitivity of greater than 1 leukemic cell in 10(5) cells. Sequence analysis of MLL/AF-4 PCR products demonstrated fusion mRNAs resulting from breaks in MLL introns 6, 7, or 8, with alternative splicing to one of three exons in the AF-4 gene. In contrast, analysis for the der(4)-derived transcript resulted in the detection of this chimeric mRNA in only 84% of the cases analyzed. These data suggest that the critical chimeric gene product involved in the establishment of the leukemic clone is derived from the der(11) chromosome. Moreover, these data demonstrate the utility of the RT-PCR assay for the der(11)- encoded message both for diagnosing t(4;11)-containing leukemia and for monitoring patients for minimal residual disease.
Reductions in physiologic shear rates lead to CD11/CD18-dependent, selectin-independent leukocyte rolling in vivo
Authors: Gaboury JP , Kubes P
Published: October 2019

In this study, we tested the hypothesis that reducing shear rates in postcapillary venules causes CD18-dependent, selectin-independent leukocyte rolling. Intravital microscopy was used to assess shear rate- dependent leukocyte rolling in 25- to 40-microns rat mesenteric venules. Pretreatment of animals with 25 mg/kg fucoidin, a carbohydrate moiety that binds to and inhibits selectin function, essentially abolished the number of spontaneously rolling leukocytes. When shear rates were reduced by 50% (from 438 +/- 36 s-1 to 222 +/- 19 s-1) in the presence of fucoidin, leukocyte rolling increased fourfold, suggesting a selectin-independent mechanism of leukocyte rolling. Administration of CL26, an anti-CD18 antibody, prevented the leukocyte rolling associated with reduced shear rates. A second objective was to determine if the integrin-mediated leukocyte rolling at reduced shear rates would lead to firm adhesion of leukocytes in the presence of a chemotactic stimulus. Animals were pretreated with fucoidin and 100 nmol/L platelet-activating factor (PAF) was superfused over the mesentery. Fucoidin prevented leukocyte rolling and subsequent PAF- induced adhesion at normal shear rates; however, when shear rates were reduced by 50%, a significant CD18-dependent increase in leukocyte rolling (10-fold) and adhesion (5-fold) was noted within 15 minutes. These data raise the possibility that, at lower shear rates, as is the case in various inflammatory conditions, selectin-independent, CD18- dependent leukocyte rolling and subsequent adhesion can occur in postcapillary venules.
Platelets inhibit fibrinolysis in vitro by both plasminogen activator inhibitor-1-dependent and -independent mechanisms
Authors: Fay WP , Eitzman DT , Shapiro AD , Madison EL , Ginsburg D
Published: October 2019

Platelet-rich thrombi are resistant to lysis by tissue-type plasminogen activator (t-PA). Although platelet alpha-granules contain plasminogen activator inhibitor-1 (PAI-1), a fast-acting inhibitor of t-PA, the contribution of PAI-1 to the antifibrinolytic effect of platelets has remained a subject of controversy. We recently reported a patient with a homozygous mutation within the PAI-1 gene that results in complete loss of PAI-1 expression. Platelets from this individual constitute a unique reagent with which to probe the role of platelet PAI-1 in the regulation of fibrinolysis. The effects of PAI-1-deficient platelets were compared with those of normal platelets in an in vitro clot lysis assay. Although the incorporation of PAI-1-deficient platelets into clots resulted in a moderate inhibition of t-PA-mediated fibrinolysis, normal platelets markedly inhibited clot lysis under the same conditions. However, no difference between PAI-1-deficient platelets and platelets with normal PAI-1 content was observed when streptokinase or a PAI-1-resistant t-PA mutant were used to initiate fibrinolysis. In addition, PAI-1-resistant t-PA was significantly more efficient in lysing clots containing normal platelets than wild-type t-PA. We conclude that platelets inhibit t-PA-mediated fibrinolysis by both PAI- 1-dependent and PAI-1-independent mechanisms. These results have important implications for the role of PAI-1 in the resistance of platelet-rich thrombi to lysis in vivo.
A phase I/II study of sequential interleukin-3 and granulocyte- macrophage colony-stimulating factor in myelodysplastic syndromes
Authors: Nand S , Sosman J , Godwin JE , Fisher RI
Published: October 2019

