bluuebunny/crossref_metadata_2025 huggingface dataset:
Sialyl Lewisx (sLex) and an sLexMimetic, CGP69669A, Disrupt E-Selectin–Dependent Leukocyte Rolling In Vivo
Authors: Norman Keith E. , Anderson Gary P. , Kolb Hartmut C. , Ley Klaus , Ernst Beat
Published: October 2019

Leukocyte rolling is the earliest observable event in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewisx (sLex) is required for function. Despite findings that sLex inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin–dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNFα)-stimulated mouse cremaster is increased following treatment with either sLex or the sLex-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti–P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin–dependent) rolling is unaffected by either sLex or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.
Evidence of a Local Mechanism for Desmopressin-Induced Tissue-Type Plasminogen Activator Release in Human Forearm
Authors: Wall Ulrika , Jern Sverker , Tengborn Lilian , Jern Christina
Published: October 2019

Systemic administration of desmopressin (DDAVP) induces increased plasma levels of tissue-type plasminogen activator (t-PA), coagulation factor VIII, and von Willebrand factor (vWF). However, the mechanisms behind these responses are not known. We tested the hypothesis that DDAVP acts as a local stimulator of acute endothelial release of t-PA and vWF independently of central pathways. Healthy, young, nonsmoking male volunteers were studied. In a first study (n = 7), DDAVP and placebo were administered as randomized single-blind stepwise intrabrachial artery infusions (0.7, 7.0, and 70 ng/min). In a another subset of subjects (n = 4), a constant-rate DDAVP infusion of 70 ng/min was administered for 20 minutes in the brachial artery of the nondominant arm with the dominant arm as control. To rule out that the observed t-PA release was flow-dependent, 4 additional subjects received stepwise intra-arterial infusions of both DDAVP (7.0, 21, and 70 ng/min) and sodium nitroprusside (SNP; 0.5, 2.5, and 10 μg/min). Brachial venoarterial plasma concentration gradients and forearm plasma flow were used to determine net release/uptake rates of t-PA and vWF. At baseline, the average net release rate of t-PA was 6.7 ng/min across the whole forearm vascular bed, whereas there was no detectable basal release of vWF. Stepwise infusion of DDAVP induced a massive regulated release of t-PA with a peak after 15 minutes on the highest dose-step (ANOVA; P < .0001). The average maximum net release rate was 178 ng/min, and the total amount of t-PA released was, on the average, 3,000 ng. The majority was released in its active form. Constant-rate DDAVP infusion again markedly increased t-PA release in the infusion arm but had no effect whatsoever in the control arm. In contrast, DDAVP did not stimulate a local release of vWF in either study. Central hemodynamics were unchanged during infusions despite a local vasodilatory response with DDAVP. Endothelium-independent flow stimulation by SNP did not elicit any local t-PA release. We conclude that DDAVP induces a massive acute flow-independent release of t-PA, without the simultaneous release of vWF, in the human forearm vascular bed. The lack of a t-PA response in the control arm, as well as the unaltered central hemodynamics with DDAVP, confirms that the observed regulated t-PA release is local and independent of central mechanisms.
The Low-Density Lipoprotein Receptor-Related Protein (LRP) Mediates Clearance of Coagulation Factor Xa In Vivo
Authors: Narita Masaaki , Rudolph Amy E. , Miletich Joseph P. , Schwartz Alan L.
Published: October 2019

Blood coagulation factor X plays a pivotal role in the clotting cascade. When administered intravenously to mice, the majority of activated factor X (factor Xa) binds to α2-macroglobulin (α2M) and is rapidly cleared from the circulation into liver. We show here that the low-density lipoprotein receptor-related protein (LRP) is responsible for factor Xa catabolism in vivo. Mice overexpressing a 39-kD receptor-associated protein that binds to LRP and inhibits its ligand binding activity displayed dramatically prolonged plasma clearance of 125I-factor Xa. Preadministration of α2M-proteinase complexes (α2M*) also diminished the plasma clearance of125I-factor Xa in a dose-dependent fashion. The clearance of preformed complexes of 125I-factor Xa and α2M was similar to that of 125I-factor Xa alone and was also inhibited by mice overexpressing a 39-kD receptor-associated protein. These results thus suggest that, in vivo, factor Xa is metabolized via LRP after complex formation with α2M.
Heterologous Promoters Fused to BCL6 by Chromosomal Translocations Affecting Band 3q27 Cause Its Deregulated Expression During B-Cell Differentiation
Authors: Chen Weiyi , Iida Shinsuke , Louie Diane C. , Dalla-Favera Riccardo , Chaganti R.S.K.
Published: October 2019

