bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Four Genes in Endothelial Cells Whose Expression Is Affected by Tumor Cells and Host Immune Status—A Study in Ex Vivo–Isolated Endothelial Cells
Authors: Liliensiek Birgit , Rocha Marian , Umansky Victor , Benner Axel , Lin Jie , Ziegler Reinhard , Nawroth Peter P. , Schirrmacher Volker
Published: October 2019

A spontaneously metastasizing, well-defined mouse lymphoma was chosen as an in vivo model to study the effect of tumor-host interaction on gene expression in liver sinusoidal endothelial cells. Forty-nine bovine aortic endothelial cell (BAEC) genes, recently isolated by a differential screening approach of a cDNA library enriched for tumor necrosis factor- (TNF-) suppressed genes, were investigated. Four of these genes were finally selected because they were affected differentially by host immuno-competence, TNF-, and tumor cells. Sequence analysis showed them to encode the bovine polyubiquitin (A4), elongation factor 1 (B2), the acidic ribosomal phosphoprotein PO (C3), and the ribosomal protein S2 (E10). Gene expression was analyzed by dot-blot or Northern blot analysis. TNF- and tumor cell conditioned supernatant suppressed the genes additive in BAEC but not in other endothelial cells except for bovine capillary endothelial cells. Ex vivo–isolated liver endothelial cells of tumor-bearing syngeneic DBA/2 mice showed strong downregulation of these four genes in comparison to normal control values. In contrast, endothelial cells of tumor-bearing immuno-incompetent Balb/c (nu/nu) mice showed no downregulation but upregulation of these genes. Consistently, all four genes were also downregulated when BAEC were incubated with supernatants derived from ex vivo–isolated liver metastases from immuno-competent but not from -incompetent mice. Thus, the expression of a group of genes involved in protein translation and processing was more profoundly altered in endothelial cells in vivo than in vitro, suggesting that microenviromental factors and cell-cell and cell-matrix interactions play an important role.

© 1998 by The American Society of Hematology.
Enhanced Adherence of Sickle Erythrocytes to Thrombin-Treated Endothelial Cells Involves Interendothelial Cell Gap Formation
Authors: Manodori Annamaria B. , Matsui Neil M. , Chen James Y. , Embury Stephen H.
Published: October 2019

The adherence of sickle erythrocytes to vascular endothelium has the capacity to initiate vasoocclusion. The known effects of thrombin on endothelial cell function and the increased activity of thrombin in sickle cell disease led us to examine the effect of thrombin on the adhesivity of cultured endothelial cells for sickle erythrocytes. In particular, we studied whether the effect of thrombin on interendothelial cell gap formation (ICGF) was involved in endothelial cell adhesivity for sickle erythrocytes. Those endothelial cell monolayers stimulated by thrombin to maximal levels of static sickle erythrocyte adherence also underwent striking cell contraction and enlargement of interendothelial cell gaps. Adhesivity also increased when gaps were induced with antilaminin antibodies or EDTA. Maximally adhesogenic thrombin conditions failed to increase adhesivity when gap formation was prevented by pretreatment of the monolayers with 8-bromo-cyclic adenosine monophosphate (bromo-cAMP) or glutaraldehyde, agents that respectively inhibit actin-myosin–dependent cell contraction or cross-link adjacent cells in the monolayer. The influence of these two agents on EDTA-enhanced adhesivity was linked to their ability to prevent gap formation. Glutaraldehyde prevented both increased adherence and gap formation; bromo-cAMP prevented neither. Interendothelial cell gap formation may contribute to vasoocclusion by facilitating sickle erythrocyte adherence.

© 1998 by The American Society of Hematology.
Chronic Hepatitis C Virus Infections in Leukemia Survivors: Prevalence, Viral Load, and Severity of Liver Disease
Authors: Paul Ian M. , Sanders Jeffrey , Ruggiero Francesca , Andrews Thomas , Ungar David , Eyster M. Elaine
Published: October 2019

The natural history of chronic hepatitis C (HCV) infections in long-term leukemia survivors has not been well characterized. We studied the prevalence of HCV infections, measured HCV RNA levels, and evaluated the severity of liver disease in patients with leukemia who achieved long-term remissions after intensive chemotherapy or bone marrow transplantation (BMT). HCV antibody tests were performed by the enzyme-linked immunosorbent assay (ELISA) and positive tests confirmed by the recombinant immunoblot assay (RIBA). HCV RNA levels were measured by the branched DNA (bDNA) assay. Seventy-five leukemia survivors with 25 or more blood donor exposures were identified. Nine (12%) were anti-HCV positive. All were infected before 1992 when second generation HCV screening tests were implemented. Mean HCV RNA levels were 10.3 ×106 eq/mL versus 3.2 × 106 eq/mL (P = .056) in a control group of 20 anti-HCV positive immunocompetent individuals of comparable age who were infected twice as long (17.8 ± 6.5 years v 9.0 ± 4.4 years in leukemia survivors, P = .001). Liver biopsies were performed on six of the nine anti-HCV positive leukemia survivors. All showed at least moderate portal inflammation and half had evidence of bridging fibrosis. We conclude that viral loads in anti-HCV positive leukemia survivors are markedly higher than in immunocompetent controls. Our results suggest that long-term leukemia survivors with chronic HCV may have more rapidly progressive liver disease than has been previously recognized.
Selective Expansion of Alveolar Macrophages In Vivo by Adenovirus-Mediated Transfer of the Murine Granulocyte-Macrophage Colony-Stimulating Factor cDNA
Authors: Worgall Stefan , Singh Ravi , Leopold Philip L. , Kaner Robert J. , Hackett Neil R. , Topf Norbert , Moore Malcolm A.S. , Crystal Ronald G.
Published: October 2019

