bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypersensitivity of circulating progenitor cells to megakaryocyte growth and development factor (PEG-rHu MGDF) in essential thrombocythemia
Authors: Axelrad Arthur A. , Eskinazi Denise , Correa Paulo N. , Amato Dominick
Published: October 2019

Hematopoietic progenitor cells in 2 myeloproliferative disorders, juvenile chronic myelomonocytic leukemia and polycythemia vera, are known to be hypersensitive to cytokines that control normal progenitor cell proliferation, differentiation, and survival in their respective granulocyte/macrophage and erythroid lineages. Because thrombopoietin controls these functions in the normal megakaryocytic lineage, we asked the question: Are megakaryocytic progenitor cells in the myeloproliferative disorder essential thrombocythemia (ET) hypersensitive to thrombopoietin? Peripheral blood mononuclear cells from patients with ET, or secondary (reactive) thrombocytosis (2°T), or healthy volunteers were grown in strictly serum-free agarose culture containing interleukin 3 (IL-3) and all-trans-retinoic acid, with various concentrations of PEG-rHu megakaryocyte growth and development factor (MGDF). The concentration of cytokine at half-maximum colony number served as a measure of progenitor cell sensitivity. Hypersensitivity to PEG-rHu MGDF was found in circulating progenitors from 18 of 20 (90%) informative patients with presumptive diagnosis ET, 1 of 8 (12.5%) 2 °T patients, and none of the 22 healthy volunteers. Median MGDF sensitivity ratio in ET patients was approximately 53 times greater than in the controls. This hypersensitivity, which was also directed to rHu thrombopoietin, was highly specific with respect to cytokine, disease, and cell lineage. We propose that, despite their single pluripotential cell origin, the different clinicopathologic phenotypes in different chronic myeloproliferative disorders are determined by lineage-restricted hypersensitivities of hematopoietic progenitor cells to endogenous cytokines. This work emphasizes the importance of stringent serum-free conditions for revealing true sensitivities to cytokines. The findings also offer a basis for evolving a positive test for ET, a diagnosis now made essentially by exclusion.
Cadmium and platinum suppression of erythropoietin production in cell culture: clinical implications
Authors: Horiguchi Hyogo , Kayama Fujio , Oguma Etsuko , Willmore William G. , Hradecky Pavel , Bunn H. Franklin
Published: October 2019

Both toxic exposure to cadmium and cancer therapy with cisplatin (CDDP) can induce anemia in patients owing to the insufficient production of erythropoietin (EPO). Therefore, the effects of cadmium chloride (Cd) and CDDP in the Hep3B human hepatoma cell line, which up-regulates EPO expression in response to hypoxia and cobalt (Co), were investigated. The induction of binding activity of the HIF-1 transcription factor and EPO mRNA expression and protein production were suppressed by Cd and CDDP in a dose-dependent manner with no apparent cell damage. Mercuric chloride also suppressed hypoxia- and Co-induced EPO production, mRNA expression, and HIF-1 binding in a manner similar to Cd and CDDP, whereas zinc chloride suppressed Co-induced EPO production, mRNA expression, and HIF-1 binding but did not affect hypoxia induction or that observed after simultaneous exposure to hypoxia and Co. In contrast, lead and tin salts had no effect on HIF-1 activation or EPO expression. These results indicate that Cd and CDDP have a strong and specific inhibitory effect on hypoxia- and Co-induced signaling and EPO induction in hepatic cells. It is likely that these agents cause anemia by directly impacting EPO production in the kidney.
Characterization of dendritic cell differentiation pathways from cord blood CD34+CD7+CD45RA+hematopoietic progenitor cells
Authors: Canque Bruno , Camus Sandrine , Dalloul Ali , Kahn Edmond , Yagello Micaël , Dezutter-Dambuyant Colette , Schmitt Daniel , Schmitt Christian , Gluckman Jean Claude
Published: October 2019

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD7+CD45RA+ progenitor population found among cord blood CD34+ cells. Unlike CD7−CD45RA+ and CD7+CD45RA− progenitors, this population displayed high natural killer (NK) cell differentiation capacity when cultured with stem cell factor (SCF), interleukin (IL)-2, IL-7, and IL-15, attesting to its lymphoid potential. In cultures with SCF, Flt3 ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α (standard condition), CD7+CD45RA+ progenitors expanded less (37- vs 155-fold) but yielded 2-fold higher CD1a+ DC percentages than CD7−CD45RA+ or CD7+CD45RA− progenitors. As reported for CD34+CD1a− thymocytes, cloning experiments demonstrated that CD7+CD45RA+ cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7−CD45RA+ and CD7+CD45RA+ progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7+CD45RA+ or thymic progenitors differentiated into Lag+S100+Langerhans cells in the absence of exogenous transforming growth factor (TGF)-β1. Analysis of the DC differentiation pathways showed that CD7+CD45RA+ progenitors generated CD1a+CD14− precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a−CD14+ precursors that readily differentiated into DCs under the standard condition. Accordingly, CD7+CD45RA+ progenitor-derived mature DCs produced 2- to 4-fold more IL-6, IL-12, and TNF-α on CD40 ligation and elicited 3- to 6-fold higher allogeneic T-lymphocyte reactivity than CD7−CD45RA+ progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD34+CD7+CD45RA+ progenitors represent an original population for their developmental pathways and function.
Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation
Authors: Sahni Abha , Francis Charles W.
Published: October 2019

