bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstration of Frequent Occurrence of Clonal T Cells in the Peripheral Blood But Not in the Skin of Patients With Small Plaque Parapsoriasis
Authors: Muche J. Marcus , Lukowsky Ansgar , Heim Jürgen , Friedrich Markus , Audring Heike , Sterry Wolfram
Published: October 2019

Clinical, immunohistological, and molecular biological data suggest the chronic dermatosis small plaque parapsoriasis (SPP) to be a precursor of mycosis fungoides (MF). However, most data are contradictory and confusing due to inexact definition of SPP. Recently, clonal T cells were detected in skin and blood samples of early MF. Because demonstration of identical T-cell clones in skin and blood of SPP patients would indicate a close relationship of SPP to MF, we investigated the clonality of skin and blood specimens from 14 well-defined SPP patients. By a polymerase chain reaction (PCR) amplifying T-cell receptor γ rearrangements and subsequent high-resolution electrophoresis, clonal T cells were detected in 9 of 14 initial and 32 of 49 follow-up blood samples, but in 0 of 14 initial skin specimens. Even a clone-specific PCR showing the persistence of the initial blood T-cell clone in 20 of 20 follow-up samples, failed to detect the T-cell clone in the skin. In 2 patients, the clonal T cells were shown to be CD4+. For the first time, the majority of SPP patients was shown to carry a T-cell clone in the peripheral blood. Although a relation between circulating clonal T cells and SPP cannot directly be proven by the applied techniques, our results indicate blood T-cell clonality to be a characteristic feature of SPP and CTCL because analysis of multiple controls and clinical workup of our SPP patients excluded other factors simulating or causing a clonal T-cell proliferation. A sufficient cutaneous antitumor response but also an extracutaneous origin of the T-cell clones might explain the failure to detect skin infiltrating clonal T cells.
Proteolytic Processing of Big Endothelin-3 by the Kell Blood Group Protein
Authors: Lee Soohee , Lin Melissa , Mele Aldo , Cao Ying , Farmar James , Russo David , Redman Colvin
Published: October 2019

Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp21-Ile22, yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp21-Val22, yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.
Promoter Elements of vav Drive Transgene Expression In Vivo Throughout the Hematopoietic Compartment
Authors: Ogilvy Sarah , Metcalf Donald , Gibson Leonie , Bath Mary L. , Harris Alan W. , Adams Jerry M.
Published: October 2019

To develop a method for targeting expression of genes to the full hematopoietic system, we have used transgenic mice to explore the transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it. Previously, we showed that a cluster of elements surrounding its promoter could drive hematopoietic-specific expression of a bacterial lacZ reporter gene, but the expression was confined to lymphocytes and was sporadically silenced. Those limitations are ascribed here to the prokaryotic reporter gene. With a human CD4 (hCD4) cell surface reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types. In multiple lines, hCD4 appeared on most, if not all, B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid cells. Moreover, high levels appeared on both lineage-committed progenitors and the more primitive preprogenitors. In the fetus, expression was evident in erythroid cells of the definitive but not the primitive type. These results indicate that a prokaryotic sequence can inactivate a transcription unit and that the vavpromoter region constitutes a potent transgenic vector for the entire definitive hematopoietic compartment.
Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro
Authors: Fujisaki Tomoaki , Berger Marc G. , Rose-John Stefan , Eaves Connie J.
Published: October 2019

Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.
The Human Platelet IIb Gene Is Not Closely Linked to Its Integrin Partner β3
Authors: Thornton M.A. , Poncz M. , Korostishevsky M. , Yakobson E. , Usher S. , Seligsohn U. , Peretz H.
Published: October 2019

IIbb3 integrin is a heterodimeric receptor facilitating platelet aggregation. Both genes are on chromosome 17q21.32. Intergenic distance between them has been reported to be 125 to 260 kilobasepairs (kb) by pulsed-field gel electrophoresis (PFGE) genomic analysis, suggesting that they may be regulated coordinately during megakaryopoiesis. In contrast, other studies suggest these genes are greater than 2.0 megabasepairs (mb) apart. Because of the potential biological implications of having these two megakaryocytic-specific genes contiguous, we attempted to resolve this discrepancy. Taking advantage of large kindreds with mutations in either IIb or β3, we have developed a genetic linkage map between the thyroid receptor hormone-1 gene (THRA1) and β3 as follows: cen-THRA1-BRCA1-D17S579/IIb-β3-qter, with a distance of 1.3 centiMorgans (cM) between IIb and β3 and the two genes being oriented in the same direction. PFGE genomic and YAC clone analysis showed that the β3 gene is distal and ≥365 kb upstream of IIb. Additional restriction mapping shows IIb is linked to the erythrocyte band 3 (EPB3) gene, and β3 to the homeobox HOX2b gene. Analysis of IIb+-BAC and P1 clones confirm that the EPB3 gene is ∼110 kb downstream of the IIb gene. Sequencing the region surrounding the human IIb locus showed the Granulin gene ∼18 kb downstream to IIb, and the KIAA0553 gene ∼5.7 kb upstream. This organization is conserved in the murine sequence. These studies show that IIb and β3 are not closely linked, with IIb flanked by nonmegakaryocytic genes, and imply that they are unlikely to share common regulatory domains during megakaryopoiesis.
A Novel Epstein-Barr Virus-Like Virus, HVMNE, in aMacaca Nemestrina With Mycosis Fungoides
Authors: Rivadeneira E.D. , Ferrari M.G. , Jarrett R.F. , Armstrong A.A. , Markham P. , Birkebak T. , Takemoto S. , Johnson-Delaney C. , Pecon-Slattery J. , Clark E.A. , Franchini G.
Published: October 2019