Based on the hypothesis that genetic modification of freshly isolated alveolar macrophages (AM) with the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA would induce AM to proliferate, this study focuses on the ability of adenoviral (Ad) vectors to transfer and efficiently express the murine (m) GM-CSF cDNA in murine AM with consequent expansion in the number of AM in vitro and in vivo. To demonstrate that an Ad vector can effectively transfer and express genes in AM, murine AM recovered by bronchoalveolar lavage from the lung of Balb/c mice were infected with an Ad vector coding for green fluorescent protein (GFP) in vitro and expressed GFP in a dose-dependent fashion. Infection of AM with an Ad vector containing an expression cassette coding for mGM-CSF led to GM-CSF expression and to AM proliferation in vitro. When AM infected with AdGFP were returned to the respiratory tract of syngeneic recipient mice, GFP-expressing cells could still be recovered by bronchoalveolar lavage 2 weeks later. In vitro infection of AM with AdmGM-CSF and subsequent transplantation of the genetically modified AM to the lungs of syngeneic recipients led to GM-CSF expression in vivo. Strikingly, the AM recovered by lavage 5 weeks after transplantation demonstrated an increased rate of proliferation, and the total number of alveolar macrophages was 1.9-fold greater than controls. Importantly, the increase in the numbers of AM was selective (ie, other inflammatory cell numbers were unchanged), and there was no modification to the lung architecture. Thus, it is feasible to genetically modify AM with Ad vectors and to use this strategy to modify the behavior of AM in vivo. Based on the importance of AM in the primary defense of the respiratory epithelial surface, this strategy may be useful in enhancing pulmonary defenses in immunodeficiency states.
Augmented Proliferation of Human Alveolar Macrophages After Allogeneic Bone Marrow Transplantation
Authors: Nakata Koh , Gotoh Hajime , Watanabe Junichi , Uetake Takeshi , Komuro Iwao , Yuasa Kazumi , Watanabe Shinya , Ieki Ryuji , Sakamaki Hisashi , Akiyama Hideki , Kudoh Shohji , Naitoh Makoto , Satoh Hitoshi , Shimada Kaoru
Published: October 2019

After allogeneic bone marrow transplantation (allo-BMT), recipient alveolar macrophages (AM) are gradually replaced by AM of the donor origin. An influx of mononuclear phagocytes of donor origin to the lung is responsible for the repopulation, but the detailed kinetics remain unclear. We therefore studied 24 BMT recipients who underwent bronchoalveolar lavage (BAL) from 24 to 83 days after BMT. AM cell number, size, morphology, proliferating ability, and genotype of AM were measured. Before day 50, the number and size of AM in BAL fluid were similar to those of normal nonsmokers. However, after day 50, the mean number of AM increased threefold and the mean cell size decreased due to the increase of small AM. These small cells are presumably of donor origin based on DNA fingerprinting analysis and based on fluorescence in situ hybridization for the Y chromosome in a sex-mismatched case. Immunohistochemistry and cell cycle analysis demonstrated that the increase in AM number coincided with a remarkable increase of AM expressing proliferating cell nuclear antigen, suggesting that small AM are proliferating. This is the first report representing that augmented proliferation of donor AM in situ may contribute to the reconstitution of AM population after BMT.
Persistence and Clinical Outcome of Hepatitis G Virus Infection in Pediatric Bone Marrow Transplant Recipients and Children Treated for Hematological Malignancy
Authors: Yamada-Osaki Maki , Sumazaki Ryo , Tsuchida Masahiro , Koike Kazutoshi , Fukushima Takashi , Matsui Akira
Published: October 2019

The natural course and the clinical significance of hepatitis G virus (HGV) infection were investigated in 106 pediatric patients who received chemotherapy for hematological malignancy or underwent bone marrow transplantation (BMT) using HGV-RNA and antibodies to the HGV-E2 protein (anti-E2). HGV markers were detected in 21 patients (19.8%; HGV-RNA in 19 and anti-E2 in 2). Longitudinal analysis of these HGV-infected patients showed that 1 had anti-E2 before the initial blood transfusion, 14 had persistent viremia, and 6 became clear of circulating HGV-RNA after completion of therapy, although 5 of the 6 HGV-cleared patients never developed anti-E2. Reactivation of HGV infection during chemotherapy was observed in two anti-E2–positive, HGV-RNA–negative patients; the reappearance of the same HGV strain was confirmed by phylogenetic analysis. Among BMT survivors without other known causes of liver dysfunction, HGV-RNA–positive patients had a higher peak serum alanine amino transferase (ALT) value than negative patients. Contrary to previous reports, immunosuppressed patients can apparently recover from HGV infection without detectable anti-E2 and some patients who supposedly recovered from HGV infection can nonetheless suffer exacerbation when subsequently immunosuppressed.
Direct Alloreactivity by Human Cytotoxic T Lymphocytes Can Be Inhibited by Altered Peptide Ligand Antagonism
Authors: Burrows Scott R. , Khanna Rajiv , Moss Denis J.
Published: October 2019