The BCL6 gene encodes a POZ/Zinc-finger protein, which acts as a sequence-specific transcriptional repressor. It is expressed in B cells within the germinal centers (GC) and is required for GC formation. In ≈40% of diffuse large cell lymphomas (DLCL) and ≈14% of follicular lymphomas (FL), the BCL6 gene is rearranged by chromosomal translocations, which juxtapose heterologous promoters and 5′ untranslated sequences derived from other chromosomes to the BCL6 coding domain or by mutations in the 5′ regulatory region. To understand the functional consequence of the chromosomal translocations, we have studied the patterns of expression of the promoters found juxtaposed to BCL6 in DLCL and FL during B-lineage differentiation. Distinct heterologous 5′ untranslated regions (IGH, IGL, TTF) were identified fused to the BCL6 coding domain by analysis ofBCL6 cDNAs in two DLCL cases and one mixed follicular lymphoma (MxFL). These three sequences, as well as three other previously identified BCL6 fusion partners (IGHG3, BOB1,H4), were studied for their pattern of expression during B-lineage differentiation by Northern blot analysis of B-cell lines representative of the pre-B, B, immunoblast, and plasma cell stages. In contrast to BCL6, whose transcription is activated only in B cells within the GC, all of the other sequences displayed a broader pattern of expression ranging from constitutive expression throughout B-cell differentiation to persistent expression in immunoblasts and plasma cells. These results indicate that the expression ofBCL6 is deregulated as a consequence of fusion to heterologous promoter regions. The persistent expression of activated BCL6may contribute to lymphomagenesis by blocking B-cell differentiation within the GC.
The Metastatic Characteristics of Murine Lymphoma Cell Lines In Vivo Are Manifested After Target Organ Invasion
Authors: Aoudjit Fawzi , Potworowski Edouard F. , St-Pierre Yves
Published: October 2019

The ability of a tumor cell to survive is critical for successful dissemination to sites distant from the primary tumor. Tumor cells must enter blood circulation, resist hemodynamic shear stress of the blood circulation, successfully extravasate, and then migrate through dense tissue stroma to a site favorable for tumor growth. Some tumor cells must therefore be endowed with peculiar abilities to successfully metastasize, whereas others, although capable of forming tumor in specific organs, cannot metastasize. This property has often been associated with the homing ability of a given tumor cell, likely through the expression of organ-specific homing receptors that are critical for the extravasation process. The present work was aimed at establishing the point at which metastatic and nonmetastatic lymphoma cells diverge. Although 164T2 and 267T2 lymphoma cell lines can successfully form thymic lymphoma when injected intrathymically, only the 164T2 clone can efficiently form tumor in kidneys, spleen, and liver after intravenous inoculation. Using the Indium-labeling technique to monitor the homing kinetic of both cell lines, we showed that the critical step for the successful metastasis of the lymphoma cell was determined in the final steps of the disseminating process, namely after homing. These results indicate that, whereas binding of tumor cells to vascular endothelium through specific adhesion mechanisms is a prerequisite for dissemination of tumor cells, the resistance of a tumor cell to the antagonist action of the host and/or its ability to grow tumor occurs only after homing to the target organ.
Suppression of Cell Proliferation and the Expression of abcr-abl Fusion Gene and Apoptotic Cell Death in a New Human Chronic Myelogenous Leukemia Cell Line, KT-1, by Interferon-α
Authors: Yanagisawa Kohsuke , Yamauchi Hayato , Kaneko Masahiko , Kohno Hidehisa , Hasegawa Hitoshi , Fujita Shigeru
Published: October 2019

A new human leukemia cell line, KT-1, was established from a patient in the blastic crisis phase of chronic myelogenous leukemia (CML). This cell line had a positive reaction for intracytoplasmic myeloperoxidase and two Philadelphia chromosomes (Ph1) [t(9;22)(q34;q11)] and lacked normal copies of chromosomes 9 and 22. Molecular characterization of the breakpoint in the t(9;22)(q34;q11) showed that KT-1 had a bcr-2/abl-2 splice junction. When the KT-1 cells were cultured with interferon (IFN)-α or IFN-γ, the growth of the cells were dose-dependently suppressed. IFN-α and IFN-γ exerted synergistic suppressive effects on the growth of KT-1 cells. Furthermore, IFN-α suppressed the expression of the bcr-ablfusion gene in KT-1 cells, and induced G1 cell-cycle arrest and apoptotic cell death. The KT-1 cell line should be a valuable tool for studying the molecular mechanism of the suppression of Ph1clone cells from CML by IFN.
Acute Regulation of Glucose Transport After Activation of Human Peripheral Blood Neutrophils by Phorbol Myristate Acetate, fMLP, and Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Tan An S. , Ahmed Nuzhat , Berridge Michael V.
Published: October 2019

Activation of human peripheral blood neutrophils by pathogens or by phorbol myristate acetate (PMA), fMLP, or myeloid growth factors generates a respiratory burst in which superoxide production plays an important role in killing invading microorganisms. Although the increased energy demands of activated neutrophils would be expected to be associated with increased glucose uptake and utilization, previous studies have shown that PMA inhibits 2-deoxyglucose (2-DOG) uptake. In this study, we show that PMA activation of neutrophils, isolated by methods not involving hypotonic lysis, increases the rate of 2-DOG uptake and results in a 1.6-fold to 2.1-fold increase in transporter affinity for glucose without changing Vmax. Increased transporter affinity in response to PMA was also observed with 3-O-methyglucose, which is not phosphorylated, and inclusion of glucose in the activation medium further increased respiratory burst activity. Increased 2-DOG uptake and increased transporter affinity for glucose were also observed with the peptide activator, fMLP, and with granulocyte-macrophage colony-stimulating factor (GM-CSF). The protein kinase C (PKC) inhibitor, calphostin C, and the tyrosine kinase inhibitor, genistein, inhibited both PMA- and fMLP-stimulated 2-DOG uptake. In contrast, genistein inhibited fMLP-induced superoxide production, but had little effect on the PMA-induced response, while staurosporine differentially inhibited PMA-induced superoxide production. These results show that neutrophil activation involves increased glucose transport and intrinsic activation of glucose transporter molecules. Both tyrosine kinases and PKC are implicated in the activation process.
A Naturally Occurring Mutation in FcγRIIA: A Q to K127 Change Confers Unique IgG Binding Properties to the R131 Allelic Form of the Receptor
Authors: Norris Cynthia F. , Pricop Luminita , Millard Sean S. , Taylor Scott M. , Surrey Saul , Schwartz Elias , Salmon Jane E. , McKenzie Steven E.
Published: October 2019

