bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Pathogenesis of Type I Congenital Plasminogen Deficiency: Expression of Recombinant Human Mutant Plasminogens in Mammalian Cells
Authors: Azuma Hiroyuki , Mima Nobuaki , Shirakawa Mitsuo , Miyamoto Kazumasa , Yamaguchi Hiroshi , Mitsui Takao , Shigekiyo Toshio , Saito Shiro
Published: October 2019

We previously reported the genetic abnormality in a Japanese family with type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation. To characterize the molecular pathogenesis of the disease in this family, we expressed recombinant human wild-type and mutant (rS572P) plasminogens in COS-1 cells. Activation-resistant wild-type and mutant plasminogen stable transfectants in CHO-K1 cells also were established. Transient transfection and metabolic labeling experiments followed by immunoprecipitation analysis showed that the mutant plasminogen was secreted from COS-1 cells in reduced amounts, compared with the wild type. Endo H digestion of the wild-type and mutant plasminogen showed no shift in their migrations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, indicating that both contain complex type oligosaccharide structures and could therefore be secreted. Furthermore, the secretion of activation-resistant mutant plasminogen was significantly reduced. Pulse-chase experiments and Northern blot analysis showed that the impaired secretion of the mutant plasminogen was the consequence of the accumulation of the mutant protein inside the cells but not of reduced plasminogen mRNA. Immunocytochemical staining of stable transfectants also revealed that CHO-K1 cells expressing the activation-resistant mutant plasminogen stained mainly in the perinuclear area, suggesting delayed processing of the mutant protein in the intracellular transport pathway. We conclude that the impaired secretion of mutant plasminogen, due to intracellular accumulation, is the molecular pathogenesis of type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation.
Interleukin-15 Triggers the Proliferation and Cytotoxicity of Granular Lymphocytes in Patients With Lymphoproliferative Disease of Granular Lymphocytes
Authors: Zambello R. , Facco M. , Trentin L. , Sancetta R. , Tassinari C. , Perin A. , Milani A. , Pizzolo G. , Rodeghiero F. , Agostini C. , Meazza R. , Ferrini S. , Semenzato G.
Published: October 2019

The recently cloned cytokine interleukin-15 (IL-15) shares several functional activities with IL-2 in different cell systems. Although IL-15 does not show sequence homology with IL-2, it uses components of the IL-2 receptor (IL-2R) for binding and signal transduction, namely, p75 (β) and the p64 (γ) chains of IL-2R. To evaluate whether IL-15 is involved in the activation of granular lymphocytes (GL) in patients with lymphoproliferative disease of granular lymphocytes (LDGL), we evaluated the ability of IL-15 to stimulate GL proliferation, cytotoxic function, and the role of IL-2R β and γ molecules on relevant cells. Our results show that IL-15 stimulates cell proliferation and cytotoxic activity of GL in LDGL patients. Reverse-transcriptase polymerase chain reaction (RT-PCR) and phenotypic analyses using the anti–IL-2R γ-chain–specific TUGh4 monoclonal antibody (MoAb) indicate that both CD3+ and CD3− GL express the p64 IL-2R, a result previously unknown. IL-15 activity was inhibited by antibodies against p75 and p64 IL-2R chains, while no inhibitory effects are detectable with anti-p55 IL-2R antibody. The association of anti-p75 and anti-p64 IL-2R MoAbs resulted in a nearly complete (95%) inhibition of IL-15–induced GL proliferation. Using RT-PCR analysis, we demonstrated that highly purified CD3+ and CD3− GL did not express mRNA for IL-15 or IL-2. By contrast, a clear-cut IL-15 mRNA signal was detected by RT-PCR in patients' peripheral blood mononuclear cells, with monocytes likely accounting for the source of IL-15 in LDGL patients. However, even in concentrated supernatants from enriched monocyte populations, we could not demonstrate the presence of IL-15 protein. Using anti–IL-15 specific MoAbs, a membrane-bound form of this cytokine was demonstrated both on CD3+ and CD3− LDGL cells. By RT-PCR analysis, purified GL from these patients were found to express the message for IL-15 receptor α chain. Taken together, these results indicate that both CD3+ and CD3− GL are stimulated by IL-15 and that this cytokine mediates its activity through the β and γ chains of the IL-2R, providing further suggestions for the interpretation of the mechanisms that lead to cell expansion in patients with LDGL.
Interleukin-6 Inhibits Fas-Induced Apoptosis and Stress-Activated Protein Kinase Activation in Multiple Myeloma Cells
Authors: Chauhan Dharminder , Kharbanda Surender , Ogata Atsushi , Urashima Mitsuyoshi , Teoh Gerrard , Robertson Michael , Kufe Donald W. , Anderson Kenneth C.
Published: October 2019