Vascular development and response to injury are regulated by several cytokines and growth factors including the members of the fibroblast growth factor and vascular endothelial cell growth factor (VEGF) families. Fibrinogen and fibrin are also important in these processes and affect many endothelial cell properties. Possible specific interactions between VEGF and fibrinogen that could play a role in coordinating vascular responses to injury are investigated. Binding studies using the 165 amino acid form of VEGF immobilized on Sepharose beads and soluble iodine 125 (125I)–labeled fibrinogen demonstrated saturable and specific binding. Scatchard analysis indicated 2 classes of binding sites with dissociation constants (Kds) of 5.9 and 462 nmol/L. The maximum molar binding ratio of VEGF:fibrinogen was 3.8:1. Further studies characterized binding to fibrin using 125I-labeled VEGF- and Sepharose-immobilized fibrin monomer. These also demonstrated specific and saturable binding with 2 classes of sites havingKds of 0.13 and 97 nmol/L and a molar binding ratio of 3.6:1. Binding to polymerized fibrin demonstrated one binding site with a Kd of 9.3 nmol/L. Binding of VEGF to fibrin(ogen) was independent of FGF-2, indicating that there are distinct binding sites for each angiogenic peptide. VEGF bound to soluble fibrinogen in medium and to surface immobilized fibrinogen or fibrin retained its capacity to support endothelial cell proliferation. VEGF binds specifically and saturably to fibrinogen and fibrin with high affinity, and this may affect the localization and activity of VEGF at sites of tissue injury.
Roles of SLP-76, phosphoinositide 3-kinase, and gelsolin in the platelet shape changes initiated by the collagen receptor GPVI/FcRγ-chain complex
Authors: Falet Hervé , Barkalow Kurt L. , Pivniouk Vadim I. , Barnes Michael J. , Geha Raif S. , Hartwig John H.
Published: October 2019

How platelet shape change initiated by a collagen-related peptide (CRP) specific for the GPVI/FcRγ-chain complex (GPVI/FcRγ-chain) is coupled to SLP-76, phosphoinositide (PI) 3-kinase, and gelsolin is reported. As shown by video microscopy, platelets rapidly round and grow dynamic filopodial projections that rotate around the periphery of the cell after they contact a CRP-coated surface. Lamellae subsequently spread between the projections. All the actin-driven shape changes require SLP-76 expression. SLP-76 is essential for the Ca++mobilization induced by CRP, whereas PI 3-kinase only modulates it. The extension of lamellae requires net actin assembly and an exposure of actin filament barbed ends downstream of PI 3-kinase. Gelsolin expression is also required for the extension of lamellae, but not for the formation of filopodia. Altogether, the data describe the role of SLP-76 in the platelet activation initiated by GPVI/FcRγ-chain and the roles of PI 3-kinase and gelsolin in lamellae spreading.
Endothelial [Ca2+]i signaling during transmigration of polymorphonuclear leukocytes
Authors: Su Wen-Hong , Chen Hsiun-ing , Huang Ji-ping , Jen Chauying J.
Published: October 2019

Vascular endothelium plays an important role in regulating the transendothelial migration of polymorphonuclear leukocytes (PMNs). In this study, the intracellular calcium ion ([Ca2+]i) signaling of endothelial cells (ECs) during PMN transmigration was examined at the single-cell level. Human umbilical vein ECs were cultured on a thin layer of collagen gel. The ECs were labeled with fura-2, immersed in formyl-Met-Leu-Phe, and subsequently perfused with fresh buffer to establish a gradient of chemoattractant across the EC monolayer. The entire process of PMN rolling on, adhering to, and transmigrating across the EC monolayer was recorded under both phase-contrast and fluorescence optics. The data showed the following: (1) At high concentration (approximately 3 × 106/mL), both PMN suspension and its supernatant stimulated frequent EC [Ca2+]i elevations across the monolayer; (2) when used at lower concentration (approximately 5 × 105/mL) to avoid the interference of soluble factors, PMN transmigration, but not rolling or adhesion, was accompanied by EC [Ca2+]i elevation; (3) the latter EC [Ca2+]i elevation occurred simultaneously in ECs adjacent to the transmigration site, but not in those that were not in direct contact with the transmigrating PMNs; (4) this EC [Ca2+]i elevation was an initial and required event for PMN transmigration; and (5) PMNs pretreated with 5,5′-dimethyl-1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid transmigrated with the accompanying EC [Ca2+]i elevation, but they became elongated in the collagen gel. In conclusion, PMNs induce adjacent EC [Ca2+]i signaling, which apparently mediates the “gating” step for their subsequent transmigration.
Thymomas alter the T-cell subset composition in the blood: a potential mechanism for thymoma-associated autoimmune disease
Authors: Hoffacker Viola , Schultz Anja , Tiesinga James J. , Gold Ralf , Schalke Berthold , Nix Wilfred , Kiefer Reinhard , Müller-Hermelink Hans Konrad , Marx Alexander
Published: October 2019