Epstein-Barr virus (EBV) infection of humans has been associated with the development of lymphoid malignancies mainly of B-cell lineage, although occasionally T-cell lymphomas have been reported. We describe here the characterization of a novel EBV-like virus (HVMNE) isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II) seronegative pigtailed macaque (Macaca nemestrina) with a cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin lesions demonstrated that the infiltrating cells were of the CD3+/CD8+ phenotype. Two primary transformed CD8+ T-cell lines were obtained from cultures of peripheral blood mononuclear cells (PBMC) and skin, and, with time, both cell lines became interleukin-2–independent and acquired the constitutive activation of STAT proteins. Polymerase chain reaction analysis of the DNA from the cell lines and tissues from the lymphomatous animal demonstrated the presence of a 536-bp DNA fragment that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HVMNE according to the existing nomenclature. In situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8+ T-cell lines as well as in the infiltrating CD8+ T cells of skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from the polymerase gene of HVMNE placed this virus within theLymphocryptovirus genus and demonstrated that HVMNEis a distinct virus, clearly related to human EBV and other EBV-like herpesviruses found in nonhuman primates.
Stealth Cells: Prevention of Major Histocompatibility Complex Class II-Mediated T-Cell Activation by Cell Surface Modification
Authors: Murad Kari L. , Gosselin Edmund J. , Eaton John W. , Scott Mark D.
Published: October 2019

Transfusion or transplantation of T lymphocytes into an allogeneic recipient can evoke potent immune responses including, in immunocompromised patients, graft-versus-host disease (GVHD). As our previous studies demonstrated attenuated immunorecognition of red blood cells covalently modified with methoxy(polyethylene glycol) (mPEG), we hypothesized that T-cell activation by foreign antigens might similarly be prevented by mPEG modification. Mixed lymphocyte reactions (MLR) using peripheral blood mononuclear cells (PBMC) from HLA class II disparate donors demonstrate that mPEG modification of PBMC effectively inhibits T-cell proliferation (measured by 3H-thymidine incorporation) in a dose-dependent manner. Even slight derivatization (0.4 mmol/L mPEG per 4 × 106 cells) resulted in a ≥75% decrease, while higher concentrations caused ≥96% decrease in proliferation. Loss of PBMC proliferation was not due to either mPEG-induced cytotoxicity, as viability was normal, or cellular anergy, as phytohemagglutinin (PHA)-stimulated mPEG-PBMC demonstrated normal proliferative responses. Addition of exogenous interleukin (IL)-2 also had no proliferative effect, suggesting that the mPEG-modified T cells were not antigen primed. Flow cytometric analysis demonstrates that mPEG-modification dramatically decreases antibody recognition of multiple molecules involved in essential cell:cell interactions, including both T-cell molecules (CD2, CD3, CD4, CD8, CD28, CD11a, CD62L) and antigen-presenting cell (APC) molecules (CD80, CD58, CD62L) likely preventing the initial adhesion and costimulatory events necessary for immune recognition and response.
In Vivo Selection of Wild-Type Hematopoietic Stem Cells in a Murine Model of Fanconi Anemia
Authors: Battaile Kevin P. , Bateman Raynard L. , Mortimer Derik , Mulcahy Jean , Rathbun R. Keaney , Bagby Grover , Fleming William H. , Grompe Markus
Published: October 2019

Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.
Genetic Modification of Human B-Cell Development: B-Cell Development Is Inhibited by the Dominant Negative Helix Loop Helix Factor Id3
Authors: Jaleco Ana C. , Stegmann Alexander P.A. , Heemskerk Mirjam H.M. , Couwenberg Franka , Bakker Arjen Q. , Weijer Kees , Spits Hergen
Published: October 2019