Alloreactive T lymphocytes that respond directly to foreign major histocompatibility complex (MHC) molecules and bound peptide are known to be central mediators of graft-versus-host disease (GVHD) and allograft rejection. We have recently identified a peptide from the human protein, cytochrome P450 (isotypes IIC9, 10, or 18), that is recognized in association with human leukocyte antigen (HLA) B*3501 by alloreactive cytotoxic T lymphocytes (CTLs). These CTLs with this specificity were isolated from several unrelated individuals and were found to express a common T-cell receptor (TCR). Synthetic analogs of the cytochrome P450 peptide were generated by introducing single amino acid substitutions at putative TCR contact positions. Four altered peptide ligands were powerful competitive antagonists of these CTL clones, reducing lysis levels of target cells expressing the alloantigen HLA B*3501 by over 80%. This first demonstration that it is possible to suppress CTL alloreactivity with structural variants of allodeterminants raises the prospect that such TCR antagonists could be exploited within the clinical arena to specifically modulate GVHD and allograft rejection.
Reconstitution of the Functional Mouse Oncostatin M (OSM) Receptor: Molecular Cloning of the Mouse OSM Receptor β Subunit
Authors: Tanaka Minoru , Hara Takahiko , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Miyajima Atsushi
Published: October 2019

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines that share the gp130 receptor subunit. Of these family members, leukemia inhibitory factor (LIF) is most closely related to OSM, and various overlapping biologic activities have been described between human LIF and OSM (hLIF and hOSM). Two types of functional hOSM receptors are known: the type I OSM receptor is identical to the LIF receptor that consists of gp130 and the LIF receptor β subunit (LIFRβ), and the type II OSM receptor consists of gp130 and the OSM receptor β subunit (OSMRβ). It is thus conceivable that common biologic activities between hLIF and hOSM are mediated by the shared type I receptor and OSM-specific activities are mediated by the type II receptor. However, in contrast to the human receptors, recent studies have demonstrated that mouse OSM (mOSM) does not activate the type I receptor and exhibits unique biologic activity. To elucidate the molecular structure of the functional mOSM receptor, we cloned a cDNA encoding mOSMRβ, which is 55.5% identical to the hOSMRβ at the amino acid level. mOSM-responsive cell lines express high-affinity mOSM receptors, as well as mOSMRβ, whereas embryonic stem cells, which are responsive to LIF but not to mOSM, do not express mOSMRβ. mOSMRβ alone binds mOSM with low affinity (kd = 13.0 nmol/L) and forms a high-affinity receptor (kd = 606 pmol/L) with gp130. Ba/F3 transfectants expressing both mOSMRβ and gp130 proliferated in response to mOSM, but failed to respond to LIF and human OSM. Thus, the cloned mOSMRβ constitutes an essential and species-specific receptor component of the functional mOSM receptor. Reminiscent of the colocalization of the mOSM and mLIF genes, the mOSMRβ gene was found to be located in the vicinity of the LIFRβ locus in the proximal end of chromosome 15.
Combined Genotypes of CCR5, CCR2, SDF1, and HLA Genes Can Predict the Long-Term Nonprogressor Status in Human Immunodeficiency Virus-1–Infected Individuals
Authors: Magierowska Magdalena , Theodorou Ioannis , Debré Patrice , Sanson Françoise , Autran Brigitte , Rivière Yves , Charron Dominique , ALT French , Groups IMMUNOCO Study , Costagliola Dominique
Published: October 2019

Human immunodeficiency virus (HIV)-1–infected long-term nonprogressors (LT-NP) represent less than 5% of HIV-1–infected patients. In this work, we tried to understand whether combined genotypes of CCR5-▵32, CCR2-64I, SDF1-3′A and HLA alleles can predict the LT-NP status. Among the chemokine receptor genotypes, only the frequency of the CCR5-▵32 allele was significantly higher in LT-NP compared with the group of standard progressors. The predominant HLA alleles in LT-NP were HLA-A3, HLA-B14, HLA-B17, HLA-B27, HLA-DR6, and HLA-DR7. A combination of both HLA and chemokine receptor genotypes integrated in a multivariate logistic regression model showed that if a subject is heterozygous for CCR5-▵32 and homozygous for SDF1 wild type, his odds of being LT-NP are increased by 16-fold, by 47-fold when a HLA-B27 allele is present with HLA-DR6 absent, and by 47-fold also if at least three of the following alleles are present: HLA-A3, HLA-B14, HLA-B17, HLA-DR7. This model allowed a correct classification of 70% of LT-NPs and 81% of progressors, suggesting that the host’s genetic background plays an important role in the evolution of HIV-1. The chemokine receptor and chemokine genes along with the HLA genotype can serve as predictors of HIV-1 outcome for classification of HIV-1–infected subjects as LT-NPs or progressors.
Downregulation of Interleukin-12 (IL-12) Responsiveness in Human T Cells by Transforming Growth Factor-β: Relationship With IL-12 Signaling
Authors: Pardoux Cécile , Ma Xiaojing , Gobert Stéphanie , Pellegrini Sandra , Mayeux Patrick , Gay Françoise , Trinchieri Giorgio , Chouaib Salem
Published: October 2019

Interleukin-12 (IL-12) is a cytokine that plays a central role in the control of cell-mediated immunity. We have previously shown that transforming growth factor-β1 (TGF-β) inhibitory effects on human primary allogeneic cytotoxicity and proliferative responses interfere with IL-12 pathway. The present study was undertaken to further elucidate the biochemical basis of the functional interaction between these two cytokines and to define the site of TGF-β action on the signaling pathway activated by IL-12. Our data indicate that TGF-β induced an inhibition of interferon-γ (IFN-γ) production without affecting the IL-12Rβ1 and IL-12Rβ2 subunits mRNA expression by activated T cells. We further show that TGF-β has a significant inhibitory effect on the early signal transduction events following interaction of IL-12 with its receptor on activated T cells, resulting in the inhibition of both JAK2 and Tyk2 phosphorylation. In addition, TGF-β was found to significantly inhibit IL-12–induced phosphorylation of the STAT4 transcription factor. Electrophoretic mobility shift assay indicated that TGF-β induced a decrease in IL-12–induced STAT4 DNA binding activity in T lymphocytes. This study suggests that TGF-β influences IL-12 responsiveness at least in part by inhibiting early signaling events essential to gene induction in IL-12–activated T cells.
Susceptibility to Childhood Acute Lymphoblastic Leukemia: Influence of CYP1A1, CYP2D6, GSTM1, and GSTT1 Genetic Polymorphisms
Authors: Krajinovic Maja , Labuda Damian , Richer Chantal , Karimi Sepideh , Sinnett Daniel
Published: October 2019