Fas belongs to the family of type-1 membrane proteins that transduce apoptotic signals. In the present studies, we characterized signaling during Fas-induced apoptosis in RPMI-8226 and IM-9 multiple myeloma (MM) derived cell lines as well as patient plasma cell leukemia cells. Treatment with anti-Fas (7C11) monoclonal antibody (MoAb) induced apoptosis, evidenced by internucleosomal DNA fragmentation and propidium iodide staining, and was associated with increased expression of c-jun early response gene. We also show that anti-Fas MoAb treatment is associated with activation of stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK); however, no detectable increase in extracellular signal-regulated kinases (ERK1 and ERK2) activity was observed. Because interleukin-6 (IL-6) is a growth factor for MM cells and inhibits apoptosis induced by dexamethasone and serum starvation, we examined whether IL-6 affects anti-Fas MoAb-induced apoptosis and activation of SAPK or p38 MAPK in MM cells. Culture of MM cells with IL-6 before treatment with anti-Fas MoAb significantly reduced both DNA fragmentation and activation of SAPK, without altering induction of p38 MAPK activity. These results therefore suggest that anti-Fas MoAb-induced apoptosis in MM cells is associated with activation of SAPK, and that IL-6 may both inhibit apoptosis and modulate SAPK activity.
Migration of Cells With Immunoglobulin/c-myc Recombinations in Lymphoid Tissues of Mice
Authors: Müller Jürgen R. , Jones Gary M. , Janz Siegfried , Potter Michael
Published: October 2019

Recombinations between c-myc and immunoglobulin (Ig) sequences that typically occur in pristane-induced mouse plasmacytomas were detected in secondary lymphoid tissues from normal mice, chiefly in the gut-associated lymphoid tissue. Based on the analysis of recombination sequences as clonotypic markers, migration of c-myc recombination-positive cells was observed between Peyer's patches and into the intestine. Treatment of plasmacytoma-susceptible BALB/cAn mice with pristane induced proliferation and migration of these cells into mesenteric lymph node, spleen, and oil granuloma within 7 days. Plasmacytoma-resistant strains of mice (DBA/2N, C3H/HeJ, C57BL/6) differed in that (1) they harbored fewer clones (Ig/c-myc recombinations were detected in 33% of resistant mice versus 91% of BALB/cAn mice after pristane treatment); (2) Ig/c-myc-positive cells were rarely detected in the oil granuloma, and (3) c-myc recombined predominantly with the Ig α locus in BALB/cAn mice (72%), but with the Igμ locus in DBA/2N and in C57BL/6 (67%). The results demonstrate that normal mice generate a large number of lymphocytes with aberrant c-myc in intestinal tissues without developing tumors.
Engagement of the Lewis X Antigen (CD15) Results in Monocyte Activation
Authors: Lo Siu K. , Golenbock Douglas T. , Sass Philip M. , Maskati Azmat , Xu Hong , Silverstein Roy L.
Published: October 2019

We previously reported that monocyte adhesion to tumor necrosis factor-α (TNF-α)–treated endothelial cells increased expression of tissue factor and CD36 on monocytes. Using immunological cross-linking to mimic receptor engagement by natural ligands, we now show that CD15 (Lewis X), a monocyte counter-receptor for endothelial selectins may participate in this response. We used cytokine production as a readout for monocyte activation and found that CD15 cross-linking induced TNF-α release from peripheral blood monocytes and cells from the monocytic cell line MM6. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed an increase in steady-state TNF-α mRNA after 3 to 4 hours of cross-linking. CD15 cross-linking also concomitantly increased interleukin-1β (IL-1β) mRNA, while no apparent change was observed in the levels of β-actin mRNA, indicating specificity. To examine transcriptional regulation of cytokine genes by CD15 engagement, a CAT plasmid reporter construct containing IL-1β promoter/enhancer sequences was introduced into MM6. Subsequent cross-linking of CD15 increased CAT activity. CD15 engagement by monoclonal antibody also attenuated IL-1β transcript degradation, demonstrating that signaling via CD15 also had posttranscriptional effects. Nuclear extracts of anti-CD15 cross-linked cells demonstrated enhanced levels of the transcriptional factor activator protein-1, minimally changed nuclear factor-κB, and did not affect SV40 promoter specific protein-1. We conclude that engagement of CD15 on monocytes results in monocyte activation. In addition to its well-recognized adhesive role, CD15 may function as an important signaling molecule capable of initiating proinflammatory events in monocytes that come into contact with activated endothelium.
Cytokine Receptor Expression on Hematopoietic Stem and Progenitor Cells
Authors: McKinstry William J. , Li Chung-Leung , Rasko John E.J. , Nicola Nicos A. , Johnson Gregory R. , Metcalf Donald
Published: October 2019