FcγRIIa is widely expressed on hematopoietic cells. There are two known allelic polymorphic forms of FcγRIIa, FcγRIIa-R131 and FcγRIIa-H131, which differ in the amino acid at position 131 in the second Ig-like domain. In contrast to FcγRIIa-R131, FcγRIIa-H131binds hIgG2 but not mIgG1, and this differential binding has clinical implications for host defense, autoimmune disease, immunohematologic disease, and response to therapeutic monoclonal antibodies. We identified a novel FcγRIIA genotype in a healthy individual homozygous for FcγRIIA R/R131 in whom a C to A substitution at codon 127 changes glutamine (Q) to lysine (K) in one of the two FcγRIIA genes. This individual's homozygosity for FcγRIIA-R/R131 leads to the prediction that the receptors on her cells would not bind hIgG2. Monocyte and neutrophil phagocytosis of hIgG2-opsonized erythrocytes was significantly higher (P < .05) for cells from this K/Q127, R/R131 individual than for Q/Q127, R/R131 donors. Platelet aggregation stimulated by an mIgG1 anti-CD9 antibody in this individual was significantly different (P < .05) from Q/Q127, H/R131 and Q/Q127, H/H131 donors and similar to Q/Q127, R/R131. Our data show that the K127/R131 receptors have a unique phenotype, binding both hIgG2 and mIgG1. Further functionally significant mutations in human Fcγ receptors and possible novel mechanisms for inherited differences in disease susceptibility should be sought with unbiased screening methods.
Mice Transgenic for the Human CGM6 Gene Express Its Product, the Granulocyte Marker CD66b, Exclusively in Granulocytes
Authors: Eades-Perner Anne-Marie , Thompson John , van der Putten Herman , Zimmermann Wolfgang
Published: October 2019

The nonspecific cross-reacting antigen-95 (NCA-95/CD66b), is a member of the human carcinoembryonic antigen (CEA) family encoded by the CGM6 gene that is exclusively expressed in neutrophils and eosinophils. No murine counterpart is known to exist. We have analyzed a cosmid containing the complete CGM6 gene. The coding sequence is contained within six exons spanning a 16.5 kb region. The main transcriptional start site was mapped to a tight cluster between nucleotides -95 and -101 relative to the translational start site. As with other members of the CEA gene family, no typical TATA or CAAT-box sequences were found in the CGM6 gene. Transgenic mice were established with the cosmid insert. CD66b expression is first seen in the fetal liver on day 12.5 of mouse embryonic development, and it first appears in the bone marrow at day 17.5. Northern blot analysis showed that CD66b transcripts are confined to the bone marrow of adult mice, whereas immunohistochemistry also showed CD66b-positive granulocytes in the spleen, thymus, and lungs. FACScan analyses of bone marrow and spleen cells showed CD66b expression to be exclusive to granulocytes. Thus, all the elements necessary for regulating granulocyte-specific expression are present within this cosmid clone. These mice could provide a model for transplantation and for inflammation studies using CD66b as a granulocyte-specific marker.
Dominantly Transmitted β-Thalassemia Arising From the Production of Several Aberrant mRNA Species and One Abnormal Peptide
Authors: Faustino Paula , Osório-Almeida Leonor , Romão Luı́sa , Barbot José , Fernandes Berta , Justiça Benvindo , Lavinha João
Published: October 2019

We describe a dominantly inherited β-thalassemia intermedia phenotype observed in a five-generation Portuguese family. Carriers are characterized by moderate anemia, hypochromia, microcytosis, elevated hemoglobin (Hb)A2 and HbF levels, splenomegaly, hepatomegaly, and inclusion bodies in pheripheral red blood cells after splenectomy. The molecular basis of this condition is a small deletion within the 5′ consensus splicing sequence of the second intron of the β-globin gene, IVS-II-4,5 (-AG). Reticulocyte RNA studies performed by reverse transcription-polymerase chain reaction (RT-PCR) and primer extension analysis showed three abnormally processed transcripts, which, upon sequencing, were shown to correspond to (1) skipping of exon 2, and (2) activation of two cryptic splice sites (between codons 59/60, and at IVS-II-47). In vitro translation studies of these patients' reticulocyte RNA have shown that at least one of these aberrant mRNA species is translated into an abnormally elongated peptide whose cytotoxic properties could, in part, be causing the atypical dominant mode of inheritance observed in this family. We suggest that this elongated β chain is unable to combine with an α-globin chain to form a functional Hb molecule. Its degradation would, then, exhaust the proteolytic defense mechanism of the erythroid precursors, leading to inefficient proteolysis of the free α chains in excess.
Absence of Immunogenicity of Diaspirin Cross-Linked Hemoglobin in Humans
Authors: Patel Mehul J. , Webb Edwin J. , Shelbourn Tina E. , Mattia-Goldberg Cynthia , George Andrew J.T. , Zhang Feng , Moore Edwin G. , Nelson Deanna J.
Published: October 2019