Transgenic and gene targeted mice have contributed greatly to our understanding of the mechanisms underlying B-cell development. We describe here a model system that allows us to apply molecular genetic techniques to the analysis of human B-cell development. We constructed a retroviral vector with a multiple cloning site connected to a gene encoding green fluorescent protein by an internal ribosomal entry site. Human CD34+CD38− fetal liver cells, cultured overnight in a combination of stem cell factor and interleukin-7 (IL-7), could be transduced with 30% efficiency. We ligated the gene encoding the dominant negative helix loop helix (HLH) factor Id3 that inhibits many enhancing basic HLH transcription factors into this vector. CD34+CD38− FL cells were transduced with Id3-IRES-GFP and cultured with the murine stromal cell line S17. In addition, we cultured the transduced cells in a reaggregate culture system with an SV-transformed human fibroblast cell line (SV19). It was observed that overexpression of Id3 inhibited development of B cells in both culture systems. B-cell development was arrested at a stage before expression of the IL-7R. The development of CD34+CD38− cells into CD14+ myeloid cells in the S17 system was not inhibited by overexpression of Id3. Moreover, Id3+ cells, although inhibited in their B-cell development, were still able to develop into natural killer (NK) cells when cultured in a combination of Flt-3L, IL-7, and IL-15. These findings confirm the essential role of bHLH factors in B-cell development and demonstrate the feasibility of retrovirus-mediated gene transfer as a tool to genetically modify human B-cell development.
Expression of pT mRNA in a Committed Dendritic Cell Precursor in the Human Thymus
Authors: Res Pieter C.M. , Couwenberg Franka , Vyth-Dreese Florry A. , Spits Hergen
Published: October 2019

We have characterized dendritic cell precursors (pre-DC) in the human thymus. These CD1a−CD3−CD4+CD8−cells express high levels of interleukin-3R (IL-3R) on the membrane and are able to develop into mature DC upon culture with IL-3 and CD40 ligation. The DC precursors are predominantly located in the thymic medulla. Interestingly, the pre-DC express pT mRNA, which is also present in CD1a+CD3−CD4+ CD8−pre-T cells. Yet, the pre-DC lack expression of recombination activating gene-1 mRNA and fail to develop into T cells in appropriate assays. The thymic pre-DC are very similar to the recently characterized pre-DC found in the T cell areas of the tonsil, and it is suggested that these pre-DC populations are of lymphoid origin.
The Wsh, W57, and Ph Kit Expression Mutations Define Tissue-Specific Control Elements Located Between −23 and −154 kb Upstream of Kit
Authors: Berrozpe Georgina , Timokhina Inna , Yukl Steven , Tajima Youichi , Ono Masao , Zelenetz Andrew D. , Besmer Peter
Published: October 2019

The Kit and PDGFRa receptor tyrosine kinases are encoded in close proximity at the murine white spotting (W) and patch (Ph) loci. Whereas W mutations affect hematopoiesis, melanogenesis, and gametogenesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. TheWsh, W57, and Phmutations diminish Kit expression in certain cell types such as mast cells and enhance it in others. The Wsh,W57, and Ph mutations arose from deletions and inversions affecting sequences in between the Kit andPDGFRa genes. We have determined the precise location of the breakpoint of the Wshinversion and the endpoints of the W57deletion upstream of the Kittranscription start site and examined the effect of these mutations on Kit expression in mast cells and hematopoietic stem cells and lineage progenitors. Our results indicate that positive elements controlling Kit expression in mast cells mapping in between −23 and −154 kb from the transcription start site can be dissociated from negative elements controlling Kit misexpression during embryonic development in the vicinity of the PDGFRa gene. In addition, we have identified two clusters of hypersensitive sites in mast cells at −23 −28 kb and −147 −154 kb from the Kit gene transcription start site. Analysis of these hypersensitive sites in mutant mast cells indicates a role for HS4-6 in Kit expression in mast cells. These findings provide a molecular basis for the phenotype of these Kit expression mutations and they provide insight into the complex mechanisms governing the regulation ofKit expression.
Activation of the Erythropoietin Receptor Is Not Required for Internalization of Bound Erythropoietin
Authors: Beckman Diana L. , Lin Lilie L. , Quinones Mary E. , Longmore Gregory D.
Published: October 2019

Erythropoietin (EPO) is required for the survival and expansion of red blood cell progenitor cells and supports continued differentiation of these committed progenitors to mature red blood cells. After binding to its cognate receptor, EPO promotes receptor homodimerization, activation of receptor-associated JAK2, subsequent receptor tyrosine phosphorylation, and transduction of signal. EPO is also internalized and degraded in lysosomes. The contribution of EPO-induced receptor internalization to modulation of EPO signals has not been determined. To examine this question, we generated a panel of hematopoietic cell lines containing progressively truncated isoforms of the erythropoietin receptor (EPO-R) and determined the rate and extent of EPO internalization and receptor downregulation. We demonstrated that a membrane-proximal domain of the cytoplasmic tail of the EPO-R was the minimal region required for EPO-induced receptor internalization. This cytoplasmic domain is also the minimal domain required for activation of JAK2, a cytosolic tyrosine kinase essential for the function of the EPO-R. However, neither EPO activation of cytosolic JAK2 tyrosine kinase activity nor tyrosine phosphorylation of the EPO-R cytoplasmic tail was required for EPO-induced receptor downregulation. Both functional and nonfunctional cell surface receptor isoforms were internalized equally. These results suggest that, for downregulation of cell surface ligand occupied EPO-R and possibly for signaling receptors of the cytokine receptor superfamily in general, internalization of cell surface ligand occupied receptors may follow a pathway distinct from signaling receptors of the receptor tyrosine kinase (RTK) family.
Influence of Fibrillar Collagen Structure on the Mechanisms of Platelet Thrombus Formation Under Flow
Authors: Savage Brian , Ginsberg Mark H. , Ruggeri Zaverio M.
Published: October 2019