Thymomas are the only tumors that are proven to generate mature T cells from immature precursors. It is unknown, however, whether intratumorous thymopoiesis has an impact on the peripheral T-cell pool and might thus be related to the high frequency of thymoma-associated myasthenia gravis. This study shows, using fluorescence-activated cell sorting-based analyses and T-cell proliferation assays, that thymopoiesis and T-cell function in thymomas correspond with immunologic alterations in the blood. Specifically, the proportion of circulating CD45RA+CD8+ T cells is significantly increased in patients with thymoma compared with normal controls, in accordance with intratumorous T-cell development that is abnormally skewed toward the CD8+ phenotype. Moreover, it is primarily the proportion of circulating CD45RA+CD8+ T cells that decreases after thymectomy. The results also demonstrate that T cells reactive toward recombinant autoantigens are distributed equally between thymomas and blood, whereas T-cell responses to foreign antigen (ie, tetanus toxoid) are seen only among circulating T cells and not among thymoma-derived T cells. These functional studies support the hypothesis that thymopoiesis occurring within thymomas alters the peripheral T-cell repertoire. Because many thymomas are enriched with autoantigen-specific T cells, a disturbance of circulating T-cell subset composition by export of intratumorous T cells may contribute to paraneoplastic autoimmune disease arising in patients with thymoma.
AML-1/ETO fusion protein is a dominant negative inhibitor of transcriptional repression by the promyelocytic leukemia zinc finger protein
Authors: Melnick Ari , Carlile Graeme W. , McConnell Melanie J. , Polinger Adam , Hiebert Scott W. , Licht Jonathan D.
Published: October 2019

The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RARα fusion protein and, in a similar manner, inhibits RARα target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RARα and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia.
Identification of alloreactive T-cell epitopes on the Rhesus D protein
Authors: Stott Lisa-Marie , Barker Robert N. , Urbaniak Stanislaw J.
Published: October 2019

Although considerable effort has been devoted to characterizing alloantibodies specific for the Rhesus D (RhD) blood group antigen, virtually nothing is known about the helper response that drives their production. Therefore, the aim of this study was to map alloreactive T-cell epitopes on the RhD protein. Peripheral blood mononuclear cells (PBMCs) were obtained from 22 RhD-negative volunteers in whom anti-D alloantibodies had developed after deliberate immunization or RhD-incompatible pregnancy. The PBMCs were stimulated with a panel of up to 68 overlapping synthetic 15-mer peptides spanning the complete sequence of the RhD protein. One or more peptides elicited proliferative responses by PBMCs from all 22 of the alloimmune volunteers but from only 2 of 8 alloantibody-negative control donors. Proliferation of PBMCs from the alloimmune donors was mediated by major histocompatibility complex class II–restricted T cells expressing the CD45RO marker of previous activation or memory. The number of peptides that induced proliferative responses was unrelated to either the frequency of, or time since, exposure to RhD-positive red blood cells, but it correlated strongly (Rs = 0.75;P < .003) with the level of anti-D antibodies in deliberately immunized donors. The patterns of stimulatory peptides varied among alloimmune volunteers, but particular sequences were commonly recognized, with 4 peptides each eliciting a response in more than 50% of these donors. Identification of such peptides containing dominant alloreactive helper epitopes is the first step in the development of improved or new approaches to preventing hemolytic disease of the newborn that are based on modulating the T-cell response to the RhD protein.
I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo
Authors: Bernardini Giovanni , Spinetti Gaia , Ribatti Domenico , Camarda Grazia , Morbidelli Lucia , Ziche Marina , Santoni Angela , Capogrossi Maurizio C. , Napolitano Monica
Published: October 2019