Diaspirin cross-linked hemoglobin (DCLHb) is an intramolecularly cross-linked hemoglobin-based oxygen carrier being developed as a therapy for acute blood loss. We report here the absence of immunogenicity of DCLHb in patients enrolled in phase II and III clinical trials of DCLHb. Two very sensitive immunoassays, an enzyme-linked immunosorbent assay (ELISA) and a Western blot assay, were developed and validated for this assessment. The DCLHb-antibodies used in these assays were raised in monkeys, had similar affinities for DCLHb and native human hemoglobin (SFHb), and showed cross-reactivity for subunits of DCLHb and SFHb on the Western blot, suggesting that these antibodies were elicited as a xenogenic response to the protein. In the ELISA, the optical density of a patient sample exposed to DCLHb-coated wells was compared with that of the patient sample exposed to carbonate buffer-coated wells; an optical density ratio of 1.4 was established for discriminating between a positive (reactive) or negative DCLHb antibody response. To date, all of the more than 300 patient specimens (preinfusion and postinfusion) from clinical trials have exhibited a ratio of less than 1.4, confirming the lack of preexisting antibodies to DCLHb and clearly showing the absence of DCLHb antibodies after exposure to this new biologic entity. There has been no requirement for use of the confirmatory Western blot assay. Taken together, the results from this study indicate DCLHb is not immunogenic in humans at doses evaluated clinically.
Expression of Granulocyte-Macrophage Colony-Stimulating Factor Receptors in Human Prostate Cancer
Authors: Rivas Coralia I. , Carlos Vera Juan , Delgado-López Fernando , Heaney Mark L. , Guaiquil Victor H. , Zhang Rong H. , Scher Howard I. , Concha Ilona I. , Nualart Francisco , Cordon-Cardo Carlos , Golde David W.
Published: October 2019

We studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, α and β. While the isolated α subunit binds GM-CSF at low-affinity, the isolated β subunit does not bind GM-CSF by itself; but complexes with the α subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the α and β subunits of the GM-CSF receptor in LNCaP cells, and the presence of the α and β proteins was confirmed by immunolocalization with anti-α and anti-β antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF α and β subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-α subunit antibodies were also reactive with anti-β subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.
Human Factor IX Corrects the Bleeding Diathesis of Mice With Hemophilia B
Authors: Kung Szu-Hao , Nathan Hagstrom J. , Cass Darrell , Jen Tai Shing , Lin Hui-Feng , Stafford Darrel W. , High Katherine A.
Published: October 2019

Mice with hemophilia B have been engineered using gene targeting techniques. These animals exhibit severe factor IX deficiency and a clinical phenotype that mirrors the human disease. We have bred the founder animals onto two different strains of mice, C57B1/6 and CD-1, and have sought to determine whether adenoviral vectors expressing human factor IX could correct the bleeding diathesis of mice with hemophilia B. Initial experiments showed that purified plasma-derived human factor IX added to murine factor IX–deficient plasma resulted in complete correction of the activated partial thromboplastin time (aPTT), and that injection of 1011 particles of an adenoviral vector expressing human factor IX resulted in normalization of a modified aPTT in mouse plasma. As an additional method of assessing the function of human factor IX in the murine coagulation system, bleeding times were performed in normal, hemophilic, and adenoviral-treated hemophilic mice. By two different bleeding-time techniques, the treated hemophilic mice gave values identical to normal littermate controls, whereas the untreated hemophilic mice exhibited heavy blood loss and prolonged bleeding. There was a marked difference in antibody formation in the two strains of mice; 100% of the hemophilic CD-1 mice formed antibodies to human factor IX, but none of the C57B1/6 mice did. These data suggest that the C57B1/6 hemophilic mice will be more useful for gene transfer studies, while the CD-1 hemophilic mice may be of greater utility in studying the development of inhibitors.
Deficient Heme and Globin Synthesis in Embryonic Stem Cells Lacking the Erythroid-Specific δ-Aminolevulinate Synthase Gene
Authors: Harigae Hideo , Suwabe Naruyoshi , Weinstock Peter H. , Nagai Mayumi , Fujita Hiroyoshi , Yamamoto Masayuki , Sassa Shigeru
Published: October 2019

The erythroid-specific isoform of δ-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for β-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.
Elevated Serum Concentrations of Hepatocyte Growth Factor in Patients With Multiple Myeloma
Authors: Seidel Carina , Børset Magne , Turesson Ingemar , Abildgaard Niels , Sundan Anders , e Nordic Myeloma Study Group Anders Waage for th
Published: October 2019