We have used real-time video microscopy to study the mechanisms of platelet adhesion to type I collagen fibrils of distinct structure exposed to flowing blood. Electron microscopy analysis by surface replication demonstrated morphological differences between acid-insoluble fibrils, displaying a regularly repeating striated pattern (banded collagen), and acid-soluble fibrils generated by pepsin treatment of insoluble collagen, smaller in size with a helical configuration (nonbanded collagen). These structural differences proved to be related to the role of platelet integrin 2β1 in stabilizing adhesion to collagen under a variety of flow conditions. Blocking 2β1 function with a monoclonal antibody had no effect on platelet adhesion to insoluble type I collagen coated at high density on a glass surface, whereas there was an absolute dependence of 2β1 function for the initial permanent arrest of platelets and subsequent thrombus formation on pepsin-solubilized type I collagen under the same conditions. In contrast, reconstituted, banded fibrils prepared from pepsin-solubilized type I collagen supported platelet adhesion and thrombus development even when platelet 2β1 function was blocked, a process that was greatly accelerated by pre-exposure of this substrate to autologous plasma under flow. These results implicate a collagen receptor(s) on platelets other than 2β1 that can selectively engage domains in banded, but not nonbanded type I collagen when 2β1 function is blocked. In addition, collagen structure may regulate the extent and affinity of the binding under flow of plasma components such as von Willebrand factor and/or other IIbβ3 ligands.
Neutrophil Adhesion on Polyurethanes Preadsorbed With High Molecular Weight Kininogen
Authors: Yung Lin-Yue L. , Colman Robert W. , Cooper Stuart L.
Published: October 2019

Interaction of biomaterials with blood components including neutrophils is responsible for some of the clinical complications that have occurred in cardiopulmonary bypass, hemodialysis, and ventricular assist procedures. The possibility of inhibiting the initial adhesion of neutrophils to biomaterials has been studied extensively, but the problem remains unsolved. In this study, we investigated the effect of HK adsorption on polyurethane, a widely used component of extracorporeal and intracorporeal devices. HK and HKa were allowed to adsorb on 4 different charged polyurethanes: noncharged (PU), cationic (NR4), anionic (SO3), and zwitterionic (GPC) polyurethanes. The effect of kininogen adsorption on neutrophil adhesion, the surface density of the adsorbed kininogen, and the exposure of HK domains 3 and 5 (D3 and D5H), which are responsible for the binding of HK to the neutrophil integrin mβ2 or Mac-1, were examined. On PU, NR4, and SO3, kininogen adsorption reached 80% of monolayer coverage when 100 pmol/mL or higher concentration of protein solutions were used. The NR4 surface adsorbed the most kininogen along with a high exposure of D3 and D5H. The availability of D3 and D5Hallowed neutrophils to bind to the surface via the Mac-1 receptor; thus, on the NR4 surface, adsorbed kininogens lost their antiadhesive property, which resulted in a high degree of neutrophil adhesion. Increasing Mac-1 expression by exposure to fMLP increased the neutrophil adhesion on this surface. In contrast, exposure of D3 and D5H on SO3 was significantly less, because HK binds to anionic surfaces with similar protein sequences used for cell binding. This low binding site exposure preserved the antiadhesive property of HK. GPC was resistant to neutrophil adhesion even in the absence of adsorbed kininogens because of its phosphorylcholine moiety. Thus, both SO3 coupled with kininogen (or kininogen peptides) and GPC have the potential to markedly reduce neutrophil adhesion to biomaterial devices.
An Inositolphosphate-Binding Immunophilin, IPBP12
Authors: Cunningham Earlene Brown
Published: October 2019