Hematopoietic stem and progenitor cell populations were obtained by fluorescence activated cell sorting of murine bone marrow (BM) cells into Rhodamine-123lo lineage−Ly6A/E+ c-kit+ (primitive stem cells highly enriched for long-term BM repopulating activity), Rhodamine-123med/hi lineage− Ly6A/E+ c-kit+ (mature stem cells highly enriched for short-term BM repopulating activity and day 13 spleen colony-forming activity) and lineage− Ly6A/E− c-kit+ (enriched for in vitro colony forming cells) populations. Neither stem cell population responds to single cytokines in vitro and each requires the synergistic action of two or more cytokines for proliferation, whereas the progenitor cell population proliferates in response to single cytokines. Since each of these cell populations was sorted as c-kit+, they express receptors for stem cell factor. Cell populations were also analyzed by autoradiography for their ability to specifically bind iodinated cytokines and this revealed that both stem cell populations expressed receptors for interleukin-1α (IL-1α), IL-3, IL-6, and granulocyte colony-stimulating factor (G-CSF ), but lacked receptors for macrophage colony-stimulating factor (M-CSF ), granulocyte-macrophage colony stimulating factor (GM-CSF ), and leukemia inhibitory factor (LIF ). Cells within the progenitor cell population specifically bound IL-3, GM-CSF, G-CSF, IL-6, and IL-1α, whereas no receptors were detected for M-CSF and LIF. Within each cell population examined, heterogeneity was observed in the percentage of cells labeled and the number of receptors per cell. These results suggest that stem cell populations can be further subdivided according to their cytokine receptor profile and it will be of interest to determine if such subpopulations have distinctive functional properties.
Transendothelial Migration of CD34+ and Mature Hematopoietic Cells: An In Vitro Study Using a Human Bone Marrow Endothelial Cell Line
Authors: Möhle Robert , Moore Malcolm A.S. , Nachman Ralph L. , Rafii Shahin
Published: October 2019

To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 μm microporous membrane inserts and placed in 6-well tissue culture plates. Granulocyte-colony stimulating factor (G-CSF )–mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 ± 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 ± 0.1 v 7.8% ± 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% ± 0.8% v 0.3% ± 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% ± 0.4%). While no difference in the expression of the β1-integrin very late activation antigen (VLA)-4 and β2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.
Expression of the Human Immunodeficiency Virus Type-1 Coreceptors CXCR-4 (fusin, LESTR) and CKR-5 in CD34+ Hematopoietic Progenitor Cells
Authors: Deichmann Martin , Kronenwett Ralf , Haas Rainer
Published: October 2019

CD34+ hematopoietic progenitor cells were assessed for mRNA expression of the human immunodeficiency virus type-1 (HIV-1) coreceptors CXCR-4, also termed fusin or LESTR, and CKR-5, also called CC-CKR-5 or CCR-5. The CD34+ cells were obtained from leukapheresis products of 17 patients after granulocyte colony-stimulating factor–supported cytotoxic chemotherapy. Using a two-step enrichment procedure including immunomagnetic bead separation and fluorescence-activated cell sorting, the CD34+ cells had a median purity of 99.8%. Assessing 9 CD34+ cell samples by polymerase chain reaction after reverse transcription (RT-PCR), CXCR-4 mRNA was found in all samples, whereas CKR-5 mRNA was only present in 3 samples, even though a nested PCR was used. Eight additional CD34+ cell samples were sorted according to CD4 expression. Based on a three-color immunofluorescence analysis, the mean relative fluorescence intensity of HLA-DR was smaller on CD34+/CD4+ cells in comparison with CD34+/CD4− cells. CXCR-4 mRNA was found in 5 of 8 CD34+/CD4+ samples and in 7 of 8 CD34+/CD4− samples, whereas CKR-5 mRNA was detected in 2 CD34+/CD4+ samples and in 1 CD34+/CD4− cell sample. Looking at the total number of CD34+ cell samples examined, the proportion of specimens containing CXCR-4 mRNA was 84% in comparison with 24% of specimens positive for CKR-5 mRNA. These data suggest that CD34+/CD4+ hematopoietic progenitor cells, including true stem cell candidates, could be susceptible to HIV-1 infection. Considering the relatively low incidence of CD34+ cell samples containing CKR-5 mRNA, CD34+/CD4+ cells appear to be particularly prone for HIV-1 infection via the CXCR-4 coreceptor. Because this chemokine receptor allows T-cell–tropic HIV-1 strains to infect cells, CD34+ cells expressing CD4 and CXCR-4 might be infected by HIV-1 during later stages of the disease, following a viral phenotype switch from macrophage- to T-cell–tropic HIV-1 strains.
Individual CD34+CD38lowCD19−CD10− Progenitor Cells From Human Cord Blood Generate B Lymphocytes and Granulocytes
Authors: Berardi Anna C. , Meffre Eric , Pflumio Françoise , Katz Andre , Vainchenker William , Schiff Claudine , Coulombel Laure
Published: October 2019

Identification of human hematopoietic stem cells and analysis of molecular mechanisms regulating their function require biological assays that permit differentiation in all hematopoietic lineages simultaneously. In this study, we established conditions that permit the joint expression of the B-lymphoid and myeloid potential from cord blood-derived CD34+CD38lowCD19−/CD10− primitive progenitors that lack B-specific markers and transcripts. When cocultured during 6 weeks with the murine stromal cells MS-5 in the absence of exogenous human cytokines, CD34+CD38lowCD19−CD10− cells generated a high number of CD19+ B cells. Virtually all of these cells expressed a CD34−CD10+CD19+cIgM− phenotype of late pro-B cells and transcripts of Pax-5, λ-like, and μ chain were detected. We further show that 7% of CD34+CD38lowCD19− cells from cord blood, when grown individually with MS-5 cells, generated both CD19+ and CD11b+ cells after 6 weeks. Efficient B-cell differentiation was also observed in vivo after transplantation of human cord blood-derived unfractionated mononuclear cells or CD34+CD19+CD10− cells into immune-deficient mice. In contrast to the in vitro situation, all stages of B-cell differentiation were observed in vivo, including pro-B, pre-B, and sIgM+ B cells. Interestingly, human progenitors with the ability to differentiate along both B-lymphoid and granulocytic pathways were also detected among human CD34+CD38low cells in the marrow of chimeric mice 6 to 7 weeks after transplantation. Both in vitro and in vivo systems will offer an invaluable tool to further identify the lymphoid and myeloid potentialities of primitive progenitor cells isolated from fetal as well as adult human hematopoietic tissues and characterize stromal-derived signals that regulate their function.
Involvement of Retinoic Acid Receptor-α–Mediated Signaling Pathway in Induction of CD38 Cell-Surface Antigen
Authors: Mehta Kapil , McQueen Teresa , Manshouri Taghi , Andreeff Michael , Collins Steven , Albitar Maher
Published: October 2019