In this phase I/II study, 9 patients with myelodysplastic syndromes (MDS) were treated with interleukin-3 (IL-3) followed by granulocyte- macrophage colony-stimulating factor (GM-CSF). Each treatment cycle was 28 days long and administered as follows: 1 microgram/kg/d IL-3 on days 1 through 7 and 3 micrograms/kg/d GM-CSF for days 8 through 21, followed by a 7-day rest period. IL-3 dose escalations were planned, but the dose of GM-CSF was fixed. Three patients had refractory anemia, 4 had refractory anemia with ringed sideroblasts, and 2 had refractory anemia with excess blasts. Six patients were dependent on red blood cell transfusions, 1 on platelet transfusions, and 2 on both. The absolute neutrophil count improved in 7 (77%) patients and the platelet count improved in 3 (33%) patients during therapy. Hemoglobin levels were unchanged. A clinically relevant response was seen in only 1 patient with thrombocytopenia, and he received five cycles of therapy. The neutrophil count decreased in 2 patients and the platelet count decreased in 4 patients during treatment. The toxicity of the treatment was significant. In the first cohort of 3 patients, 1 patient developed supraventricular tachycardia and congestive heart failure. In the second group, 1 patient developed progressive granulocytopenia and died of gram-negative septicemia. Because of the disparate toxicity, 3 more patients were treated at the same dose level. One of these experienced a high fever and bone pain requiring hospitalization. Because of these adverse effects, the IL-3 dose was not escalated and all patients received 1 microgram/kg/d for 7 days. We believe that sequential therapy with IL-3 and GM-CSF at these dose levels causes unacceptable toxicity in patients with MDS. The major toxic events occurred during weeks 4 and 5 after starting treatment and may have been primarily caused by GM-CSF therapy. Although neutrophil counts improve in most patients, the effect on red blood cells and platelets is minimal. At present, this form of therapy remains problematic and appears to have a limited potential in the management of MDS.
Structural changes in platelet glycoprotein IIb/IIIa by plasmin: determinants and functional consequences
Authors: Pasche B , Ouimet H , Francis S , Loscalzo J
Published: October 2019

Plasmin exposure modulates platelet aggregation responses, but a direct effect of plasmin on the platelet fibrinogen receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), has never been conclusively shown in a plasma milieu. To examine this issue, we incubated platelets in platelet-rich plasma with plasmin and measured the effect of this treatment on platelet aggregation, fibrinogen binding, and the structural integrity of GPIIb/IIIa. Plasmin treatment reduced maximal reversible fibrinogen binding in a dose-dependent fashion, and this reduction in binding was accompanied by a correlative reduction in the maximal rate of aggregation. Immunoblots performed with polyclonal antibodies against GPIIb/IIIa showed that GPIIIa had been cleaved by plasmin, but this cleavage was detected only after subsequent degradation of the solubilized GPIIb/IIIa with Staphylococcus aureus V8 (Glu-C) endoprotease. Peptide sequence analysis showed that cleavage occurred at the lys444-pro445 bond in the first cysteine-rich repeat domain of GPIIIa a unique proteolytic event observed only in the presence of plasma fibrinogen. These observations suggest that plasmin modifies GPIIIa by a unique proteolytic event in plasma that is dependent on fibrinogen binding and, consequently, is accompanied by significant reductions in fibrinogen binding and aggregation response.
Cyclosporin A and cyclosporin SDZ PSC 833 enhance anti-CD5 ricin A- chain immunotoxins in human leukemic T cells
Authors: Jaffrezou JP , Sikic BI , Laurent G
Published: October 2019

Recent studies have shown that cyclosporin A (CsA) may affect ricin A- chain immunotoxin (RTA-IT) therapy. In this study, we evaluated the ability of CsA and its nonimmunosuppressive analog, SDZ PSC 833, to enhance anti-CD5 T101 RTA-ITs in vitro. Both 4 mumol/L CsA and 4 mumol/L SDZ PSC 833 significantly and specifically enhanced the cytotoxic activity of T101 RTA-IT on the human lymphoblastic T-cell line, CEM III (101-fold and 105-fold, respectively). Furthermore, these Cs also enhanced the cytotoxicity of the more potent T101 F(ab')2 RTA- IT (ninefold and eightfold, respectively). The effect of human plasma, originating from four patients enrolled in a phase I high-dose CsA regimen, was examined on T101 RTA-IT cytotoxicity on CEM III cells. In each case, with plasma CsA levels between 3,090 and 4,860 ng/mL (2.5 to 4 mumol/L), a significant increase in T101 RTA-IT-mediated cytotoxicity was observed ranging from 31% to 60%. Neither CsA nor SDZ PSC 833 affected the rate of RTA-IT binding, internalization, intracellular trafficking, or degradation. Analysis of internalized T101 RTA-IT molecules showed that these were essentially intact, which suggests that these enhancers may act only on a small population of RTA-ITs that escapes present investigational techniques. In conclusion, because the concentrations used are clinically achievable, Cs appear to be promising agents for in vivo enhancement of RTA-ITs.
Autosomal recessive chronic granulomatous disease with absence of the 67-kD cytosolic NADPH oxidase component: identification of mutation and detection of carriers
Authors: de Boer M , Hilarius-Stokman PM , Hossle JP , Verhoeven AJ , Graf N , Kenney RT , Seger R , Roos D
Published: October 2019