A novel inositolphosphate-binding protein has been identified and shown to be an immunophilin. This protein, which was isolated from human erythrocyte membranes and from K562 (human erythroleukemia) cell membranes, has robust peptidylprolyl cis-trans isomerase activity that is strongly inhibited by nanomolar concentrations of FK506 or rapamycin, indicating a member of the FKBP (FK506-binding protein) class. However, unlike the cytosolic FKBP12, the isomerase activity of this membrane-associated immunophilin is strongly inhibited by nanomolar concentrations of inositol 1,4,5-trisphosphate (IP3), inositol 1,3,4,5-tetrakisphosphate (IP4), and phosphatidylinositol 4- and 4,5-phosphates, which are suggested to be physiological ligands. The demonstration of a single 12-kD protein that binds both IP4 or IP3and anti-FKBP12 provides strong support for the inositolphosphate-binding immunophilin having an apparent mass of 12 kD, and it is suggested that the protein might be called IPBP12 for 12-kD inositol phosphate binding protein. When an internal tryptic peptide derived from IPBP12 was sequenced, a sequence also present in human cytokeratin 10 was identified, suggesting a cytoskeletal localization for the immunophilin. While purifying IPBP12, it was found that it is immunoprecipitated with specific proteins that include a protein kinase and a phosphoprotein phosphatase. The latter is indicated to be phosphoprotein phosphatase 2A (PP-2A). It is suggested that immunophilins promote the assembly of multiprotein complexes that often include a protein kinase or a phosphoprotein phosphatase or both.
A Deletion in the Gene for Transforming Growth Factor β Type I Receptor Abolishes Growth Regulation by Transforming Growth Factor β in a Cutaneous T-Cell Lymphoma
Authors: Schiemann William P. , Pfeifer Walther M. , Levi Edi , Kadin Marshall E. , Lodish Harvey F.
Published: October 2019

Spontaneous regression of skin lesions is characteristic of lymphomatoid papulosis (LyP), a clonal cutaneous lymphoproliferative disorder. A minority of LyP patients progress to anaplastic large cell lymphoma (ALCL) in which skin lesions no longer regress and extracutaneous dissemination often occurs. In 1 such case, we developed a tumor cell line, JK cells, and show that these cells are resistant to the growth inhibitory effects of transforming growth factor β (TGF-β) due to the loss of cell surface expression of the TGF-β type I receptor (TβR-I). Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing of JK cell TβR-I cDNA clones identified a deletion that spanned the last 178 bp of exon 1, including the initiating methionine. Hybridization of a radiolabeled fragment internal to the deletion was detected in the genomes of TGF-β–responsive cells, but not in JK cells, indicating that they contain no wild-type TβR-I gene. PCR primers that flanked the deleted TβR-I region amplified a single band from JK cell genomic DNA that lacked the last 178 bp of exon 1 and all of the ≈ 5 kb of intron 1. This JK cell-specific genomic TβR-I PCR product was distinct from products amplified from TGF-β–responsive cells and was also readily detected in tumor biopsies obtained before the establishment of the JK cell line. Our results identify the first inactivating mutation in TβR-I gene in a human lymphoma that renders it insensitive to growth inhibition by TGF-β.
Involvement of Interleukin-10 (IL-10) and Viral IL-6 in the Spontaneous Growth of Kaposi’s Sarcoma Herpesvirus-Associated Infected Primary Effusion Lymphoma Cells
Authors: Jones Karen D. , Aoki Yoshiyasu , Chang Yuan , Moore Patrick S. , Yarchoan Robert , Tosato Giovanna
Published: October 2019

Primary effusion lymphoma (PEL) is a distinct type of lymphoma associated with Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. To determine the factors responsible for the unrestrained proliferation of PEL, we have studied the growth factor requirements of the PEL-derived BCBL-1 and BC-1 cell lines. Both cell lines were found to be autocrine growth factor dependent and to release human interleukin-6 (IL-6), viral IL-6 (vIL-6), and human IL-10 in the culture supernatant. To establish whether these cytokines contribute to autocrine growth, neutralizing antibodies against human IL-6, vIL-6, human IL-10, and soluble IL-10 receptor were used. These experiments showed that human IL-10 and, to a lesser degree, vIL-6 serve as autocrine growth factors for BCBL-1 and BC-1 cells. Thus, human IL-10 and vIL-6 are growth factors released and used by PEL cells for autonomous proliferation and may be critical to the development and progression of PEL.
Interleukin-10 (IL-10) Selectively Enhances CIS3/SOCS3 mRNA Expression in Human Neutrophils: Evidence for an IL-10–Induced Pathway That Is Independent of STAT Protein Activation
Authors: Cassatella Marco A. , Gasperini Sara , Bovolenta Chiara , Calzetti Federica , Vollebregt Marieke , Scapini Patrizia , Marchi Martina , Suzuki Ritsu , Suzuki Asuka , Yoshimura Akihiko
Published: October 2019