Human leukocyte antigen CD38, a 45-kD single-chain, transmembrane glycoprotein, is a bifunctional ectoenzyme that participates in signal transduction pathways involved in the regulation of cell growth and differentiation. In this study, we demonstrate the nature of retinoid receptors involved in retinoic acid–induced expression of CD38 protein in the human myeloblastic leukemia cell line HL-60. We used a variant HL-60 cell line, HL-60R, in which retinoid receptor function has been abrogated by a trans-dominant negative mutation. We introduced the normal retinoic acid receptors (RAR)-α, -β, and -γ or retinoid X receptor (RXR)-α into HL-60R cells by retroviral vector-mediated gene transfer. Based on experiments using these cell lines and receptor-specific synthetic retinoids that preferentially bind to one of the RARs or RXRs, we conclude that RAR-α is involved in retinoid-induced CD38 expression in HL-60 cells. Further evidence included our demonstration that blocking of RAR-α with the antagonist Ro 41-5253 completely suppressed the retinoid-induced expression of CD38 mRNA transcript and the production of CD38 protein in HL-60 cells. Various tissues from transgenic mice that expressed an antisense construct of RAR-α lacked or produced very low levels of CD38. As expected, the tissues from transgenic mice contained 50% to 80% reduced levels of RAR-α. These results suggest that regulation of CD38 expression, both in vitro and in vivo, is under the direct control of RAR-α retinoid receptors.
A Role for the Wnt Gene Family in Hematopoiesis: Expansion of Multilineage Progenitor Cells
Authors: Austin Timothy W. , Solar Gregg P. , Ziegler Francis C. , Liem Linda , Matthews William
Published: October 2019

The microenvironment is a key regulator of hematopoietic stem cells (HSCs) and is a likely source of extracellular factors that control stem cell fate. A better understanding of these microenvironmental factors may come from investigations of developmental cell fate determination in which the critical roles of cell-cell interactions of multipotential cells have been shown. The Wnt gene family is known to regulate the cell fate and cell-cell interactions of multipotential cells in a variety of tissues. Expression of Wnts and of their putative receptors encoded by murine homologs of the Drosophila frizzled gene in hematopoietic tissues was examined by reverse transcriptase-polymerase chain reaction. Wnt-5a and Wnt-10b were expressed in day-11 murine yolk sac, day-14 fetal liver, and fetal liver AA4+ cells. The expression profiles of four murine frizzled homologs, Mfz3-7, were nearly identical to that of Wnt-5a and Wnt-10b. Notably, Wnt-10b was expressed in the fetal liver AA4+ Sca+ c-kit+ (flASK) HSC population. A role for Wnts in HSC fate determination was studied by treatment of HSC populations in culture with soluble WNT proteins. The addition of conditioned media from cells transfected with Wnt-1, Wnt-5a, or Wnt-10b cDNAs to cultures of flASK cells stimulated a sevenfold, eightfold, and 11-fold expansion in cell number, respectively, relative to control media. Removal of WNT-5a from this media by immunodepletion depleted the stimulatory activity from the media, whereas addition of a partially purified WNT-5a stimulated a fivefold expansion relative to control cells. Transduction of flASK cells with a retrovirus bearing a Wnt-5a cDNA enhanced proliferation. We conclude that WNTs stimulate the survival/proliferation of hematopoietic progenitors, demonstrating that WNTs comprise a novel class of hematopoietic cell regulators.
Isolation of a T-Cell Clone Showing HLA-DRB1*0405-Restricted Cytotoxicity for Hematopoietic Cells in a Patient With Aplastic Anemia
Authors: Nakao Shinji , Takami Akiyoshi , Takamatsu Hideyuki , Zeng Weihua , Sugimori Naomi , Yamazaki Hiroto , Miura Yuji , Ueda Mikio , Shiobara Shintaro , Yoshioka Takeshi , Kaneshige Toshihiko , Yasukawa Masaki , Matsuda Tamotsu
Published: October 2019

The existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vβ21+ T-cell clone that was most dominant among Vβ21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti–HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.
Distinctions Between CD8+ and CD4+ T-Cell Regenerative Pathways Result in Prolonged T-Cell Subset Imbalance After Intensive Chemotherapy
Authors: Mackall Crystal L. , Fleisher Thomas A. , Brown Margaret R. , Andrich Mary P. , Chen Clara C. , Feuerstein Irwin M. , Magrath Ian T. , Wexler Leonard H. , Dimitrov Dimiter S. , Gress Ronald E.
Published: October 2019