Serum from 398 myeloma patients at diagnosis and serial samples from 29 patients were analysed for hepatocyte growth factor (HGF). HGF was elevated at diagnosis in 43% of myeloma patients compared with healthy controls (median 1.00 ng/mL and 0.44 ng/mL, respectively;P < .00001). In the group with elevated HGF levels 46% of the patients reached plateau phase, as compared with 60% of the patients with low HGF levels (P = .005), and the median survival time was 21 and 32 months, respectively (P = .002). In a univariate Cox regression analysis, HGF was a significant predictor of mortality (P = .02). In the subgroup of patients with β2-microglobulin levels less than or equal to 6 mg/L, high versus low HGF was a prognostic factor when a multivariate Cox regression analysis was performed. In serial samples HGF was higher at the time of diagnosis and relapse (median 0.57 ng/mL and 0.52 ng/mL, respectively; P = .0018) than at response (median 0.24 ng/mL, P = .008). We conclude that HGF may be a useful follow-up parameter in myeloma patients. Measurement of HGF may identify a group of patients with poor response to melphalan-prednisone treatment and short survival. HGF was a prognostic factor in patients with high levels of β2-microglobulin.
Overexpression of Protein Kinase C Isoform  but not δ in Human Interleukin-3–Dependent Cells Suppresses Apoptosis and Induces bcl-2 Expression
Authors: Gubina E. , Rinaudo M.S. , Szallasi Z. , Blumberg P.M. , Mufson R.A.
Published: October 2019

Hematopoietic progenitor cells die by apoptosis after removal of the appropriate colony-stimulating factor (CSF). Recent pharmacologic data have implicated protein kinase C (PKC) in the suppression of apoptosis in interleukin-3 (IL-3) and granulocyte-macrophage (GM)-CSF–dependent human myeloid cells. Because IL-3 and GM-CSF induce increases in diacylglycerol without mobilizing intracellular Ca++, it seemed that one of the novel Ca++ independent isoforms of PKC was involved. We report here that overexpression of PKC in factor-dependent human TF-1 cells extends cell survival in the absence of cytokine. Overexpression of PKCδ does not have this effect. By 72 to 96 hours after cytokine withdrawal, the PKC transfectants remain distributed in all phases of the cell cycle, as shown by fluorescence-activated cell sorting (FACS) analysis, while little intact cellular DNA is detectable in vector or PKCδ transfectants. PKC induces bcl-2 protein expression fivefold to sixfold over the levels in empty vector transfectants, whereas the levels in PKCδ transfectants are similar to those in vector controls.
Cloning and Characterization of the Human Homolog of Mouse Jak2
Authors: Dalal Ilan , Arpaia Enrico , Dadi Harjit , Kulkarni Shaila , Squire Jerami , Roifman Chaim M.
Published: October 2019

Members of the Jak family play a critical role in signal transduction mediated by cytokine and hormone receptors. In this study, we report the cloning and characterization of human Jak2. The predicted amino acid sequence shows 91% homology to the described murine Jak2, but with a significant difference in the extreme C-terminal sequence. Using the human cDNA as a probe, we localized the gene for human Jak2 to chromosome 9p23-24. Human Jak2 mRNA is highly expressed in the spleen, lymph nodes, and peripheral blood lymphocytes (PBLs). A polyclonal antibody raised against the unique C-terminus of human Jak2 was used to characterize Jak2 protein. Levels of Jak2 protein expression increased significantly in mitogen- and anti-IgM–stimulated B cells and to a lesser degree in activated T cells. In addition, high levels of Jak2 protein were detected in pre-B leukemia cells.
Redundant and Selective Roles for Erythropoietin Receptor Tyrosines in Erythropoiesis In Vivo
Authors: Longmore Gregory D. , You Yun , Molden Jaime , Liu Kathleen D. , Mikami Aki , Lai Stephen Y. , Pharr Pamela , Goldsmith Mark A.
Published: October 2019

Cytokine receptors have been shown in cell culture systems to use phosphotyrosine residues as docking sites for certain signal transduction intermediates. Studies using various cellular backgrounds have yielded conflicting information about the importance of such residues. The present studies were undertaken to determine whether or not tyrosine residues within the erythropoietin receptor (EPOR) are essential for biologic activity during hematopoiesis in vivo. A variant of the EPOR was constructed that contains both a substitution (R129C) causing constitutive receptor activation as well as replacement of all eight cytoplasmic tyrosines by phenylalanines (cEPORYF). A comparison between animals exposed to recombinant retroviruses expressing cEPOR and cEPORYF showed that efficient red blood cell (RBC) development in vivo is dependent on the presence of tyrosine residues in the cytoplasmic domain of the EPOR. In addition, an inefficient EPOR tyrosine independent pathway supporting RBC development was detected. Tyrosine add-back mutants showed that multiple individual tyrosines have the capacity to restore full erythropoietic potential to the EPOR as determined in whole animals. The analysis of primary erythroid progenitors transduced with the various cEPOR tyrosine mutants and tyrosine add-backs showed that only tyrosine 343 (Y1) and tyrosine 479 (Y8) were capable of supporting immature burst-forming unit–erythroid progenitor development. Thus, this receptor is characterized by striking functional redundancy of tyrosines in a biologically relevant context. However, selective tyrosine residues may be uniquely important for early signals supporting erythroid development.
Signaling Through the Interaction of Membrane-Restricted Stem Cell Factor and c-kit Receptor Tyrosine Kinase: Genetic Evidence for a Differential Role in Erythropoiesis
Authors: Kapur Reuben , Majumdar Manus , Xiao Xiangli , McAndrews-Hill Monica , Schindler Karen , Williams David A.
Published: October 2019