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to kill certain bacteria and fungi. This is caused by deficiencies in one of the components of NADPH oxidase, the enzyme in phagocytic leukocytes that generates superoxide. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of NADPH oxidase (p67-phox) is missing. Until now, mutations in the gene coding for this protein have not been identified. We now report on a 10-year- old girl with lymph node and liver abscesses who was recognized as an A67(0) CGD patient by lack of NADPH oxidase activity in her granulocytes, a cytosolic defect in a cell-free oxidase system, and lack of immunoreactive material with an antiserum against the p67-phox protein. mRNA for this protein was present in normal amounts in her monocytes. This p67-phox mRNA was reverse-transcribed, and the coding region was amplified by polymerase chain reaction in six overlapping fragments and was sequenced. The patient appeared to be homozygous for a G-233-->A mutation, resulting in a nonconservative amino acid change (78Gly-->Glu). This mutation was also found in the genomic DNA of this patient but not in that of 38 normal donors. Both parents and a sister proved to be carriers of the disease, as deduced from the mutation in only one allele. The carrier state was also manifested by intermediate superoxide production by their intact granulocytes and in the cell-free system.
Serum erythropoietin and erythropoiesis in high- and low-fetal hemoglobin beta-thalassemia intermedia patients
Authors: Galanello R , Barella S , Turco MP , Giagu N , Cao A , Dore F , Liberato NL , Guarnone R , Barosi G
Published: October 2019

Clinical data suggest that in beta-thalassemia-intermedia patients, higher levels of circulating fetal hemoglobin (HbF) are associated with greater disease severity at comparable degrees of anemia. We assessed the influence of the amount of circulating HbF on serum erythropoietin (s-Epo) levels and on serum transferrin receptor, a measure of erythropoiesis, in 30 beta-thalassemia-intermedia patients. Twenty-four showed more than 40% HbF (21 of whom with beta (0)-thalassemia) and 6 presented lower HbF levels (beta(+)-thalassemia). The two groups of patients did not differ in age (15.3 v 19 years, respectively) or degree of anemia (Hb = 8.8 g/dL in both groups). Log (s-Epo) was correlated inversely with Hb (r = -0.47; P < .01), and directly with HbF (r = .55; P < .001). Multivariate regression analysis showed that Hb and HbF were independently correlated with s-Epo levels. High-HbF patients had greater s-Epo values at the same Hb level than low-HbF patients. Considering that iron-deficiency anemia control patients represented the predicted physiologic response of s-Epo to anemia, the observed/predicted s-Epo ratio in low-HbF thalassemic patients was no different from controls, but was increased in the high-HbF group. High- HbF patients also showed an expansion of erythropoiesis as much as four to nine times the normal value at the same Hb level as low-HbF patients. We conclude that HbF exerts an independent regulatory effect on erythropoietin production and erythropoiesis that is detectable only when HbF levels exceed 40%.
Oligoclonal expansion of CD8+ CD57+ T cells with restricted T-cell receptor beta chain variability after bone marrow transplantation
Authors: Gorochov G , Debre P , Leblond V , Sadat-Sowti B , Sigaux F , Autran B
Published: October 2019