Rapid recovery of CD4+ T cells after intensive chemotherapy is limited by an age-dependent decline in thymopoiesis. Here we sought to determine whether similar limitations exist for CD8+ T-cell regeneration. After intensive chemotherapy, CD8+ T cells had a faster effective doubling time than CD4+ T cells (median, 12.6 v 28.2 days, P < .05). Accordingly, at 3 months posttherapy, mean CD8+ T-cell number had returned to baseline, whereas mean CD4+ T-cell number was only 35% of pretherapy values (P < .05). These differences were primarily due to very rapid expansion of CD8+CD57+ and CD8+CD28− subsets. At 3 months posttherapy, there was no relationship between age and CD8+ T-cell number (R = −.02), whereas CD4+ T-cell number was inversely related to age (R = −.66) and there were no discernible differences in CD8+ recovery among patients with or without thymic enlargement, whereas CD4+ recovery was enhanced in patients with thymic enlargement after chemotherapy (P < .01). Therefore thymic-independent pathways of T-cell regeneration appear to rapidly regenerate substantial numbers of CD8+, but not CD4+ T cells, resulting in prolonged T-cell subset imbalance after T-cell depletion. These inherent distinctions between CD4+v CD8+ T-cell regeneration may have significant implications for immunotherapeutic strategies undertaken to eradicate minimal residual neoplastic disease after cytoreductive chemotherapy.
Isolation of Human Blood Dendritic Cells Using the CMRF-44 Monoclonal Antibody: Implications for Studies on Antigen-Presenting Cell Function and Immunotherapy
Authors: Fearnley D.B. , McLellan A.D. , Mannering S.I. , Hock B.D. , Hart D.N.J.
Published: October 2019

Dendritic cells (DC) are potent antigen-presenting cells (APC) with the capacity to stimulate a primary T lymphocyte immune response and are therefore of interest for potential immunotherapeutic applications. Freshly isolated DC or DC precursors may be preferable for studies of antigen uptake and the potential control of APC costimulator activity. In this report, we report that the monoclonal antibody CMRF-44 can be used to detect early DC differentiation. The majority of DC circulating in blood do not express any known DC lineage specific markers, but can be identified by CMRF-44 labeling after a brief period of in vitro culture. The sequential acquisition of DC activation antigens allows the identification of two stages of DC maturation/activation. Cytokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF ) and tumor necrosis factor (TNF )α, enhance both phases of this process, whereas CD40-ligand trimer preferentially enhances the final DC maturation to a fully mature, activated phenotype. DC positively selected using CMRF-44 possess potent allostimulatory activity and are efficient at the uptake, processing, and presentation of soluble antigens for both primary and secondary immune responses. CMRF-44+ DC are also more potent than other APC types at restimulation of a chronic myeloid leukemia peptide specific T-cell clone. The use of a purified population of freshly isolated DC may be advantageous in attempts to initiate, maintain, and direct immune responses for immunotherapeutic applications.
Immunolocalization of the ICE/Ced-3–Family Protease, CPP32 (Caspase-3), in Non-Hodgkin's Lymphomas, Chronic Lymphocytic Leukemias, and Reactive Lymph Nodes
Authors: Krajewski Stanislaw , Gascoyne Randy D. , Zapata Juan M. , Krajewska Maryla , Kitada Shinichi , Chhanabhai Mukesh , Horsman Douglas , Berean Ken , Piro Lawrence D. , Fugier-Vivier Isabelle , Liu Yong-Jun , Wang Hong-Gang , Reed John C.
Published: October 2019

Immunohistochemical analysis of the apoptosis-effector protease CPP32 (Caspase-3) in normal lymph nodes, tonsils, and nodes affected with reactive hyperplasia (n = 22) showed strong immunoreactivity in the apoptosis-prone germinal center B-lymphocytes of secondary follicles, but little or no reactivity in the surrounding long-lived mantle zone lymphocytes. Immunoblot analysis of fluorescence-activated cell sorted germinal center and mantle zone B cells supported the immunohistochemical results. In 22 of 27 (81%) follicular small cleaved cell non-Hodgkin's B-cell lymphomas, the CPP32-immunopositive germinal center lymphocytes were replaced by CPP32-negative tumor cells. In contrast, the large cell component of follicular mixed cells (FMs) and follicular large cell lymphomas (FLCLs) was strongly CPP32 immunopositive in 12 of 17 (71%) and in 8 of 14 (57%) cases, respectively, whereas the residual small-cleaved cells were poorly stained for CPP32 in all FLCLs and in 12 of 17 (71%) FMs, suggesting that an upregulation of CPP32 immunoreactivity occurred during progression. Similarly, cytosolic immunostaining for CPP32 was present in 10 of 12 (83%) diffuse large cell lymphomas (DLCLs) and 2 of 3 diffuse mixed B-cell lymphomas (DMs). Immunopositivity for CPP32 was also found in the majority of other types of non-Hodgkin's lymphomas studied. Plasmacytomas were CPP32 immunonegative in 4 of 12 (33%) cases, in contrast to normal plasma cells, which uniformly contained intense CPP32 immunoreactivity, implying downregulation of CPP32 in a subset of these malignancies. All 12 peripheral blood B-cell chronic lymphocyte leukemia specimens examined were CPP32 immunopositive, whereas 3 of 3 small lymphocytic lymphomas were CPP32 negative, suggesting that CPP32 expression may vary depending on the tissue compartment in which these neoplastic B cells reside. The results show dynamic regulation of CPP32 expression in normal and malignant lymphocytes.
Lack of Fc-ε Receptors on Murine Eosinophils: Implications for the Functional Significance of Elevated IgE and Eosinophils in Parasitic Infections
Authors: de Andres Belen , Rakasz Eva , Hagen Michael , McCormik Mike L. , Mueller Allen L. , Elliot David , Metwali Ahmed , Sandor Matyas , Britigan Bradley E. , Weinstock Joel V. , Lynch Richard G.
Published: October 2019