Several chemokines have been shown to act as angiogenic molecules or to modulate the activity of growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF). The detection of the CC chemokine receptor (CCR) 8 message in human umbilical vein endothelial cells (HUVECs) by reverse transcription– polymerase chain reaction (RT-PCR) and RNase protection assay (RPA), prompted us to investigate the potential role exerted by the CC chemokine I-309, a known ligand of such receptor, in both in vitro and in vivo angiogenesis assays. We show here that I-309 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, and enhances HUVEC differentiation into capillary-like structures in an in vitro Matrigel assay. Furthermore, I-309 is an inducer of angiogenesis in vivo in both the rabbit cornea and the chick chorioallantoic membrane assay (CAM).
Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group study
Authors: Slovak Marilyn L. , Kopecky Kenneth J. , Cassileth Peter A. , Harrington David H. , Theil Karl S. , Mohamed Anwar , Paietta Elizabeth , Willman Cheryl L. , Head David R. , Rowe Jacob M. , Forman Stephen J. , Appelbaum Frederick R.
Published: October 2019

The associations of cytogenetics with complete remission (CR) rates, overall survival (OS), and outcomes after CR were studied in 609 previously untreated AML patients younger than 56 years old in a clinical trial comparing 3 intensive postremission therapies: intensive chemotherapy, autologous transplantation (ABMT), or allogeneic bone marrow transplantation (alloBMT) from matched related donors. Patients were categorized into favorable, intermediate, unfavorable, and unknown cytogenetic risk groups based on pretreatment karyotypes. CR rates varied significantly (P < .0001) among the 4 groups: favorable, 84% (95% confidence interval [CI], 77%-90%); intermediate, 76% (CI, 71%-81%); unfavorable, 55% (CI, 48%-63%); and unknown, 54% (CI, 33%-74%). There was similar significant heterogeneity of OS (P < .0001), with the estimated relative risk of death from any cause being 1.50 (CI, 1.10-2.05), 3.33 (CI, 2.43-4.55), and 2.66 (CI, 1.59-4.45) for the intermediate, unfavorable, and unknown risk groups, respectively, compared with the favorable group. In multivariate analyses, the effects of cytogenetic risk status on CR rate and OS could not be explained by other patient or disease characteristics. Among postremission patients, survival from CR varied significantly among favorable, intermediate, and unfavorable groups (P = .0003), with significant evidence of interaction (P = .017) between the effects of treatment and cytogenetic risk status on survival. Patients with favorable cytogenetics did significantly better following ABMT and alloBMT than with chemotherapy alone, whereas patients with unfavorable cytogenetics did better with alloBMT. Cytogenetic risk status is a significant factor in predicting response of AML patients to therapy; however, to tighten treatment correlates within genetically defined AML subsets, a significantly larger leukemia cytogenetic database is warranted.
Primitive hematopoietic stem cell function in vivo is uniquely high in the CXB-12 mouse strain
Authors: Chen Jichun , Astle Clinton M. , Müller-Sieburg Christa E. , Harrison David E.
Published: October 2019

Bone marrow cells (BMCs) from CXB-12/HiaJ (CXB-12) mice had 14 times the total long-term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. BMCs from each RI line donor were mixed with genetically marked standard competitor BMCs from the BALB/cBy×C57BL/6 F1 (CByB6F1) hybrid, the mice used to produce the RI lines, and the mixtures repopulated lethally irradiated CByB6F1 recipients. Percentages of donor-type erythrocytes and lymphocytes measured the actual long-term repopulating functions of the donor RI lines relative to the standard competitor. CXB-12 BMCs repopulated better after 3 or 6 months than after 1 month, suggesting that the most primitive precursors were involved. Compared to CByB6F1 standard competitor cells, CXB-12 cells repopulated 3 to 12 times as well, with their advantage increasing when higher doses of cells were transplanted, probably because of hybrid resistance of the recipient against low doses. This was far better than expected, because F1 cells normally function 2 to 3 times as well as cells from an inbred strain. In competitive dilution, the advantage resulted from 2 factors: more precursor cells and more function per precursor. In the model that best fit the data, CXB-12 donors had 2.4 times the concentration of hematopoietic stem cells (HSCs) as the CByB6F1 standard, and each HSC repopulated 1.4 times as well. CXB-12 mice did not have elevated erythrocyte and lymphocyte numbers in blood and marrow and did not have unusually elevated concentrations of colony-forming unit spleen, cobblestone colonies, and long-term colony-initiating cells in marrow.
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0
Authors: Glimm Hanno , Oh IL-Hoan , Eaves Connie J.
Published: October 2019

An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G1fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G0/G1and S/G2/M. Interestingly, the G0 cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle–associated change in in vivo stem cell homing, the cultured G0/G1 and S/G2/M CD34+ CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor β1 that increased the G0/G1 fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle–associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo–manipulated grafts.
Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3–independent mechanism
Authors: Gwynn Babette , Ciciotte Steven L. , Hunter Susan J. , Washburn Linda L. , Smith Richard S. , Andersen Sabra G. , Swank Richard T. , Dell'Angelica Esteban C. , Bonifacino Juan S. , Eicher Eva M. , Peters Luanne L.
Published: October 2019