Mutations of the receptor tyrosine kinase c-kit or its ligand stem cell factor (SCF), which is encoded as a soluble and membrane-associated protein by the Steel gene in mice, lead to deficiencies of germ cells, melanocytes, and hematopoiesis, including the erythroid lineage. In the present study, we have used genetic methods to study the role of membrane or soluble presentation of SCF in hematopoiesis. Bone marrow–derived stromal cells expressing only a membrane-restricted (MR) isoform of SCF induced an elevated and sustained tyrosine phosphorylation of both c-kit and erythropoietin receptor (EPO-R) and significantly greater proliferation of an erythrocytic progenitor cell line compared with stromal cells expressing soluble SCF. Transgene expression of MR-SCF inSteel-dickie (Sld) mutants resulted in a significant improvement in the production of red blood cells, bone marrow hypoplasia, and runting. In contrast, overexpression of the full-length soluble form of SCF transgene had no effect on either red blood cell production or runting but corrected the myeloid progenitor cell deficiency seen in these mutants. These data provide the first evidence of differential functions of SCF isoforms in vivo and suggest an abnormal signaling mechanism as the cause of the severe anemia seen in mutants of the Sl gene.
Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects Most-Primitive Human Hematopoietic Stem Cells as Assessed in Long-Term Bone Marrow Cultures
Authors: Weichold Frank F. , Zella Davide , Barabitskaja Oxana , Maciejewski Jaroslaw P. , Dunn Daniel E. , Sloand Elaine M. , Young Neal S.
Published: October 2019

Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.
Development of Autologous, Oligoclonal, Poorly Functioning T Lymphocytes in a Patient With Autosomal Recessive Severe Combined Immunodeficiency Caused by Defects of the Jak3 Tyrosine Kinase
Authors: Brugnoni Duilio , Notarangelo Luigi D. , Sottini Alessandra , Airò Paolo , Pennacchio Marta , Mazzolari Evelina , Signorini Simona , Candotti Fabio , Villa Anna , Mella Patrizia , Vezzoni Paolo , Cattaneo Roberto , Ugazio Alberto G. , Imberti Luisa
Published: October 2019

Defects of the common gamma chain subunit of the cytokine receptors (γc) or of Jak3, a tyrosine kinase required for γc signal transduction, result in T−B+ severe combined immunodeficiency (SCID). However, atypical cases, characterized by progressive development of T lymphocytes, have been also reported. We describe a child with SCID caused by Jak3 gene defects, which strongly but not completely affect Jak3 protein expression and function, who developed a substantial number (>3,000/μL) of autologous CD3+CD4+ T cells. These cells showed a primed/activated phenotype (CD45R0+ Fas+HLA-DR+ CD62Llo), defective secretion of T-helper 1 and T-helper 2 cytokines, reduced proliferation to mitogens, and a high in vitro susceptibility to spontaneous (caused by downregulation of bcl-2 expression) as well as activation-induced cell death. A restricted T-cell receptor repertoire was observed, with oligoclonal expansion within each of the dominant segments. These features resemble those observed in γc-/y and in Jak3−/−mice, in which a population of activated, anergic T cells (predominantly CD4+) also develops with age. These results suggest that residual Jak3 expression and function or other Jak3-independent signals may also permit the generation of CD4+ T cells that undergo in vivo clonal expansion in humans; however, these mechanisms do not allow development of CD8+ T cells, nor do they fully restore the functional properties of CD4+ T lymphocytes.
γ-Interferon Production in Peripheral Blood Mononuclear Cells and Tumor Infiltrating Lymphocytes From Kaposi's Sarcoma Patients: Correlation With the Presence of Human Herpesvirus-8 in Peripheral Blood Mononuclear Cells and Lesional Macrophages
Authors: Sirianni Maria Caterina , Vincenzi Laura , Fiorelli Valeria , Topino Simone , Scala Enrico , Uccini Stefania , Angeloni Antonio , Faggioni Alberto , Cerimele Decio , Cottoni Francesca , Aiuti Fernando , Ensoli Barbara
Published: October 2019