Chronic infection with Schistosoma mansoni induces in humans and mice a Th2-dominant immune response in which eosinophils and IgE are conspicuously elevated. Human eosinophils express IgE receptors that participate in an IgE-dependent eosinophil-mediated ADCC reaction against Schistosomula larvae in vitro. To investigate the expression of IgE receptors on murine eosinophils, they were purified (<95% pure by Giemsa-stained cytospin preparations) from liver granulomas of Schistosoma-infected mice. Flow cytometric analysis showed the absence of the low-affinity IgE receptor Fc-ε RII (CD23) and Mac-2 and the absence of binding of murine IgE. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of granuloma eosinophil mRNA did not detect transcripts for Fc-ε RII or the α-chain of the high-affinity IgE receptor Fc-ε RI, but did detect transcripts that encode Mac-2 and the low-affinity IgG receptors Fc-γ RIIb2, Fc-γ RIII, and the FcR-associated γ-chain. In vitro stimulation of granuloma eosinophils with interleukin-4 (IL-4) did not induce IgE binding, surface expression of Mac-2, or the transcription of Fc-ε receptors (Fc-ε RI, Fc-ε RII/CD23). To investigate normal murine eosinophils, we cultured normal mouse bone marrow cells with recombinant IL-3, recombinant IL-5, and recombinant granulocyte-macrophage colony-stimulating factor, conditions that promote eosinophil differentiation. Flow cytometric analysis of bone marrow-derived eosinophils failed to detect IgE binding or cell surface expression of Fc-ε RII and Mac-2, and RT-PCR analysis of fluorescence-activated cell sorted bone marrow-derived eosinophils failed to detect transcripts that encode Fc-ε RI or Fc-ε RII. These findings show that, in contrast to human eosinophils, murine eosinophils do not express cell surface receptors that bind IgE. However, because IgG receptors (Fc-γ RIIb2, Fc-γ RIII) were present on eosinophils purified from granulomas, we investigated whether they might be involved in eosinophil activation. We found that an oxidative burst in eosinophils could be triggered through their IgG receptors.
Molecular Characterization of PK-LR Gene in Pyruvate Kinase–Deficient Italian Patients
Authors: Zanella Alberto , Bianchi Paola , Baronciani Luciano , Zappa Manuela , Bredi Elena , Vercellati Cristina , Alfinito Fiorella , Pelissero Giovanni , Sirchia Girolamo
Published: October 2019

We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(−2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(−2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3′ untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(−2)c lead to an alteration of the 5′ and 3′ splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.
Cytomegalovirus-Specific T-Cell Immunity in Recipients of Autologous Peripheral Blood Stem Cell or Bone Marrow Transplants
Authors: Reusser Pierre , Attenhofer Rudolf , Hebart Holger , Helg Claudine , Chapuis Bernard , Einsele Herrmann
Published: October 2019

The cytomegalovirus (CMV)-specific CD8+ cytotoxic T-lymphocyte (CTL) and CD4+ T-helper cell (Th) functions were characterized in 15 CMV seropositive recipients of autologous peripheral blood stem cell or bone marrow transplants. These immune functions were evaluated in peripheral blood specimens obtained before and at 1, 2, and 3 months after transplant. For study of CTL activity, blood mononuclear cells were cocultured with CMV-infected autologous fibroblasts for 2 weeks and then tested for cytotoxicity against CMV-infected or mock-infected autologous and HLA-mismatched fibroblasts. The Th response to CMV antigen was assessed by standard lymphoproliferative assay. CMV-specific CD8+ CTL and CD4+ Th responses were detectable in 12 (80%) and 14 (93%) patients, respectively, in the first 3 months after transplantation. A Th response to CMV was always present by the time of first CTL detection. During the posttransplant period, CMV infection occurred in 6 (40%) patients, and detection of CMV-specific CD8+ CTL activity was associated with protection from subsequent CMV infection (P = .002). Among CMV seropositive autograft recipients, CMV-specific CD8+ CTL and CD4+ Th responses are restored in a large proportion of patients in the first 3 months after transplantation, and the presence of a specific CD8+ CTL activity affords protection from CMV infection.
Transplantation of Marrow Cells From Unrelated Donors for Treatment of High-Risk Acute Leukemia: The Effect of Leukemic Burden, Donor HLA-Matching, and Marrow Cell Dose
Authors: Sierra Jorge , Storer Barry , Hansen John A. , Bjerke Jean W. , Martin Paul J. , Petersdorf Effie W. , Appelbaum Frederick R. , Bryant Eileen , Chauncey Thomas R. , Sale George , Sanders Jean E. , Storb Rainer , Sullivan Keith M. , Anasetti Claudio
Published: October 2019