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes ofcno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3–independent mechanism critical to the biogenesis of lysosome-related organelles.
Human CLP36, a PDZ-domain and LIM-domain protein, binds to α-actinin-1 and associates with actin filaments and stress fibers in activated platelets and endothelial cells
Authors: Bauer Kristin , Kratzer Michael , Otte Marcus , de Quintana Karin Luber , Hagmann Jörg , Arnold Georg J. , Eckerskorn Christoph , Lottspeich Friedrich , Siess Wolfgang
Published: October 2019

A 38-kd protein that associates with F-actin structures in activated platelets and endothelial cells was purified, cloned, and characterized. The protein contains an N-terminal PDZ motif, a large intervening sequence, and a C-terminal LIM domain and was identified as the human homolog of rat CLP36. The study showed that CLP36 associates with actin filaments and stress fibers that are formed during shape change and spreading of platelets and during migration and contraction of endothelial cells. CLP36 binds to α-actinin-1 as shown by coimmunoprecipitation, pull-down experiments, yeast 2-hybrid analysis, and blot overlay assays and colocalizes with α-actinin-1 along endothelial actin stress fibers. In contrast to α-actinin-1, CLP36 was absent from focal adhesions in both activated platelets and endothelial cells. The N-terminal part of CLP36 containing the PDZ domain and the intervening region, but not the LIM domain, targeted enhanced green fluorescent protein fusion proteins to stress fibers in endothelial cells. Yeast 2-hybrid analysis demonstrated that the intervening sequence, but not the PDZ or the LIM domain of CLP36, binds to the spectrinlike repeats 2 and 3 of α-actinin-1. The study further shows that CLP36 binds to α-actinin in resting platelets and translocates as a CLP36/α-actinin complex to the newly formed actin cytoskeleton in activated platelets. The results indicate that CLP36 binds via α-actinin-1 to actin filaments and stress fibers in activated human platelets and endothelial cells. The study suggests that CLP36 may direct α-actinin-1 to specific actin structures and at this position might modulate the function of α-actinin-1.
Antiadhesive function of 130-kd glycoform of CD43 expressed in CD4 T-lymphocyte clones and transfectant cell lines
Authors: Fukuoka Mami , Fukudome Kenji , Yamashita Yoshio , Tokushima Miyoko , Miyake Kensuke , Kimoto Masao
Published: October 2019

Conflicting findings regarding proadhesion and antiadhesion in cell-to-cell interactions were previously reported for CD43. We examined possible differences in the role of the 130-kd glycoform and the 115-kd glycoform of CD43 in cellular adhesion in vitro. We generated a monoclonal antibody (MFT3) that discriminates between helper and nonhelper murine T-cell clones. Characterization of MFT3 with use of biochemical analysis and complementary DNA (cDNA) transfection experiments showed that it is specific for the 130-kd glycoform of CD43. T-cell clones that expressed the 130-kd CD43 glycoform showed decreased homocytic aggregation and decreased adhesion to spleen cells, B-lymphoma cell lines, and fibroblastic cell lines compared with T-cell clones negative for the 130-kd glycoform. Expression of core 2 β-1, 6-N-acetylglucosaminyltransferase (C2GnT) cDNA together with CD43 cDNA resulted in expression of both the 130-kd CD43 glycoform and the 115-kd CD43 glycoform in fibroblastic cell lines. Using these cell lines, we showed that the 130-kd glycoform but not the 115-kd glycoform of CD43 has an antiadhesive function in cellular interactions. Our findings suggest that the antiadhesive function of CD43 is primarily carried out by the 130-kd glycoform.
Ligation of E-cadherin on in vitro–generated immature Langerhans-type dendritic cells inhibits their maturation
Authors: Riedl Elisabeth , Stöckl Johannes , Majdic Otto , Scheinecker Clemens , Knapp Walter , Strobl Herbert
Published: October 2019

Epithelial tissues of various organs contain immature Langerhans cell (LC)-type dendritic cells, which play key roles in immunity. LCs reside for long time periods at an immature stage in epithelia before migrating to T-cell–rich areas of regional lymph nodes to become mature interdigitating dendritic cells (DCs). LCs express the epithelial adhesion molecule E-cadherin and undergo homophilic E-cadherin adhesion with surrounding epithelial cells. Using a defined serum-free differentiation model of human CD34+hematopoietic progenitor cells, it was demonstrated that LCs generated in vitro in the presence of transforming growth factor β1 (TGF-β1) express high levels of E-cadherin and form large homotypic cell clusters. Homotypic LC clustering can be inhibited by the addition of anti–E- cadherin monoclonal antibodies (mAbs). Loss of E-cadherin adhesion of LCs by mechanical cluster disaggregation correlates with the rapid up-regulation of CD86, neo-expression of CD83, and diminished CD1a cell surface expression by LCs—specific phenotypic features of mature DCs. Antibody ligation of E-cadherin on the surfaces of immature LCs after mechanical cluster disruption strongly reduces the percentages of mature DCs. The addition of mAbs to the adhesion molecules LFA-1 or CD31 to parallel cultures similarly inhibits homotypic LC cluster formation, but, in contrast to anti–E-cadherin, these mAbs fail to inhibit DC maturation. Thus, E-cadherin engagement on immature LCs specifically inhibits the acquisition of mature DC features. E-cadherin–mediated LC maturation suppression may represent a constitutive active epithelial mechanism that prevents the uncontrolled maturation of immature LCs.
Similar pattern of thymic-dependent T-cell reconstitution in infants with severe combined immunodeficiency after human leukocyte antigen (HLA)–identical and HLA-nonidentical stem cell transplantation
Authors: Müller Susanna M. , Kohn Thomas , Schulz Ansgar S. , Debatin Klaus-Michael , Friedrich Wilhelm
Published: October 2019