Evidence indicates that, at least in the early stage, Kaposi's sarcoma (KS) is a cytokine-mediated disease and that it is consistently associated with a novel herpesvirus termed human herpesvirus-8 (HHV-8). To gain insights into the mechanisms by which cytokines and HHV-8 may cooperate in disease pathogenesis, we examined the phenotype, the Th1 (γ-interferon [γIFN]) and Th2 (interleukin-4 [IL-4]) cytokine profile and the presence of HHV-8 in peripheral blood mononuclear cells (PBMC), tumor-infiltrating lymphocytes (TIL), and spindle cell cultures derived from skin lesions of patients affected by classical KS (C-KS) and acquired immunodeficiency syndrome (AIDS)-associated KS (AIDS-KS). TIL and spindle cell cultures were examined at day 0 or after culture in conditioned media from activated T cells (TCM) that contain the same cytokines increased in KS tissues. No differences were found in the immunophenotype of PBMC from C-KS patients versus controls, except for AIDS-KS patients who showed a T-CD8+ expansion. However, a preferential infiltration of T-CD8+ cells was found in all KS lesions examined, which was maintained after culture of TIL in TCM. γIFN production was found in both PBMC and cultures derived from all KS examined; some IL-4 positive supernatants were found only in three AIDS-KS cases. Uninvolved skin did not show appreciable lymphocyte infiltration or cytokine production. The culture conditions of the lesional skin allowed also the appearance of adherent, spindle-like cells bearing markers of tissue macrophages. Finally, most or all of the PBMC, lesions, and macrophagic cell cultures from the skin lesions were found to be positive for HHV-8 infection by nested polymerase chain reaction (PCR). These findings indicate that patients with KS express a Th1 phenotype with a prevalent γIFN production, likely accounted for by the local T-CD8+ infiltration. By analogy with other viral infections (ie, Epstein-Barr virus), this suggests that in loco recruitment of lymphoid cells and the subsequent γIFN production may be in response to or elicited by HHV-8 that was found in both PBMC and macrophagic cell cultures from the lesions of the same patients.
Shortened Telomeres Involved in a Case With a Jumping Translocation at 1q21
Authors: Hatakeyama Shinji , Fujita Kazuhiro , Mori Hiraku , Omine Mitsuhiro , Ishikawa Fuyuki
Published: October 2019

The jumping translocation (JT) is a rare chromosomal abnormality in which a specific chromosomal segment translocates onto the ends of various chromosomes (jumps). In most cases, the region distal to 1q21 jumps onto numerous different telomeres. Here we report a molecular study of the JT involving 1q21 found in a patient with acute myelomonocytic leukemia that had transformed from myelodysplastic syndrome (MDS). This is the first report describing the analysis of the molecular structure of the JT. We demonstrated the presence of a stretch of telomeric repeats at the breakpoint by means of a fluorescence in situ hybridization experiment, molecular cloning, and nucleotide sequencing of the fused region. A significant amount of variant telomeric repeats (a telomeric sequence having one-base mismatch within the authentic telomeric repeat TTAGGG) was found in this region. The variant telomeric repeat has been shown to be present in the proximal region of telomeres and does not perform telomeric functions by itself. Therefore, these results indicated that the telomeres had already been critically shortened when the jumps occurred. We suggest that the extended proliferation of cancer cells during the premalignant stage, such as MDS, results in chromosomal instability due to the loss of telomeric functions.
Prion Protein Expression in Human Leukocyte Differentiation
Authors: Dodelet Vincent C. , Cashman Neil R.
Published: October 2019

The cellular isoform of the prion protein (PrPC) is a small glycoprotein attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol anchor. This molecule is involved in the pathogenesis of prion diseases in both humans and animals. We have characterized the expression patterns of PrPC during human leukocyte maturation by flow cytometry with monoclonal antibodies to PrPC, the glycan moiety CD15, and the stem cell marker CD34. We observe that prion protein is present on CD34+bone marrow (BM) stem cells. Although lymphocytes and monocytes maintain PrPC expression throughout their differentiation, PrPC is downregulated upon differentiation along the granulocyte lineage. In vitro retinoic acid–induced differentiation of the premyeloid line HL-60 into granulocyte-like cells mimics the suppression of PrPC in granulocyte differentiation, as both PrPC mRNA and protein are downregulated. These data suggest that selected BM cells and peripheral mononuclear cells may support prion agent replication, because this process is dependent on availability of PrPC. Additionally, retinoic acid–induced extinction of PrPC expression in HL-60 cells provides a potential model to study PrP gene regulation and protein function. Finally, these data suggest the existence of cell-specific glycoforms of PrPC that may determine cellular susceptibility to infection by the prion agent.
Active Site Inhibited Factor VIIa (DEGR VIIa) Attenuates the Coagulant and Interleukin-6 and -8, but not Tumor Necrosis Factor, Responses of the Baboon to LD100Escherichia coli
Authors: Taylor F.B. , Chang A.C.K. , Peer G. , Li A. , Ezban M. , Hedner U.
Published: October 2019