Although acute lymphoblastic leukemia (ALL) is the most common childhood cancer, factors governing susceptibility to this disease have not yet been identified. As such, ALL offers a useful opportunity to examine the glutathione S-transferase and cytochrome P450 genes in determining susceptibility to pediatric cancers. Both enzymes are involved in carcinogen metabolism and have been shown to influence the risk a variety of solid tumors in adults. To determine whether these genes played a similar role in childhood leukemogenesis, we compared the allele frequencies of 177 childhood ALL patients and 304 controls for the CYP1A1, CYP2D6, GSTM1, and GSTT1 genes. We chose the French population of Quebec as our study population because of its relative genetic homogeneity. The GSTM1 null and CYP1A1*2A genotypes were both found to be significant predictors of ALL risk (odds ratio [OR] = 1.8). Those possessing both genotypes were at an even greater risk of developing the disease (OR = 3.3). None of the other alleles tested for proved to be significant indicators of ALL risk. Unexpectedly, girls carrying the CYP1A1∗4 were significantly underrepresented in the ALL group (OR = 0.2), suggesting that a gender-specific protective role exists for this allele. These results suggest that the risk of ALL may indeed be associated with xenobiotics-metabolism, and thus with environmental exposures. Our findings may also explain, in part, why ALL is more prevalent among males than females.
Autografting With Philadelphia Chromosome–Negative Mobilized Hematopoietic Progenitor Cells in Chronic Myelogenous Leukemia
Authors: Carella Angelo M. , Lerma Enrica , Corsetti Maria T. , Dejana Anna , Basta Palmina , Vassallo Franca , Abate Monica , Soracco Monica , Benvenuto Federica , Figari Osvaldo , Podestà Marina , Piaggio Giovanna , Ferrara Raimondo , Sessarego Mario , Parodi Caterina , Pizzuti Michele , Rubagotti Alessandra , Occhini Domenico , Frassoni Francesco
Published: October 2019

Intensive chemotherapy given in early chronic phase of chronic myelogenous leukemia (CML) has resulted in high numbers of circulating Philadelphia (Ph) chromosome–negative hematopoietic progenitor cells (HPC). We have autografted 30 consecutive patients with CML in chronic phase with HPC collected in this way to facilitate restoration of Ph-negative hematopoiesis in bone marrow after high-dose therapy. Hematopoietic recovery to greater than 0.5 ×109/L neutrophils and to greater than 25 × 109/L platelets occurred in all patients, a median of 13 (range, 9 to 32) days and 16 (range, 6 to 106) days postautograft, respectively. Regenerating marrow cells were Ph-negative in 16 (53%) patients and greater than 66% Ph-negative in 10 (33%) patients. Twenty-eight patients are alive 6 to 76 months (median, 24 months) after autografting. Three patients have developed blast crisis from which 2 have died. Eight patients are in complete cytogenetic remission at a median of 20 (range, 6 to 44) months with a median ratio BCR-ABL/ABL of 0.002 (range, <0.001 to 0.01). Eight patients are in major cytogenetic remission at a median of 22 (range, 6 to 48) months. No patient died as a consequence of the treatment. All patients had some degree of stomatitis that was severe in 15 (50%) patients. Gastrointestinal and hepatic toxicities were observed in about one fourth of patients. Thus, autografting with Ph-negative mobilized HPC can result in prolonged restoration of Ph-negative hematopoiesis for some patients with CML; moreover, most autograft recipients report normal or near normal activity levels, suggesting that this procedure need not to be associated either with prolonged convalescence or with chronic debility.
Role of Folylpolyglutamate Synthetase and Folylpolyglutamate Hydrolase in Methotrexate Accumulation and Polyglutamylation in Childhood Leukemia
Authors: Rots Marianne G. , Pieters Rob , Peters Godefridus J. , Noordhuis Paul , van Zantwijk Christina H. , Kaspers Gertjan J.L. , Hählen Karel , Creutzig Ursula , Veerman Anjo J.P. , Jansen Gerrit
Published: October 2019