Donor T cells after stem cell transplantation reconstitute by 2 different pathways: by expansion from grafted, mature T cells and by intrathymic maturation from progenitor cells. This study characterized thymic-dependent reconstitution of CD4+ T cells following different transplant modalities in patients with severe combined immunodeficiency (SCID). Three groups of patients were studied: one group after transplantation from human leukocyte antigen (HLA)–identical siblings with unmanipulated grafts without conditioning, a second group after transplantation from HLA-nonidentical parents with T-cell–depleted grafts without preconditioning, and a third group with prior conditioning. Reconstitution of the T-cell compartment was monitored by determining the expression of CD45 isoforms by developing CD4+ cells in the peripheral blood and in discriminating expanded (CD45RO+) and newly generated (CD45RA+) T cells. Concomitantly, changes in the size of the thymus were evaluated sequentially by ultrasonography. Reconstitution of CD4+CD45RA+ cells was delayed in all patients for several months, including patients after HLA-identical transplantation, and was always paralleled by normalization of the size of the thymus. No engraftment of donor progenitor cells was observed, as studied in one patient transplanted without conditioning. CD4+CD45RO+ cells were detected early after transplantation only in patients given unmanipulated grafts. The study showed that thymic-dependent T-cell maturation in these patients with SCID runs an autonomous course, independent of graft manipulation, of major HLA disparities, and of whether conditioning is used or not. In addition, thymic maturation may not require engraftment of donor-derived CD34+ cells in the marrow.
Abnormalities of primitive myeloid progenitor cells expressing granulocyte colony-stimulating factor receptor in patients with severe congenital neutropenia
Authors: Nakamura Kazuhiro , Kobayashi Masao , Konishi Nakao , Kawaguchi Hiroshi , Miyagawa Shin-ichiro , Sato Takashi , Toyoda Hidemi , Komada Yoshihiro , Kojima Seiji , Katoh Osamu , Ueda Kazuhiro
Published: October 2019

To define the basis for faulty granulopoiesis in patients with severe congenital neutropenia (SCN), the expression of granulocyte colony-stimulating factor receptor (G-CSFR) in primitive myeloid progenitor cells and their responsiveness to hematopoietic factors were studied. Flow cytometric analysis of bone marrow cells based on the expression of CD34, Kit receptor, and G-CSFR demonstrated a reduced frequency of CD34+/Kit+/ G-CSFR+cells in patients with SCN. The granulocyte-macrophage colony formation of CD34+/Kit+/G-CSFR+ cells in patients was markedly decreased in response to G-CSF alone and to the combination of stem cell factor, the ligand for flk2/flt3, and IL-3 with or without G-CSF in serum-deprived semisolid culture. In contrast, no difference in the responsiveness of CD34+/Kit+/G-CSFR− cells was noted between patients with SCN and subjects without SCN. These results demonstrate that the presence of qualitative and quantitative abnormalities of primitive myeloid progenitor cells expressing G-CSFR may play an important role in the impairment of granulopoiesis in patients with SCN.
Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia
Authors: Kitada Shinichi , Zapata Juan M. , Andreeff Michael , Reed John C.
Published: October 2019

