bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibodies to VLA4 Integrin Mobilize Long-Term Repopulating Cells and Augment Cytokine-Induced Mobilization in Primates and Mice
Authors: Craddock Charles F. , Nakamoto Betty , Andrews Robert G. , Priestley Gregory V. , Papayannopoulou Thalia
Published: October 2019

Although the use of cytokine-mobilized peripheral blood stem cells has gained a significant momentum in clinical transplantation, the mobilization schemes practiced are guided by a great deal of empiricism. The mechanism(s) by which cytokines or chemokines, alone or in combination, bring about redistribution of stem/progenitor cells from bone marrow to peripheral blood are poorly understood. Likewise the fate of mobilized stem/progenitor cells and their biological properties are incompletely defined. One of the leading hypotheses to explain the mechanism of cytokine-induced mobilization encompasses the view that cytokines disrupt, directly or indirectly, cytoadhesive interactions of stem/progenitor cells with their bone marrow stroma. Compatible with this view are changes in the expression and/or function of several cytoadhesion molecules, especially integrins, postmobilization, and extensive in vitro experimentation supporting the concept of cytokine/integrin interactions. To provide a further insight on the cytokine/integrin interplay in vivo, we have combined cytokine treatments with anti-integrin treatments for mobilization in primates and mice. We found that anti-VLA4 treatment combined with either granulocyte colony-stimulating factor (G-CSF ) treatment or kit ligand treatment leads to significant enhancement of mobilization efficiency (fivefold to eightfold) well above the levels produced by either cytokine alone or anti-VLA4 treatment alone. Similar enhancement was seen when combinations of cytokines, ie, G-CSF plus kit ligand or G-CSF plus Flt3-ligand were used with anti-VLA4 in primates and mice. Furthermore, when anti-VLA4 was given in 5-Fluorouracil–treated primates, significant numbers of progenitor cells were circulating for several days during the recovery period only in the anti-VLA4 treated animals. These data suggest that (1) the effect of anti-VLA4 on mobilization, when used alone, is unlikely to be mediated by secondary cytokine elaboration in vivo; (2) three different cytokines and their combinations do not appear to influence the in vivo responsiveness to anti-VLA4 in coadministration schemes; (3) even if cytokine treatments on their own exert downmodulation of VLA4 function, the target progenitor cells influenced by anti-VLA4 or by cytokines may not necessarily overlap; and (4) augmentation of mobilization in cytokine/anti-VLA4 treatments is most likely caused by an amplification of the pool of target cells on which anti-VLA4 exerts its effects. Because cytokines or anti-VLA4 are each capable of mobilizing long-term repopulating cells and because we show with the present studies that anti-VLA4 in an autologous bone marrow cell transplantation setting does not cause any delay in engraftment, the combination of cytokine/anti-integrin treatment enhancing mobilization may have a clinical use.
Glycoprotein Ib-von Willebrand Factor Interactions Activate Tyrosine Kinases in Human Platelets
Authors: Asazuma Naoki , Ozaki Yukio , Satoh Kaneo , Yatomi Yutaka , Handa Makoto , Fujimura Yoshihiro , Miura Shuji , Kume Shoji
Published: October 2019

von Willebrand factor (vWF ) in the presence of botrocetin induces p72syk activation, assessed as its autophosphorylated level and in vitro kinase assays, the transient association of p72syk with p60c-src, and the translocation of p60c-src and p54/58lyn to cytoskeletal fractions. Jararaca glycoprotein Ib-binding protein (GPIb-BP), which specifically binds to GPIb, abolished these phenomena, suggesting that they are mediated by the vWF-GPIb interaction. These tyrosine kinase-related events were not inhibited by GRGDS peptide (plus EGTA), indicating that GPIIb/IIIa is not involved in the observed responses. Shc, an adaptor protein, was also tyrosine phosphorylated by the botrocetin-vWF activation. When GPIb was immunoprecipitated with nonfunctional monoclonal antibodies (MoAbs) directed against GPIb, a kinase activity was found to associate with GPIb upon botrocetin-vWF activation. On the other hand, anti-GPIb MoAbs that inhibit the vWF-GPIb interaction did not coprecipitate a kinase activity. Because the recovery of GPIb did not differ significantly, it is suggested that the excessive presence of inhibitory anti-GPIb MoAb dissociated a kinase activity from GPIb. Phosphoamino acid analysis showed that the kinase activity was that of a tyrosine kinase. The identity of the tyrosine kinase and the mode of interaction with the cytoplasmic region of GPIb await to be determined. Our findings suggest that the tyrosine kinase associated with GPIb serves at a most proximal step in the signal transduction pathway involved in the vWF-GPIb-induced platelet activation, which leads to other tyrosine kinase-related intracellular signals.
Multiple Quinine-Dependent Antibodies in a Patient With Episodic Thrombocytopenia, Neutropenia, Lymphocytopenia, and Granulomatous Hepatitis
Authors: Hou Ming , Horney Eva , Stockelberg Dick , Jacobsson Stefan , Kutti Jack , Wadenvik Hans
Published: October 2019