Inefficient polyglutamylation is a mechanism of resistance to methotrexate (MTX) in childhood T-lineage acute lymphoblastic leukemia (T-ALL) and in acute myeloid leukemia (AML) in comparison with childhood c/preB-ALL. We analyzed the profile of MTX polyglutamylation in childhood c/preB-ALL, T-ALL, and AML (n = 45, 15, and 14, respectively), the activity of the MTX-polyglutamate synthesizing enzyme folylpolyglutamate synthetase (FPGS) (n = 39, 11, and 19, respectively) and of the MTX-polyglutamate breakdown enzyme folylpolyglutamate hydrolase (FPGH) (n = 98, 25, and 34, respectively). MTX-Glu4-6 accumulation after 24 hours exposure to 1 μmol/L [3H]-MTX in vitro was lower in T-ALL (threefold) and AML (fourfold) compared with c/preB-ALL (P ≤ .001). The FPGS activity was twofold lower in T-ALL and AML than in c/preB-ALL samples (P < .01). FPGH activity was not different between c/preB-ALL and T-ALL, but threefold higher in AML (P < .001). FPGS, FPGH, and the ratio FPGS/FPGH were correlated with MTX-Glu4-6 accumulation (r = .49, r = −.34 and r = .61, respectively). Multivariate analysis showed that FPGS, but not FPGH, was an independent contributor for MTX-Glu1-6 accumulation, but not for MTX-Glu4-6 accumulation. In conclusion, low FPGS activity is associated with low accumulation of MTX-Glu4-6 in T-ALL and AML. For the group of AML as compared with the group of ALL, a high FPGH activity can play an additional role.
Overexpression of the Receptor for Hyaluronan-Mediated Motility (RHAMM) Characterizes the Malignant Clone in Multiple Myeloma: Identification of Three Distinct RHAMM Variants
Authors: Crainie Mary , Belch Andrew R. , Mant Michael J. , Pilarski Linda M.
Published: October 2019

The receptor for hyaluronan (HA)-mediated motility (RHAMM) controls motility by malignant cells in myeloma and is abnormally expressed on the surface of most malignant B and plasma cells in blood or bone marrow (BM) of patients with multiple myeloma (MM). RHAMM cDNA was cloned and sequenced from the malignant B and plasma cells comprising the myeloma B lineage hierarchy. Three distinct RHAMM gene products, RHAMMFL, RHAMM−48, and RHAMM−147, were cloned from MM B and plasma cells. RHAMMFL was 99% homologous to the published sequence of RHAMM. RHAMM−48 and RHAMM−147 variants align with RHAMMFL, but are characterized by sequence deletions of 48 bp (16 amino acids [aa]) and 147 bp (49 aa), respectively. The relative frequency of these RHAMM transcripts in MM plasma cells was determined by cloning of reverse-transcriptase polymerase chain reaction (RT-PCR) products amplified from MM plasma cells. Of 115 randomly picked clones, 49% were RHAMMFL, 47% were RHAMM−48, and 4% were RHAMM−147. All of the detected RHAMM variants contain exon 4, which is alternatively spliced in murine RHAMM, and had only a single copy of the exon 8 repeat sequence detected in murine RHAMM. RT-PCR analysis of sorted blood or BM cells from 22 MM patients showed that overexpression of RHAMM variants is characteristic of MM B cells and BM plasma cells in all patients tested. RHAMM also appeared to be overexpressed in B lymphoma and B-chronic lymphocytic leukemia (CLL) cells. In B cells from normal donors, RHAMMFL was only weakly detectable in resting B cells from five of eight normal donors or in chronically activated B cells from three patients with Crohn’s disease. RHAMM−48 was detectable in B cells from one of eight normal donors, but was undetectable in B cells of three donors with Crohn’s disease. RHAMM−147 was undetectable in normal and Crohn’s disease B cells. In situ RT-PCR was used to determine the number of individual cells with aggregate RHAMM transcripts. For six patients, 29% of BM plasma cells and 12% of MM B cells had detectable RHAMM transcripts, while for five normal donors, only 1.2% of B cells expressed RHAMM transcripts. This work suggests that RHAMMFL, RHAMM−48, and RHAMM−147 splice variants are overexpressed in MM and other B lymphocyte malignancies relative to resting or in vivo–activated B cells, raising the possibility that RHAMM and its variants may contribute to the malignant process in B-cell malignancies such as lymphoma, CLL, and MM.
Heparin-Binding Epidermal Growth Factor–Like Growth Factor/Diphtheria Toxin Receptor Expression by Acute Myeloid Leukemia Cells
Authors: Vinante Fabrizio , Rigo Antonella , Papini Emanuele , Cassatella Marco A. , Pizzolo Giovanni
Published: October 2019

Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is an EGF family member expressed by numerous cell types that binds to EGF receptor 1 (HER-1) or 4 (HER-4) inducing mitogenic and/or chemotactic activities. Membrane-bound HB-EGF retains growth activity and adhesion capabilities and the unique property of being the receptor for diphtheria toxin (DT). The interest in studying HB-EGF in acute leukemia stems from these mitogenic, chemotactic, and receptor functions. We analyzed the expression of HB-EGF in L428, Raji, Jurkat, Karpas 299, L540, 2C8, HL-60, U937, THP-1, ML-3, and K562 cell lines and in primary blasts from 12 acute myeloid leukemia (AML) cases, by reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blot and by the evaluation of sensitivity to DT. The release of functional HB-EGF was assessed by evaluation of its proliferative effects on the HB-EGF–sensitive Balb/c 3T3 cell line. HB-EGF was expressed by all myeloid and T, but not B (L428, Raji), lymphoid cell lines tested, as well as by the majority (8 of 12) of ex vivo AML blasts. Cell lines (except for the K562 cell line) and AML blasts expressing HB-EGF mRNA underwent apoptotic death following exposure to DT, thus demonstrating the presence of the HB-EGF molecule on their membrane. Leukemic cells also released a fully functional HB-EGF molecule that was mitogenic for the Balb/c 3T3 cell line. Factors relevant to the biology of leukemic growth, such as tumor necrosis factor- (TNF-), 1,25-(OH)2D3, and especially all-trans retinoic acid (ATRA), upregulated HB-EGF mRNA in HL-60 or ML-3 cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced HB-EGF mRNA and acquisition of sensitivity to DT in one previously HB-EGF–negative leukemia case. Moreover, the U937 and Karpas 299 cell lines expressed HER-4 mRNA. This work shows that HB-EGF is a growth factor produced by primary leukemic cells and regulated by ATRA, 1,25-(OH)2D3, and GM-CSF.
Filgrastim for Cladribine-Induced Neutropenic Fever in Patients With Hairy Cell Leukemia
Authors: Saven Alan , Burian Carol , Adusumalli Jasvant , Koziol James A.
Published: October 2019