Compounds that inhibit protein kinases are currently undergoing clinical evaluation for the treatment of a variety of malignancies. The kinase inhibitors flavopiridol and 7 hydroxy-staurosporine (UCN-01) were examined for their effects on B-cell chronic lymphocytic leukemia (B-CLL) cells in vitro (n = 49). Flavopiridol and UCN-01 induced concentration-dependent apoptosis of most B-CLL samples tested, with greater than 50% cell killing occurring at concentrations of less than 1 μmol/L, and with flavopiridol displaying more potent activity than UCN-01. Flavopiridol (0.1 μmol/L) and UCN-01 (1 μmol/L) also induced striking decreases in the levels of the antiapoptosis proteins Mcl-1, X-linked inhibitor of apoptosis (XIAP), and BAG-1 in nearly all cases of B-CLL and of Bcl-2 in approximately half of B-CLL specimens evaluated. In contrast, expression of the proapoptotic proteins Bax and Bak was not significantly influenced by these kinase inhibitors. Flavopiridol-induced decreases in the levels of antiapoptosis proteins Mcl-1 and XIAP preceded apoptosis and were not substantially affected by the addition of caspase inhibitors to cultures. In contrast, UCN-01–stimulated decreases in antiapoptosis proteins were slower, occurred concurrently with apoptosis, and were partially prevented by caspase inhibitors. The findings suggest that flavopiridol and UCN-01 induce apoptosis of B-CLL cells through different mechanisms. The potent apoptotic activities of flavopiridol and UCN-01 against cultured B-CLL cells suggest that they may be effective as single agents in the treatment of B-CLL or for sensitizing B-CLL cells to conventional cytotoxic drugs.
The C-class chemokine, lymphotactin, impairs the induction of Th1-type lymphokines in human CD4+ T cells
Authors: Cerdan Chantal , Serfling Edgar , Olive Daniel
Published: October 2019

Chemokines are involved in the regulation of leukocyte migration and for some of them, T-cell costimulation. To date, the only direct property of lymphotactin (Lptn), the unique member of the C class of chemokines, consists of T-cell chemoattraction. This report describes a novel function for Lptn in human T-lymphocyte biology, by demonstrating the direct ability of Lptn to both inhibit and costimulate CD4+ and CD8+ T-cell activation, respectively. Lptn but not RANTES inhibited CD4+ T-cell proliferation, through a decreased production of Th1 (interleukin [IL]-2, interferon [IFN]-γ) but not Th2 (IL-4, IL-13) lymphokines, and decreased IL-2R expression. Transfections in Jurkat cells showed a Lptn-mediated transcriptional down-regulation of gene-promoter activities specific for Th1-type lymphokines, as well as of nuclear factor of activated T cells (NF-AT) but not AP-1 or NF-ΚB enhancer activities. This suppressive action of Lptn could be compensated by overexpression of NF-ATc but not NF-ATp. CD4+ T-cell proliferation was completely restored by exogenous IL-2 or reversed by pertussis toxin, wortmannin, and genistein, suggesting the involvement of multiple partners in Lptn signaling. In contrast to CD4+ cells, Lptn exerted a potent costimulatory activity on CD8+ T-cell proliferation and IL-2 secretion. These data provide important insights into the role of Lptn in differential regulation of normal human T-cell activation and its possible implication in immune response disorders.
A case-controlled study on the use of HBsAg-positive donors for allogeneic hematopoietic cell transplantation
Authors: Lau George K. K. , Lie Albert K. W. , Kwong Yok Lam , Lee Cheuk Kwong , Hou Jinlin , Lau Yu Lung , Lim Wei Ling , Liang Raymond
Published: October 2019

To compare the clinical and serological outcomes of patients receiving donors' marrow positive or negative for hepatitis B surface antigen (HBsAg), we studied 18 patients of allogeneic hematopoietic cell transplantation receiving HBsAg-positive marrow (group 1) and 18 receiving HBsAg-negative marrow (group 2). The recipients of the 2 groups were matched for hepatitis B virus (HBV) serology, sex, age, underlying hematological diseases, conditioning regimen, and prophylaxis against graft-versus-host diseases. Eight (44.4%) recipients in group 1 and 2 (11.1%) in group 2 suffered from HBV-related hepatitis posttransplant (P = .03). Furthermore, HBV-related hepatic failure was seen in 6 group 1 patients, but in none of the group 2 patients (P = .007). Five of the 9 (55.5%) HBsAg-negative recipients in group 1 became positive after receiving HBsAg-positive marrow. Serum HBV DNA was positive in all 5 donors of these patients, but in none of the donors of recipients who remained HBsAg negative (P = .008). Group 1 patients developing HBV-related hepatitis posttransplant were more likely to have a donor carrying a precore A1896 and/or core promoter T1762/A1764 HBV variant (62.5% versus 0%, P = .007). This study has demonstrated that a high incidence of HBV-related hepatitis was associated with the use of HBsAg-positive marrow for transplant, and a high viral load in the donor appeared to predispose recipients to the development of HBV-related hepatitis posttransplant. Further clinical trials will be necessary to determine the optimal management approach to this problem, including the use of the antiviral agents in the donors and the recipients.
Myeloid specific human CD33 is an inhibitory receptor with differential ITIM function in recruiting the phosphatases SHP-1 and SHP-2
Authors: Paul Sujatha P. , Taylor Lynn S. , Stansbury Eryn K. , McVicar Daniel W.
Published: October 2019