We have recently shown that, in human neutrophils, interleukin-10 (IL-10) fails to induce specific DNA-binding activities to the gamma-interferon response region (GRR), a regulatory element located in the FcγRI gene promoter, which is required for transcriptional activation by IL-10 and interferon γ (IFNγ) in monocytic cells. In this study, we report that IL-10 is also unable to induce the binding of STAT1 or STAT3 to the serum-inducible element (hSIE/m67), despite the fact that both proteins are expressed in neutrophils. Whereas IFNγ and granulocyte colony-stimulating factor (G-CSF) are efficient inducers of STAT1 and STAT3 tyrosine phosphorylation in polymorphonuclear neutrophils (PMN), IL-10 fails to trigger STAT1 and STAT3 tyrosine and serine phosphorylation, therefore explaining its inability to induce the FcγRI expression in these cells. By contrast, we demonstrate that IL-10 alone represents an efficient stimulus of CIS3/SOCS3 mRNA expression in neutrophils. CIS3/SOCS3 belongs to the recently cloned cytokine-inducible SH2-containing protein (CIS) gene family (which also includes CIS1, CIS2, CIS4, CIS5, and JAB) that is believed to be, at least in part, under the control of STAT transcription factors and whose products are potential modulators of cytokine signaling. Moreover, IL-10 synergizes with lipopolysaccharide (LPS) in upregulating CIS3/SOCS3 mRNA expression in PMN through a mechanism that involves mRNA stabilization. In contrast to CIS3/SOCS3, mRNA transcripts encoding other family members are unaffected by IL-10 in neutrophils. Finally, transfection of CIS3/SOCS3 in murine M1 myeloid cells suppresses LPS-induced growth arrest, macrophage-like differentiation, and nitric oxide synthesis, but not IL-6 mRNA expression. Collectively, our data suggest that, in neutrophils, the activation of STAT1 and STAT3 phosphorylation is neither required for CIS3/SOCS3 induction by IL-10 nor involved in the regulatory effects of IL-10 on cytokine production.
Correlation Between Disparity for the Minor Histocompatibility Antigen HA-1 and the Development of Acute Graft-Versus-Host Disease After Allogeneic Marrow Transplantation
Authors: Tseng Li-Hui , Lin Ming-Tseh , Hansen John A. , Gooley Ted , Pei Ji , Smith Anajane G. , Martin Emily G. , Petersdorf Effie W. , Martin Paul J.
Published: October 2019

Results of a previous study suggested that recipient mismatching for the minor histocompatibility antigen HA-1 is associated with acute graft-versus-host disease (GVHD) after allogeneic marrow transplantation. In that study, most patients received either cyclosporine or methotrexate for GVHD prophylaxis, and a cytotoxic T-cell clone was used to test for HA-1 disparity. To facilitate large-scale testing, we developed a method that uses genomic DNA to identify HA-1 alleles. A retrospective study was conducted to correlate HA-1 disparity and the occurrence of acute GVHD in 237 HLA-A2–positive white patients who had received a marrow or peripheral blood stem cell transplant from an HLA-identical sibling. All patients received both methotrexate and cyclosporine for GVHD prophylaxis. The presence of HLA-A*0201 was confirmed in 34 of the 36 HA-1 disparate pairs by sequencing the HLA-A locus. Grades II-IV GVHD occurred in 22 (64.7%) of these 34 patients, compared with 86 (42.8%) of the 201 patients without HA-1 disparity (odds ratio, 2.45; 95% confidence interval [CI], 1.15 to 5.23; P = .02). Recipient HA-1 disparity showed a trend for association with acute GVHD (odds ratio, 2.1; 95% CI, 0.91 to 4.68; P = .08) when a multivariable logistic regression model was used to include additional risk factors. These data are consistent with results of the previous study, suggesting an association between HA-1 disparity and risk of acute GVHD, but the strength of this association may be lower in patients who received both methotrexate and cyclosporine than in those who received methotrexate or cyclosporine alone.
Viral Hyperinfection of the Central Nervous System and High Mortality After Hematopoietic Stem Cell Transplantation for Treatment of Theiler’s Murine Encephalomyelitis Virus-Induced Demyelinating Disease
Authors: Burt Richard K. , Padilla Josette , Dal Canto Mauro C. , Miller Stephen D.
Published: October 2019

Theiler’s murine encephalomyelitis virus (TMEV) establishes a persistent infection in the central nervous system (CNS) leading to an inflammatory demyelinating disease of the CNS in which the histology and clinical course is similar to multiple sclerosis (MS). Disease pathogenesis is primarily due to T-cell–mediated destruction of myelin, which has been attributed to cytopathic effects of the virus, but immune-mediated destruction of myelin mediated via both virus-specific and myelin-specific T cells appear to play the major role. To determine if bone marrow transplantation would be an effective therapy for a virus-initiated autoimmune disease and to better separate viral cytopathic effects from immune-mediated demyelination, we ablated the immune system of TMEV-infected animals with 1,100 cGy total body irradiation, and then the animal’s immunity was reconstituted by transplantation of disease-susceptible SJL/J mice with syngeneic marrow or disease-susceptible DBA/2J with marrow from disease-resistant (C57Bl/6 × DBA/2)F1 (B6D2) donors. Hematopoietic transplant performed after onset of disease resulted in 42% mortality in SJL/J syngeneic transplants, 47% mortality in diseased DBA2 recipients restored with marrow from naive B6D2 donors, and 12% in diseased DBA2 recipients receiving marrow from B6D2 donors previously infected with TMEV. Delayed type hypersensitivity (DTH) to both virion and myelin proteins was decreased in surviving mice that underwent transplantation; however, CNS viral titers were significantly elevated compared with nontransplanted controls. We conclude that a functional immune system with appropriate T-cell responses are important in prevention of lethal cytopathic CNS effects from TMEV. Relevant to the clinical use of bone marrow transplantation, attempts to ablate the immune system in viral-mediated immune diseases or virus-initiated autoimmune disease may have acute and lethal consequences. Our results raise concern about the attempted use of autologous hematopoietic transplantation in patients with MS, an autoimmune disease with a suspected virus etiology, particularly if the graft is aggressively depleted of lymphocytes.
Expansion of human cord blood CD34+CD38−cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function
Authors: Dorrell Craig , Gan Olga I. , Pereira Daniel S. , Hawley Robert G. , Dick John E.
Published: October 2019