Cladribine treatment of hairy cell leukemia (HCL) is complicated by neutropenic fever in 42% of patients despite documented infections being relatively uncommon. We performed a study of priming filgrastim followed by cladribine and then filgrastim again to determine if filgrastim would lead to a reduction of neutropenia and febrile episodes. Thirty-five patients received filgrastim and cladribine and were compared with 105 historic controls treated with cladribine alone. Cladribine was administered at 0.1 mg/kg/d by continuous infusion for 7 days. Filgrastim was administered at 5 μg/kg/d subcutaneously on days −3, −2, and −1 and then again after the completion of cladribine until the absolute neutrophil count (ANC) was ≥2 × 109/L on 2 consecutive days (days +8, +9, etc). After filgrastim priming, the median ANC increased from 0.9 × 109/L to 2.26 × 109/L (2.5-fold increase), and after cladribine, the median nadir ANC in the filgrastim-treated group was 0.53 × 109/L compared with 0.29 × 109/L among historic controls (P = .04). The median number of days to an ANC greater than 1.0 × 109/L was 9 days in the filgrastim-treated group versus 22 days among historic controls (P < 10−5). The percentage of febrile patients, number of febrile days, and frequency of admissions for antibiotics were not statistically different in the two groups. Filgrastim regularly increases the ANC in patients with HCL and shortens the duration of severe neutropenia after cladribine. This phase II study, with comparison to historical controls, failed to detect any clinical advantage from the use of filgrastim and cladribine in the treatment of HCL. Accordingly, the routine adjunctive use of filgrastim with cladribine in the treatment of HCL cannot be recommended.
Clinical Characteristics and Outcome of Young Chronic Lymphocytic Leukemia Patients: A Single Institution Study of 204 Cases
Authors: Mauro Francesca R. , Foa Robert , Giannarelli Diana , Cordone Iole , Crescenzi Sabrina , Pescarmona Edoardo , Sala Roberta , Cerretti Raffaella , Mandelli Franco
Published: October 2019

A retrospective analysis on chronic lymphocytic leukemia (CLL) patients ≤55 years observed at a single institution was performed with the purpose of characterizing the clinical features and outcome of young CLL and of identifying patients with different prognostic features. Over the period from 1984 to 1994, 1,011 CLL patients (204 [20%] ≤55 years of age and 807 [80%] >55 years of age) were observed. At diagnosis, younger and older patients displayed a similar distribution of clinical features, except for a significantly higher male/female ratio in younger patients (2.85 v 1.29;P < .0001). Both groups showed an elevated rate of second primary cancers (8.3% v 10.7%), whereas the occurrence of Richter’s syndrome was significantly higher in younger patients (5.9% v 1.2%; P < .00001). Younger and older patients showed a similar overall median survival probability (10 years) but were characterized by a different distribution of causes of deaths: CLL unrelated deaths and second primary malignancies predominated in the older age group, whereas the direct effects of leukemia were prevalent in the younger age group. Although younger and older patients displayed a similar survival, the evaluation of the relative survival rates showed that the disease had a greater adverse effect on the expected survival probability of the younger population. Multivariate analysis showed that for young CLL patients only dynamic parameters, such as lymphocyte doubling time and other signs of active disease, were the independent factors that significantly influenced survival probability (P = .00001). A prolonged clinico-hematologic follow-up allowed us to identify two subsets of young CLL patients with a different prognostic outcome: a group of patients (40%) with long-lasting stable disease without treatment and an actuarial survival probability of 94% at 12 years from diagnosis and another group (60%) with progressive disease and a median survival probability of 5 years after therapy. For the latter patients, the therapeutic effect of innovative therapies with curative intents needs to be investigated in prospective, comparative clinical trials.
Microsatellite Instability and p53 Mutations Are Associated With Abnormal Expression of the MSH2 Gene in Adult Acute Leukemia
Authors: Zhu Y.-M. , Das-Gupta E.P. , Russell N.H.
Published: October 2019

Microsatellite instability (MSI) and p53 mutations have been reported to occur in a significant proportion of patients with therapy-related acute myeloid leukemia (AML). MSH2 is one of the genes involved in DNA mismatch repair to maintain fidelity of genomic replication, and defects of MSH2 are directly involved in MSI in hereditary nonpolyposis colorectal tumors and other human tumors. We have examined the expression of MSH2 protein by Western blotting in 43 adult leukemia samples, including 42 AML and 1 acute lymphoblastic leukemia (ALL) using the antibody MSH2 (Ab-1) (Calbiochem, La Jolla, CA). Abnormal expression of MSH2 protein was found in 14 of 43 (32.6%) cases; a control antibody to actin was always positive. Of the 14 patients that had abnormal expression of MSH2, 2 had therapy-related acute leukemia and 9 were elderly patients (>60 years of age). Expression of MSH2 mRNA was further examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Deletion of MSH2 mRNA was found in 1 of 14 cases with deficient MSH2 protein expression. This group of patients was also screened for loss of heterozygosity (LOH) at the MSH2 locus using a panel 4 microsatellite markers (D2S367, D2S288, D2S391, and D2S2294). LOH was found in 5 of 11 cases examined. There was no evidence of LOH in 14 patients with normal MSH2 expression who were examined using the same markers. Functional evidence for defective DNA mismatch repair in leukemic cells lacking MSH2 as manifest by MSI was found in 7 of 11 cases studied. Mutations of the p53 gene in these 43 samples were also investigated by direct sequencing of full-length p53 cDNA. Mutations of p53 were found in 6 of 43 cases, including 5 of the 14 (35.7%) cases that did not express MSH2 protein. In contrast, mutation of p53 was only found in 1 of 29 (3.4%) cases with normal MSH2 protein expression (χ2 = 5.720, P < .02). These results suggest that abnormalities of DNA mismatch repair due to defective MSH2 expression could play a key role in leukemogenesis, in particular in AML arising in elderly patients or secondary to previous chemotherapy.
Keratinocyte Growth Factor Separates Graft-Versus-Leukemia Effects From Graft-Versus-Host Disease
Authors: Krijanovski Oleg I. , Hill Geoffrey R. , Cooke Kenneth R. , Teshima Takanori , Crawford James M. , Brinson Yani S. , Ferrara James L.M.
Published: October 2019