Antitissue factor antibody attenuated the coagulopathic and lethal responses to LD100Escherichia coli, whereas active site inhibited factor Xa inhibited only the coagulopathic response. In this study, we wished to determine: (1) whether active site inhibited factor VIIa blocks the coagulopathic and/or attenuates the lethal effects of LD100E coli and (2) whether these effects are accompanied by attenuation of the inflammatory cytokine response to LD100E coli. Eight baboons infused for 2 hours with LD100E coli also were given five bolus infusions of DEGR VIIa of 280 μg/kg at T = −10 minutes, +2, 4, 6, and 8 hours and observed for changes in vital signs, and the concentrations of hemostatic components (fibrinogen, platelets, fibrin degradation products) and inflammatory mediators (tumor necrosis factor [TNF], interleukin-6 [IL-6], IL-8) at T = 0, 1, 2, 4, 6, and 8 hours. Eight control baboons were also infused with LD100E coli alone and followed as described above. Four of the eight baboons treated with DEGR VIIa were permanent 7-day survivors versus none in the control group. The mean survival times for the treated and control groups were 116 ± 22 and 26 ± 8 hours, respectively. These values differed significantly from each other, (P = .0008). The decrease in platelet and fibrinogen concentrations and the increase in fibrin degradation products observed in the control group were significantly attenuated in the treated group, as was thrombosis of renal glomerular capillaries. Treatment with DEGR VIIa showed no effect on the peak TNF response to LD100E coli at T = 2 hours (170 ± 32 v120 ± 35 ng/mL). DEGR VIIa, however, did attenuate the IL-6 and IL-8 responses at T = 8 hours (ie, the IL-6 concentrations were 81 ± 10 for treated and 1,256 ± 236 for the control groups and the IL-8 concentrations were 28 ± 3.9 for the treated and 60 ± 8.2 for the control group). These values for IL-6 and IL-8 differed significantly from each other between the treated and control groups (P = .0001 and .0074, respectively). It should be noted that the initial responses of IL-6 and IL-8 up to T = 4 hours were not attenuated. We concluded that DEGR VIIa treatment attenuates inflammatory, as well as hemostatic system responses to LD100E coli. We hypothesize that this occurs through interference with the assembly and/or interactions of tissue factor/VIIa complexes.
A Proteasome Inhibitor, an Antioxidant, or a Salicylate, but not a Glucocorticoid, Blocks Constitutive and Cytokine-Inducible Expression of P-Selectin in Human Endothelial Cells
Authors: Xia Lijun , Pan Junliang , Yao Longbiao , McEver Rodger P.
Published: October 2019

Proteasome inhibitors, antioxidants, salicylates, or glucocorticoids block the cytokine-induced expression of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. These pharmacological agents have been assumed to inhibit the expression of adhesion molecules primarily by blocking activation of the transcription factor NF-κB. We found that the proteasome inhibitor ALLN, the antioxidant PDTC, or sodium salicylate, but not the glucocorticoid dexamethasone, inhibited both the constitutive and the interleukin-4– or oncostatin M–induced expression of the adhesion molecule P-selectin in human endothelial cells. ALLN, PDTC, or sodium salicylate decreased P-selectin expression without a detectable requirement for inhibition of NF-κB activation or for an intact κB element in the P-selectin gene. These results extend the potential anti-inflammatory utility of such drugs to inhibition of P-selectin expression and suggest that they have important actions that do not involve the NF-κB system.
The Relationship of Blood Lymphocytes to the Recirculating Lymphocyte Pool
Authors: Andrade William N. , Johnston Miles G. , Hay John B.
Published: October 2019

Lymphocyte recirculation facilitates the detection and elimination of pathogens and the dissemination of immunologic memory. It is generally assumed that all small lymphocytes in the blood are actively recirculating, yet there is little quantitative data directly comparing the migration of this population with actively recirculating, lymph-derived lymphocytes. In this study blood lymphocytes were labeled with fluorescein isothiocyanate (FITC), and lymph lymphocytes were labeled with CM-DiI, reinfused intravenously, and monitored in blood and lymph. After equilibration the concentration of blood lymphocytes was several times higher in blood than in lymph, whereas lymph lymphocytes displayed the opposite behavior. This suggested that blood lymphocytes did not recirculate as efficiently as lymph lymphocytes, so we examined the following blood lymphocyte subsets in greater detail: B cells, CD4+, CD8+, and γδ T cells. Within 4 hours postinjection the percentage of FITC+CD8+ and CD4+ lymphocytes fell in the blood and remained significantly lower than the injected sample. In contrast, the concentration of FITC+ γδ T cells did not change, and the percentage of FITC+ B cells increased. These data suggest that subpopulations of B and perhaps γδ T lymphocytes in the blood do not recirculate efficiently through lymph nodes.
FLT-3 Ligand and Marrow Stroma-Derived Factors Promote CD3γ, CD3δ, CD3ζ, and RAG-2 Gene Expression in Primary Human CD34+LIN−DR− Marrow Progenitors
Authors: Gaffney Patrick M. , Lund Jeanne , Miller Jeffrey S.
Published: October 2019

We hypothesize that early lymphoid commitment from primitive hematopoietic marrow progenitors is governed by signals from the marrow microenvironment leading to sequential induction of lineage-specific genes. Using expression of lymphoid genes as markers of differentiation, we characterize a highly purified population (>99.8% by double sorting) of primary human CD34+Lin−DR− progenitors. This population was then used to evaluate the effects of supplemental cytokines (interleukin-2 [IL-2], IL-3, IL-7, c-kit ligand), FLT-3 ligand (FL), and stroma-derived factors on lymphoid differentiation in vitro. CD3, RAG-1, Ikaros, CD10, and TdT transcripts were detected in the starting CD34+Lin−DR− population. By contrast, CD3γ, CD3δ, CD3ζ, and RAG-2 transcripts were not present in any samples tested. The presence of supplemental cytokines alone at culture initiation permitted stimulation of the expression of CD3ζ, but not of CD3γ or CD3δ. However, when FL and stroma-derived factors were added to cytokines, CD3 gene expression was induced in all samples. The predominant CD3 transcripts induced by optimal culture conditions were alternatively spliced isoforms lacking transmembrane sequences (CD3δ and CD3γ) and portions of the intracellular and extracellular domains (CD3γ). The combination of cytokines, FL, and stromal factors also provided a potent stimulus for RAG-2 gene expression. These findings show that FL in combination with stroma-derived factors provide important signals to promote early events required for lymphoid differentiation.