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin− CD34+CD38−cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin−CD34+ CD38− cells and SRC, CD34+–enriched lineage–depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34+CD38− cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP+ human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34+CD38− phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34+CD38− phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34+CD38+ cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)
Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor β subunit
Authors: McCormack Matthew P. , Gonda Thomas J.
Published: October 2019

Several activating mutations have recently been described in the common β subunit for the human interleukin(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (hβc). Two of these, FIΔ and I374N, result, respectively, in a 37–amino acid duplication and an isoleucine-to-asparagine substitution in the extracellular domain. A third, V449E, leads to valine-to–glutamic acid substitution in the transmembrane domain. Previous studies have shown that when expressed in murine hemopoietic cells in vitro, the extracellular mutants can confer factor independence on only the granulocyte-macrophage lineage while the transmembrane mutant can do so to all cell types of the myeloid and erythroid compartments. To further study the signaling properties of the constitutively active hβc mutants, we have used novel murine hemopoietic cell lines, which we describe in this report. These lines, FDB1 and FDB2, proliferate in murine IL-3 and undergo granulocyte-macrophage differentiation in response to murine GM-CSF. We find that while the transmembrane mutant, V449E, confers factor-independent proliferation on these cell lines, the extracellular hβc mutants promote differentiation. Hence, in addition to their ability to confer factor independence on distinct cell types, transmembrane and extracellular activated hβc mutants deliver distinct signals to the same cell type. Thus, the FDB cell lines, in combination with activated hβc mutants, constitute a powerful new system to distinguish between signals that determine hemopoietic proliferation or differentiation. (Blood. 2000;95:120-127)
Development of dendritic cells in vitro from murine fetal liver–derived lineage phenotype-negative c-kit+hematopoietic progenitor cells
Authors: Zhang Yanyun , Zhang Yi , Wang Yong , Ogata Masafumi , Hashimoto Shin-ichi , Onai Nobuyuki , Matsushima Kouji
Published: October 2019

We describe here that lineage phenotype- negative (Lin)−c-kit+ hematopoietic progenitor cells (HPCs) from day 13 postcoitus (dpc) murine fetal liver (FL) can generate dendritic cell (DC) precursors when cultured in vitro in the presence of PA6 stromal cells plus granulocyte/macrophage colony-stimulating factor (GM-CSF) + stem cell factor (SCF) + Flt3 ligand (Flt3L) for 12 to 14 days, and develop into mature DCs when stimulated with GM-CSF plus mouse tumor necrosis factor  (mTNF) for an additional 3 to 5 days. A transwell culture system showed that the generation of DC precursors depended on the support of PA6 cell-secreted soluble factor(s). The mature DCs derived from 13 dpc FL Lin−c-kit+ HPCs showed characteristic morphology and function of DCs and expressed high levels of Ia, CD86, and CD40 molecules, low levels of DEC205, E-cadherin, and F4/80 molecules, but barely detectable CD11c antigen. Once FL-derived HPCs were cultured without GM-CSF, NK1.1+ cells developed in the presence of PA6 cells + SCF + Flt3L. These NK1.1+ cells could develop into DC precursors at an earlier stage of differentiation by reculturing with PA6 cells + SCF + Flt3L + GM-CSF, but they would be irreversibly committed to NK cell precursors without GM-CSF after 3 days, suggesting that GM-CSF plays a critical role in controlling the transition of DC and NK cell precursors from 13 dpc FL-derived Lin−c-kit+ HPCs. This study represents the first success in generating mature DCs in vitro from murine FL HPCs. (Blood. 2000;95:138-146)
Application of the ELISPOT assay to the characterization of CD8+ responses to Epstein-Barr virus antigens
Authors: Yang Jie , Lemas Victor M. , Flinn Ian W. , Krone Chris , Ambinder Richard F.
Published: October 2019