The major obstacles to successful outcome after allogeneic bone marrow transplantation (BMT) for leukemia remain graft-versus-host disease (GVHD) and leukemic relapse. Improved survival after BMT therefore requires more effective GVHD prophylaxis that does not impair graft-versus-leukemia (GVL) effects. We studied the administration of human recombinant keratinocyte growth factor (KGF) in a well- characterized murine BMT model for its effects on GVHD. KGF administration from day -3 to +7 significantly reduced GVHD mortality and the severity of GVHD in the gastrointestinal (GI) tract, reducing serum lipopolysaccharide (LPS) and tumor necrosis factor (TNF) levels, but preserving donor T-cell responses (cytotoxic T lymphocyte [CTL] activity, proliferation, and interleukin [IL]-2 production) to host antigens. When mice received lethal doses of P815 leukemia cells at the time of BMT, KGF treatment significantly decreased acute GVHD compared with control-treated allogeneic mice and resulted in a significantly improved leukemia-free survival (42%v 4%, P < .001). KGF administration thus offers a novel approach to the separation of GVL effects from GVHD.
Activation of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-3 Receptor Subunits in a Multipotential Hematopoietic Progenitor Cell Line Leads to Differential Effects on Development
Authors: Evans Caroline A. , Pierce Andrew , Winter Sandra A. , Spooncer Elaine , Heyworth Clare M. , Whetton Anthony D.
Published: October 2019

Activation of specific cytokine receptors promotes survival and proliferation of hematopoietic progenitor cells but their role in the control of differentiation is unclear. To address this issue, the effects of human interleukin-3 (hIL-3) and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) on hematopoietic development were investigated in hematopoietic progenitor cells. Murine multipotent factor-dependent cell-Paterson (FDCP)-mix cells, which can self-renew or differentiate, were transfected with the genes encoding the unique  and/or shared βc human hIL-3 receptor (hIL-3 R) or hGM-CSF receptor (hGM R) subunits by retroviral gene transfer. Selective activation of hIL-3 R,βc or hGM R,βc transfects by hIL-3 and hGM-CSF promoted self-renewal and myeloid differentiation, respectively, over a range of cytokine (0.1 to 100 ng/mL) concentrations. These qualitatively distinct developmental outcomes were associated with different patterns of protein tyrosine phosphorylation and, thus, differential signaling pathway activation. The cell lines generated provide a model to investigate molecular events underlying self-renewal and differentiation and indicate that the  subunits act in combination with the hβc to govern developmental decisions. The role of the  subunit in conferring specificity was studied by using a chimeric receptor composed of the extracellular hIL-3 R and intracellular hGM R subunit domains. This receptor promoted differentiation in response to hIL-3. Thus, the  subunit cytosolic domain is an essential component in determining cell fate via specific signaling events.
Growth Characteristics of Acute Myelogenous Leukemia Progenitors That Initiate Malignant Hematopoiesis in Nonobese Diabetic/Severe Combined Immunodeficient Mice
Authors: Ailles Laurie E. , Gerhard Brigitte , Kawagoe Hiroyuki , Hogge Donna E.
Published: October 2019

The use of immunodeficient mice, particularly of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) strain, has allowed detection of very primitive malignant progenitors from patients with acute myelogenous leukemia (AML). To define the sensitivity and reproducibility with which the engraftment of different AML cells can be detected, 61 different samples from patients with newly diagnosed AML representing a variety of cytogenetic and French-American-British (FAB) subtypes were injected into NOD/SCID mice. Eight weeks after intravenous injection of 107 AML cells, the average percent of human cells in mouse bone marrow was 13.3%, with 70% of samples showing easily detectable engraftment of CD45+ cells. AML samples with cytogenetic changes associated with a poor clinical prognosis tended to engraft to higher levels than those with changes associated with a good prognosis. Cells with FAB subtypes M3 and, to a lesser extent, M2, engrafted more poorly (P = .002 and .06, respectively) than those from other subtypes. Intraperitoneal injection of human interleukin-3 and Steel factor thrice weekly for 4 weeks did not enhance the levels of AML cell engraftment. However, AML samples that showed cytokine-independent colony growth in methylcellulose assay or expressed growth-factor mRNA in malignant blasts achieved significantly higher levels of engraftment than those which were cytokine dependent in culture or failed to express cytokine message (P < .03 and P < .02, respectively). In 6 patient samples, the frequency of NOD/SCID leukemia-initiating cells (NOD/SL-IC) varied from 0.7 to 45 per 107 cells, which was 200- to 800-fold lower than the frequency of AML long-term culture-initiating cells (AML LTC-IC) in the same samples. Each NOD/SL-IC will produce more than 106leukemic blasts as well as many AML-CFC and AML LTC-IC as detected 8 weeks postinjection into mice. Serial transplant experiments showed the ability of NOD/SL-IC to maintain their own numbers over at least 3 to 4 weeks in vivo. The ability of these progenitors to self-renew combined with their potential to differentiate to produce large numbers of more mature progenitors and leukemic blasts suggests that the NOD/SL-IC assay identifies leukemic ‘stem cells’ that may maintain the malignant clone in human patients. The further use of this assay should facilitate studies of AML stem cell biology and the evolution of novel therapeutic strategies.
Enhanced B7-2 Gene Expression by Interferon-γ in Human Monocytic Cells Is Controlled Through Transcriptional and Posttranscriptional Mechanisms
Authors: Curiel R.E. , Garcia C.S. , Rottschafer S. , Bosco M.C. , Espinoza-Delgado I.
Published: October 2019