Transplantation of hematopoietic stem cells from an HLA-compatible unrelated volunteer is an option for patients with acute leukemia lacking a family match. However, criteria for patient and donor selection and the most effective transplant procedures, including the number of hematopoietic cells, remain to be defined. We tested factors influencing outcome of 174 patients with primary acute leukemia receiving non–T-cell depleted marrow from unrelated donors. Median patient age was 20 years (range, 0.5 to 54 years). A multivariable analysis found that leukemia in remission at the time of transplantation was associated with improved leukemia-free survival (relative risk [RR] of treatment failure: 0.5, confidence interval [CI]: 0.3 to 0.7), and presence of blasts in the peripheral blood, as opposed to marrow involvement only or isolated extramedullary relapse, was associated with impaired outcome (RR of treatment failure: 2.5, CI: 1.7 to 5.0). The use of donors with a limited HLA-mismatch was associated with decreased leukemic relapse (RR: 0.5, CI: 0.3 to 0.9) but no improvement in leukemia-free survival compared with HLA-matched unrelated donors. Transplantation of a marrow cell dose above the median value of 3.65 × 108/kg was associated with faster neutrophil (RR: 1.5, CI: 1.1 to 2.0) and platelet (RR: 4.5, CI: 2.7 to 7.5) engraftment, and decreased incidence of severe acute graft-versus-host disease (RR: 0.6, CI: 0.4 to 0.9). In patients transplanted in remission, the use of a marrow cell dose above the median translated into less nonleukemic death (RR: 0.2, CI: 0.1 to 0.4) and better leukemia-free survival (RR of treatment failure: 0.3, CI: 0.2 to 0.6). Transplant in remission with a high dose of marrow cells was associated with the best outcome in both children and adults.
Bone Marrow and Thymus Expression of Interferon-γ Results in Severe B-Cell Lineage Reduction, T-Cell Lineage Alterations, and Hematopoietic Progenitor Deficiencies
Authors: Young Howard A. , Klinman Dennis M. , Reynolds Della A. , Grzegorzewski Krzysztof J. , Nii Aisuke , Ward Jerrold M. , Winkler-Pickett Robin T. , Ortaldo John R. , Kenny James J. , Komschlies Kristin L.
Published: October 2019

Interferon-γ (IFN-γ) is an immunoregulatory lymphokine that is primarily produced by T cells and natural killer cells. It has effects on T-cell, B-cell, and macrophage differentiation and maturation. We have developed transgenic mice that express elevated levels of IFN-γ mRNA and protein by inserting multiple copies of murine IFN-γ genomic DNA containing an Ig λ-chain enhancer in the first intron. The founder line carrying eight copies of this transgene has eightfold to 15-fold more IFN-γ–producing cells in the bone marrow and spleen than do nontransgenic littermates. Transgenic mice show a pronounced reduction in B-lineage cells in the bone marrow, spleen, and lymph nodes. In addition, single positive (CD4+,CD8− and CD4−,CD8+) thymocyte numbers are increased twofold, yet the number of splenic T cells is reduced by 50%. There is also a twofold to threefold decrease in the frequency and total number of myeloid progenitors in the bone marrow. Granulomatous lesions and residual degenerating cartilaginous masses are also present in the bones of these mice. Overall, our data show that the abnormal expression of IFN-γ in these transgenic mice results in multiple alterations in the immune system. These animals provide an important model to examine the role of IFN-γ expression on lymphoid and myeloid differentiation and function.
Inactivation of Recombinant Monocyte cAMP-Specific Phosphodiesterase by cAMP Analog, 8-[(4-Bromo-2,3-Dioxobutyl)thio]Adenosine 3′,5′-Cyclic Monophosphate
Authors: Omburo George A. , Torphy Theodore J. , Scott Gilbert , Jacobitz Susanne , Colman Roberta F. , Colman Robert W.
Published: October 2019

Two cAMP analogs, 8- and 2- [(4-bromo-2,3-dioxobutyl) thio]adenosine 3′,5′-cyclic monophosphate (8- and 2-BDB-TcAMP) have been used in probing the catalytic site of recombinant monocyte cAMP-specific phosphodiesterase (PDE4a). 2-BDB-TcAMP is a reversible and competitive inhibitor (Ki = 5.5 μmol/L) of cAMP hydrolysis by PDE4a. 8-BDB-TcAMP irreversibly inactivates the enzyme in a time- and concentration-dependent manner with a second order rate constant of 0.022 mmol/L−1min−1. The rate of inactivation of PDE4a is reduced by the presence of the substrate cAMP and specific inhibitors, rolipram and denbufylline, but not by cGMP or AMP. Reduction of the enzyme-inhibitor complex with sodium [3H]borohydride shows that 1.2 mol of the affinity label/mol of enzyme was incorporated. The radiolabeled peptide is composed of 10 amino acid residues (697 to 706) located near the carboxyl end of the proposed catalytic domain. The peptide (GPGHPPLPDK) has seven nonpolar and aliphatic residues, of which four are proline, giving the peptide a highly structured conformation. This peptide is the first to be identified in the putative catalytic domain involved in substrate recognition.
Glucocorticoids Induce Kaposi's Sarcoma Cell Proliferation Through the Regulation of Transforming Growth Factor-β
Authors: Cai Jie , Zheng Tong , Lotz Martin , Zhang Ya , Masood Rizwan , Gill Parkash
Published: October 2019