CD8+ cells have an important role in controlling Epstein-Barr virus (EBV) infection. We adapted the interferon-γ ELISPOT assay to the quantitative analysis of EBV-specific CD8+ cells. Using peripheral blood mononuclear cells (PBMCs) from healthy donors, we measured both the aggregate response to the virus, using EBV-transformed lymphoblastoid cell lines (LCLs) as stimulators, and the specific responses to 2 A2-restricted peptide epitopes: the subdominant latency membrane protein-2 (LMP2) peptide CLGGLLTMV and the early lytic BMLF1 peptide GLCTLVAML. LCL-responsive CD8+ cells were detected in all EBV-seropositive donors (range 954 to 37 830 spots/106CD8+ cells). LMP2 peptide-responsive CD8+cells were detected in 10 of 11 healthy seropositive A2 donors (range 11 to 83 spots/106 PBMC). BMLF1 peptide-responsive CD8+ cells were detected in all seropositive A2 donors examined (range 13 to 943 spots/106 PBMC). Cytotoxic T-lymphocyte (CTL) lines generated with weekly stimulation of LCLs for therapeutic purposes were also studied. Relative to PBMCs, these CTL lines showed a marked increase in the level of LCL-responsive and LMP2 peptide-responsive CD8+ cells and a lesser degree of expansion of BMLF1 peptide-responsive CD8+ cells. Finally, we applied the ELISPOT assay to monitor adoptive infusion of EBV CTL lines. In 2 patients examined, a transient increase in LCL-responsive CD8+ cells could be detected after infusion. Thus, the ELISPOT assay can be applied to the analysis of CD8+responses to EBV antigens in PBMCs, in ex vivo expanded CTL lines, and in PBMCs from patients treated with ex vivo expanded CTL lines. (Blood. 2000;95:241-248)
Evidence of increased angiogenesis in patients with acute myeloid leukemia
Authors: Hussong Jerry W. , Rodgers George M. , Shami Paul J.
Published: October 2019

Angiogenesis plays a key role in solid tumor growth. The purpose of this work was to study angiogenesis in acute myeloid leukemia (AML). We stained bone marrow samples from 20 adult patients with untreated AML and 20 normal controls using endothelial cell markers (ULEX-E and von Willebrand factor [vWF]). The number of vessels per millimeter length of bone marrow core biopsy specimen was scored by light microscopy. Using ULEX-E staining, AML marrows had (average ± SEM) 8.3 ± 3.6 vessels/mm (range, 3.7-19.3), whereas normal marrows had 4.3 ± 1.8 vessels/mm (range, 1.6-7.9). A similar difference was noted using vWF staining (8.6 ± 3.0 vessels/mm vs 4.9 ± 2.2 vessels/mm in AML vs normal bone marrows, respectively). The differences between the numbers of vessels/mm in AML and normal marrows were highly significant (P < .0001 for both ULEX-E and vWF staining). When analyzed by FAB category, there was no difference in the average number of vessels/mm among the different subgroups of AML. Using reverse transcriptase polymerase chain reaction, we observed that the HL-60 and U937 human AML cell lines and 4 of 4 freshly isolated AML cells from untreated patients expressed mRNA for vascular endothelial growth factor (VEGF). Both cell lines as well as all fresh AML isolates tested expressed VEGF protein. Basic fibroblast growth factor was expressed only in HL-60 cells and in only 3 of 4 fresh AML samples. These observations suggest that angiogenesis may play a role in the pathogenesis of AML. Inhibition of angiogenesis could constitute a novel strategy for the treatment of AML. (Blood. 2000;95:309-313).
β2 Microglobulin-deficient (B2mnull) NOD/SCID mice are excellent recipients for studying human stem cell function
Authors: Kollet Orit , Peled Amnon , Byk Tamara , Ben-Hur Herzl , Greiner Dale , Shultz Leonard , Lapidot Tsvee
Published: October 2019

Human SCID repopulating cells (SRC) are defined based on their functional ability to repopulate the bone marrow of NOD/SCID mice with both myeloid and lymphoid cell populations. The frequency of SRC in umbilical cord blood cells is 1 in 9.3 × 105mononuclear cells. We report that as few as 8 × 104 human cord blood mononuclear cells transplanted into NOD/SCID/B2mnull mice resulted in mutlilineage differentiation in the murine bone marrow, revealing a more than 11-fold higher SRC frequency than in NOD/SCID mice. Moreover, as few as 2 to 5 × 103 CD34+ cells recovered from the bone marrow of primary transplanted NOD/SCID mice were sufficient for engrafting secondary NOD/SCID/B2mnull mice with SRC, suggesting SRC self-renewal. Thus, by using NOD/SCID/B2mnull mice as recipients, we established a functional assay for human stem cells capable of engrafting the bone marrow of primary and secondary transplanted immune-deficient mice with SRC, providing a model that better resembles autologous stem cell transplantation.