B7-2 is a costimulatory molecule expressed on professional antigen-presenting cells that provides T cells with a critical signal resulting in T-cell activation. Interferon-γ (IFN-γ) enhances B7-2 protein expression in monocytic cells. However, the molecular mechanisms controlling the enhanced expression of B7-2 are poorly understood. Northern blot and flow cytometry analysis revealed that human monocytes and the human monocytic cell line MonoMac6 (MM6) constitutively expressed B7-2 mRNA and protein and IFN-γ treatment further enhanced the expression of both molecules. The ability of IFN-γ to enhance B7-2 mRNA was evident at the dose of 31 U/mL and reached plateau levels at 500 U/mL. The effects of IFN-γ on B7-2 mRNA expression were time dependent and occurred within 3 hours of treatment and increased through 24 hours. In vitro transcription assays and mRNA stability experiments showed that IFN-γ increases both transcriptional activity and the stability of B7-2 mRNA. Treatment of MM6 cells with cycloheximide showed that de novo protein synthesis was not required for the IFN-γ–enhanced expression of B7-2 mRNA. Overall, these studies show for the first time that IFN-γ–enhanced expression of B7-2 protein in human monocytic cells is controlled at the gene level through a dual mechanism involving transcriptional and posttranscriptional mechanisms.
Functional Association of FcɛRIγ With Arginine632 of Paired Immunoglobulin-Like Receptor (PIR)-A3 in Murine Macrophages
Authors: Taylor Lynn S. , McVicar Daniel W.
Published: October 2019

Paired immunoglobulin-like receptors (PIR) are expressed on B cells and macrophages and include inhibitory and putative activating receptors referred to as PIR-B and PIR-A, respectively. Although PIR-B’s inhibitory pathway has been described, it is unknown whether PIR-A receptors can deliver activation signals to macrophages, and if so, through what mechanism. Here we use chimeric receptors to address the mechanisms of PIR-A signaling. Cotransfection of chimeric receptors comprised of the extracellular region of human CD4 and the transmembrane and cytoplasmic domains of murine PIR-A3 showed the ability of PIR-A3 to physically interact with the FcɛRIγ chain in 293T cells. This interaction is dependent on Arg632 within the PIR-A3 transmembrane domain. We also demonstrate PIR-A3 interaction with the endogenous FcɛRIγ of the ANA-1 macrophage cell line, again in an Arg632-dependent manner. Furthermore, we show that crosslinking of these chimeric receptors synergizes with IFN-γ in the production of nitric oxide. Our data are the first to show the potential of PIR-A3 to deliver activation signals to macrophages and establish its dependence on Arg632. These findings suggest that further study of the PIR-A receptors should be aggressively pursued toward a complete understanding of the intricate regulation of macrophage biology.
Increased Sensitivity to Complement and a Decreased Red Blood Cell Life Span in Mice Mosaic for a Nonfunctional Piga Gene
Authors: Tremml G. , Dominguez C. , Rosti V. , Zhang Z. , Pandolfi P.P. , Keller P. , Bessler M.
Published: October 2019

The gene PIGA encodes one of the protein subunits of the 1-6-N acetylglucosaminyltransferase complex, which catalyses an early step in the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. PIGA is somatically mutated in blood cells from patients with paroxysmal nocturnal hemoglobinuria (PNH), leading to deficiency of GPI-linked proteins on the cell surface. To investigate in detail how inactivating mutations of the PIGA gene affect hematopoiesis, we generated a mouse line, in whichloxP-mediated excision of part of exon 2 occurs on the expression of Cre. After crossbreeding with EIIa-cre transgenic mice, recombination occurs early in embryonic life. Mice that are mosaics for the recombined Piga gene are viable and lack GPI-linked proteins on a proportion of circulating blood cells. This resembles the coexistence of normal cells and PNH cells in patients with an established PNH clone. PIGA(−) blood cells in mosaic mice have biologic features characteristic of those classically seen in patients with PNH, including an increased sensitivity toward complement mediated lysis and a decreased life span in circulation. However, during the 12-month follow-up, the PIGA(−) cell population did not increase, clearly showing that a Piga gene mutation is not sufficient to cause the human disease, PNH.
Transgenic Analysis of a 100-kb Human β-Globin Cluster–Containing DNA Fragment Propagated as a Bacterial Artificial Chromosome
Authors: Kaufman Richard M. , Pham Christine T.N. , Ley Timothy J.
Published: October 2019

To date, the normal transcriptional regulation of the human β-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human β-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human β-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F1 mice showed that 2 lines express human β globin at levels approximately equivalent to the endogenous mouse β-major genes. One line expresses no human β globin, while the remaining 6 lines show intermediate expression levels. Complete γ→β-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic→adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size.