CD33 is a myeloid specific member of the sialic acid-binding receptor family and is expressed highly on myeloid progenitor cells but at much lower levels in differentiated cells. Human CD33 has two tyrosine residues in its cytoplasmic domain (Y340 and Y358). When phosphorylated, these tyrosines could function as docking sites for the phosphatases, SHP-1 and/or SHP-2, enabling CD33 to function as an inhibitory receptor. Here we demonstrate that CD33 is tyrosine phosphorylated in the presence of the phosphatase inhibitor, pervanadate, and recruits SHP-1 and SHP-2. Co-expression studies suggest that the Src-family kinase Lck is effective at phosphorylating Y340, but not Y358, suggesting that these residues may function in the selective recruitment of adapter molecules and have distinct functions. Further support for overlapping, but nonredundant, roles for Y340 and Y358 comes from peptide-binding studies that revealed the recruitment of both SHP-1 and SHP-2 to Y340 but only SHP-2 to Y358. Analysis using mutants of SHP-1 demonstrated that binding Y340 of CD33 was primarily to the amino Src homology-2 domain of SHP-1. The potential of CD33 to function as an inhibitory receptor was demonstrated by its ability to down-regulate CD64-induced calcium mobilization in U937. The dependence of this inhibition on SHP-1 was demonstrated by blocking CD33-mediated effects with dominant negative SHP-1. This result implies that CD33 is an inhibitory receptor and also that SHP-1 phosphatase has a significant role in mediating CD33 function. Further studies are essential to identify the receptor(s) that CD33 inhibits in vivo and its function in myeloid lineage development.
Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells
Authors: Cancelas Jose A. , Koevoet Wendy L. M. , de Koning Alexandra E. , Mayen Angelique E. M. , Rombouts Elwin J. C. , Ploemacher Rob E.
Published: October 2019

Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area–forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells.
VLA-4 (α4β1) engagement defines a novel activation pathway for β2 integrin–dependent leukocyte adhesion involving the urokinase receptor
Authors: May Andreas E. , Neumann Franz-Josef , Schömig Albert , Preissner Klaus T.
Published: October 2019

During acute inflammatory processes, β2 and β1 integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (4β1) engagement on β2 integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)–Fc or by 2 anti-CD29 (β1 chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via β2 integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of β2 integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1–transfected Chinese hamster ovary cells, but not to ligands of β1 integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1–Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1–Fc was required for β2 integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-β1 integrin mAb were ineffective. Activation of β2 integrins by 4β1 integrin ligation (VCAM-1–Fc or anti-β1 mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1–Fc or anti-β1 integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of β2 integrin–dependent cell-to-cell adhesion that requires 4β1 integrin ligation for initiation and uPAR as activation transducer.
Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor–dependent interactions
Authors: Chavakis Triantafyllos , Kanse Sandip M. , Lupu Florea , Hammes Hans-Peter , Müller-Esterl Werner , Pixley Robin A. , Colman Robert W. , Preissner Klaus T.
Published: October 2019

Proteolytic cleavage of single-chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind 2-chain high molecular weight kininogen (HKa) that has been previously reported to exert antiadhesive properties as well as to bind to the urokinase receptor (uPAR) on endothelial cells. In this study we defined the molecular mechanisms for the antiadhesive effects of HKa related to disruption of integrin- and uPAR-mediated cellular interactions. Vitronectin (VN) but not fibrinogen or fibronectin-dependent vβ3 integrin–mediated adhesion of endothelial cells was blocked by HKa or its isolated domain 5. In a purified system, HKa but not HK competed for the interaction of VN with vβ3 integrin, because HKa and the isolated domain 5 but not HK bound to both multimeric and native VN in a Zn2+-dependent manner. The interaction between HKa or domain 5 with VN was prevented by heparin, plasminogen activator inhibitor-1, and a recombinant glutathione-S-transferase (GST)-fusion peptide GST-VN (1-77) consisting of the amino terminal portion of VN (amino acids 1-77), but not by a cyclic arginyl-glycyl-aspartyl peptide, indicating that HKa interacts with the amino terminal portion of VN (“somatomedin B region”). Furthermore, we have confirmed that HKa but not HK bound to uPAR and to the truncated 2-domain form of uPAR lacking domain 1 in a Zn2+-dependent manner. Through these interactions, HKa or its recombinant His-Gly-Lys–rich domain 5 completely inhibited the uPAR-dependent adhesion of myelomonocytic U937 cells and uPAR-transfected BAF-3 cells to VN and thereby promoted cell detachment. By immunogold electron microscopy, both VN and HK/HKa were found to be colocalized in sections from human atherosclerotic coronary artery, indicating that the described interactions are likely to take place in vivo. Taken together, HK and HKa inhibit different VN-responsive adhesion receptor systems and may thereby influence endothelial cell- or leukocyte-related interactions in the vasculature, particularly under inflammatory conditions.