A major expansion of CD8+57+ lymphocytes expressing an alpha-beta T- cell receptor (TCR) is frequent after bone marrow transplantation (BMT). We examined the clonality of the TCR beta gene repertoire in these expanded CD8+57+ cells after allogeneic or autologous BMT. We performed a polymerase chain reaction (PCR) analysis of the V beta chain usage in CD8+57+ cells purified from nine BMT recipients with a series of oligonucleotides specific for 20 V beta gene families. PCR products from selected TCR beta gene rearrangements were sequenced. The CD8+57+ cells from eight of nine patients used a restricted set of V beta families, with a marked predominance of two to three V beta gene families per patient, whereas the control autologous CD57- subset expressed the whole 20 V beta families. A direct sequencing analysis confirmed the V beta 16 and V beta 17 clonality in six patients, showing a striking homology in the CDR3 sequences of the V beta 16 products. The CD8+57+ cells, but not the CD57- cells, displayed an oligoclonal pattern of TCR rearrangements as shown by PCR analysis of TCR gamma gene rearrangements. Such an oligoclonal expansion of CD8+57+ cells, using a restricted set of the V beta gene families, may result from a specific TCR stimulation of a limited number of T-cell clones in BMT recipients.
Severe combined immunodeficiency, interleukin-2 (IL-2), and the IL-2 receptor: experiments of nature continue to point the way [see comments]
Authors: Voss SD , Hong R , Sondel PM
Published: October 2019

The recent discovery of molecular defects in three forms of X-linked immunodeficiency has quickly transformed the study of immunodeficiency into one of the most exciting in basic and clinical immunology. The identification of defects in the IL-2R gamma chain in the etiology of X- linked SCID has suggested a heretofore unanticipated functional role of the gamma chain in immunologic development. While new and novel cytokines and cytokine receptors continue to be identified, it has become clear that our knowledge of IL-2, one of the best understood cytokine/receptor systems, is far from complete. Clarifying the molecular interactions between IL-2 and its receptor complex will improve the sophistication with which these interactions are manipulated in the clinic for the treatment of autoimmune disorders and allograft rejection, treatment of lymphoid malignancies, and cytokine- based therapies for immunotherapeutic treatment of nonlymphoid cancers. Recent gene therapy approaches to the treatment of children with the ADA-deficient form of SCID offers yet another exciting path for investigation. The use of retrovirally infected cord blood hematopoietic progenitor cells in attempts to reconstitute the immune system of ADA-deficient SCID children with ADA-producing cells raises the possibility of similarly “correcting” the defect in X-linked SCID. Such approaches almost certainly loom on the near horizon for other diseases. However, in view of the complexity and potentially pleiomorphic nature of defects in the IL-2R gamma chain, both in terms of their identification and correction, gene therapy for treatment of X- linked SCID will require a thorough understanding of the molecular nature of the respective defects. Effective therapy will require precise knowledge of the defects, in terms of their influence on the ligand, receptor, and signaling apparatus, as well as their potential effects on cells of multiple lineages. However, these caveats aside, the potential for understanding and correcting a disease that robs infants at so early an age of the potential for a normal life will continue to make these exciting and extraordinarily rewarding pursuits.
CD44 mediates hyaluronan binding by human myeloid KG1A and KG1 cells
Authors: Morimoto K , Robin E , Le Bousse-Kerdiles MC , Li Y , Clay D , Jasmin C , Smadja- Joffe F
Published: October 2019

Hyaluronan-binding function of the CD44 molecule has not been so far detected in myeloid cells. To study pure populations of primitive myeloid cells, we investigated the hyaluronan-binding function of the CD44 molecule from three myeloid cell lines: KG1a, KG1, and HL60. Both KG1a and KG1 cells express the CD34 antigen characteristic of the hematopoietic stem cells and HL60 cells do not; accordingly, KG1a and KG1 cells are generally considered as the most primitive and HL60 cells as the most mature of these cell lines. Measurement of cell adhesion to hyaluronan-coated surfaces (using 51Cr-labeled cells) and of aggregate formation in hyaluronan-containing solutions, showed that 45% of KG1 cells and 22% to 24% of KG1a spontaneously bind to hyaluronan, whereas HL60 cells do not either spontaneously or after treatment with a phorbol ester. Hyaluronan binding by KG1a and KG1 cells is mediated by CD44, because it is specifically abolished by monoclonal antibodies (MoAbs) to this molecule. The binding might require phosphorylation by protein kinase C and perhaps also by protein kinase A, because it is prevented by staurosporine, which inhibits these enzymes. 12-O- tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C, rises to 80% the proportion of KG1 and KG1a cells that bind hyaluronan; this activation is dependent on protein synthesis, for it is abrogated by cyclophosphamide, a protein synthesis inhibitor. Binding of TPA- treated cells to hyaluronan is only partly inhibited by MoAb to CD44: this suggests that TPA may induce synthesis of a hyaluronan-binding protein distinct from CD44. Considering the abundance of hyaluronan in human bone marrow, these results suggest that CD44 may be involved in mediating precursor-stroma interaction.