Glucocorticoid (GC) use is known to induce or enhance the growth of Kaposi's sarcoma (KS) in many clinical settings including human immunodeficiency virus infection, collagen vascular disease, lymphoproliferative disorders, and renal transplantation. Because GCs may induce immune suppression and thus tumor growth, we determined whether GCs had a direct effect on KS growth. We found that GCs directly induce the growth of KS cell lines. In examining the mechanism of action of GCs, we did not observe induction of known autocrine growth factors for KS including interleukin-1 (IL-1), IL-6, oncostatin-M, basic fibroblast growth factor (bFGF ), and vascular endothelial growth factor (VEGF ). We thus examined factor(s) that inhibit KS growth. Transforming growth factor-β (TGF-β) is produced by KS cells and has pleiotropic effects, including inhibiting the growth of hematopoietic and endothelial cells. We show that TGF-β is produced by KS cells in both the latent and active forms, and that TGF-β is an autocrine growth inhibitory factor. We then studied the effects of GCs on the regulation of TGF-β and found that GCs do not inhibit TGF-β transcription, but significantly inhibit TGF-β activation. This effect is mediated through regulation of the TGF-β activation pathway. TGF-β is activated by plasmin which is positively regulated by plasminogen activator (PA) and PA receptor (PAR), and negatively regulated by plasminogen activator inhibitor (PAI). GCs downregulated PAR and upregulated PAI. Thus, glucocorticoids enhance KS cell growth through the regulation of TGF-β activation.
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6-Benzylguanine Potentiates the In Vivo Toxicity and Clastogenicity of Temozolomide and BCNU in Mouse Bone Marrow
Authors: Chinnasamy Nachimuthu , Rafferty Joseph A. , Hickson Ian , Ashby John , Tinwell Helen , Margison Geoffrey P. , Dexter T. Michael , Fairbairn Leslie J.
Published: October 2019

The effects of treatment of mice with O6-benzylguanine (O6-BeG) on the levels of O6-alkylguanine-DNA alkyltransferase (ATase) in the hematopoietic compartment and on the in vivo sensitivity of hematopoietic progenitor cells to the toxic and clastogenic effects of the antitumor agents 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) and temozolomide were studied. When the overall effects of BCNU alone or with O6-BeG pretreatment were compared, dose potentiating factors of 4.17 for marrow cellularity, 4.57 for granulocyte macrophage-colony forming cells (GM-CFC) and 8.25 for colony forming unit-spleen (CFU-S) in O6-BeG pretreated versus nonpretreated animals were observed. A similar trend of dose potentiation was observed for temozolomide, although it was of lower magnitude: 1.20 for marrow cellularity, 1.63 for GM-CFC, and 1.68 for CFU-S. When the clastogenic effects of BCNU and temozolomide were examined in the mouse bone marrow micronucleus assay, a significantly (P < .05 to .001) higher frequency of micronuclei formation was observed in mice that received O6-BeG pretreatment compared with mice that received no pretreatment. These data suggest that the use of O6-BeG as a tumor-sensitizing agent before treatment of patients with O6-alkylating agents may lead to more severe hematological toxicity and possibly to an increased incidence of secondary leukemias as a result of elevated mutation frequencies in these patients.
Loss of Inducible Virus in CD45RA Naive Cells After Human Immunodeficiency Virus-1 Entry Accounts for Preferential Viral Replication in CD45RO Memory Cells
Authors: Woods Toni C. , Roberts Beverly D. , Butera Salvatore T. , Folks Thomas M.
Published: October 2019

Controversy exists concerning the preferential infection and replication of human immunodeficiency virus-1 (HIV-1) within naive (CD45RA+) and memory (CD45RO+) subsets of CD4+ lymphocytes. To explore the susceptibility of these subsets to HIV-1 infection, we purified CD45RA+/CD4+ (RA) and CD45RO+/CD4+ (RO) cells from normal donors and subjected them to a novel monokine activation culture scheme. Following HIV-1 infection and interleukin-2 (IL-2) induction, viral production measured on day 13 was 19-fold greater in RO cultures compared with RA cultures. IL-2–stimulated proliferation in uninfected control cultures was equivalent. To explore the mechanisms by which RA cells were reduced in viral production capacity, RA and RO cells were exposed to HIV-1 followed by treatment with trypsin, and then phytohemagglutinin antigen (PHA)-stimulated at days 4, 7, and 10 postinfection. HIV-1 production in day 4 postinfection RA and RO cultures was analogous, indicating that viral fusion and entry had occurred in both cell types. However, whereas similarly treated day 7 and 10 postinfection RO cultures produced virus, HIV-1 was markedly reduced or lost in the corresponding RA cultures. These results suggest that a temporally labile postfusion HIV-1 complex exists in unstimulated RA cells that requires cellular activation signals beyond that provided by IL-2 alone for productive infection.