A 58-year-old man experienced episodes of fever, vomiting, and diarrhea over a 2-year period. The laboratory evaluation during these attacks consistently disclosed thrombocytopenia, leukopenia, and elevated liver enzymes. A liver biopsy performed at one of these attacks showed a typical picture of granulomatous hepatitis. In retrospect, all episodes seemed to be associated with the ingestion of quinine. Indeed, such a correlation was established by a challenge with quinine. By using flow cytometry, quinine-dependent IgG antibodies to platelets were detected in the patient serum. By a two-color flow cytometric assay, the patient serum was also found to hold quinine-dependent antibodies specific for neutrophils, T lymphocytes, and B lymphocytes. Moreover, serum absorbed with neutrophils in the presence of quinine continued to react with platelets, T lymphocytes, and B lymphocytes; serum that was absorbed with mononuclear cells continued to react with neutrophils and platelets. These experiments indicated that the antigen targets were different on platelets, neutrophils, and lymphocytes. Further, the patient serum in the presence of quinine immunoprecipitated surface-labeled platelet proteins with electrophoretic mobilities closely resembling those of glycoprotein (GP) Ib/IX and GPIIb/IIIa. By a modified monoclonal antibody–specific immobilization of platelet antigens assay, the patient serum in the presence of quinine reacted with platelet GPIb/IX and GPIIb/IIIa. Also, the patient serum in the presence of quinine immunoprecipitated an uncharacterized 15-kD double-band from surface-labeled granulocyte proteins. We conclude that our patient's thrombocytopenia, neutropenia, and lymphocytopenia were caused by quinine-dependent antibodies and that these antibodies recognized cell lineage-specific epitopes.
Expression of Cytokine Receptors on Follicular Dendritic Cells
Authors: Yamada Kazuhiko , Yamakawa Mitsunori , Imai Yutaka , Tsukamoto Masaru
Published: October 2019

Follicular dendritic cells (FDCs) in the lymphoid follicle (LF) are essential to the sequential processes of B-cell proliferation, selection, and differentiation. Although the importance of some cytokines in these processes has been pointed out, there is little information about the follicular localization of their receptors. We investigated, with special reference to FDCs, the localization of cytokine receptors as well as cytokines themselves in human tonsils by several means, including immunochemistry, immunoelectron microscopy, reverse transcriptase polymerase chain reaction, and in situ hybridization. FDCs in the follicular apical light zone expressed transforming growth factor-β receptor II (TGF-βR II), granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα; CDw116), tumor necrosis factor receptor I (TNFR I; CD120a), interleukin-1 receptor II (IL-1R II; CDw121b), IL-2 receptor β (IL-2Rβ; CD122), IL-4 receptor (IL-4R; CDw124), and IL-6 receptor (IL-6R; CD126), among the 10 receptors examined. Those in the basal light zone expressed strongly TNFR I and weakly GM-CSFR α, IL-1R II, IL-2Rβ, IL-4R, and IL-6R, and often those in the outer and mantle zones expressed GM-CSFR α, IL-4R, and IL-6R. FDCs in the apical light zone expressed only TGF-β among the 7 cytokines examined. On the other hand, follicular lymphocytes mainly in the light zone expressed 9 kinds of receptors, with the exception being TGF-βR II; expression was rather frequent for TNF-α, IL-1α, and IL-2 and less frequent for TGF-β, GM-CSF, IL-4, and IL-6. These data indicate that only FDCs mainly in the light zone express many cytokine receptors, although FDCs may produce the cytokine, TGF-β. Cytokines may act not only on some follicular lymphocytes but also on most FDCs in the light zone expressing cytokine receptors.
Induction of Dendritic Cell Differentiation by Granulocyte-Macrophage Colony-Stimulating Factor, Stem Cell Factor, and Tumor Necrosis Factor α In Vitro From Lineage Phenotypes-Negative c-kit+ Murine Hematopoietic Progenitor Cells
Authors: Zhang Yi , Mukaida Naofumi , Wang Jian-bin , Harada Akihisa , Akiyama Mariko , Matsushima Kouji
Published: October 2019

To elucidate the capacity of murine early hematopoietic progenitor cells (HPCs) to differentiate into dendritic cells (DCs), lineage phenotypes (Lin)−c-kit+ HPCs were highly purified from either wild-type or tumor necrosis factor (TNF) receptor p55 (TNF-Rp55)-deficient mice. Upon culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) for 14 days, wild-type mouse Lin−c-kit+ HPCs did not exhibit characteristic features of DC such as sheet-like projections and veil processes. Moreover, these cells expressed a marginal level of DC markers such as DEC-205, CD86, and barely supported allogenic MLR. However, the addition of mouse TNFα generated a large number of cells with typical DC morphology, expression of high levels of Ia, DEC-205, CD86, and function of stimulating allogenic MLR. Moreover, a proportion of these mature DCs and thymic DCs expressed Thy-1 mRNA as well as Thy-1 antigen, whereas freshly isolated splenic DCs did not. These results suggested that DCs generated in our culture system phenotypically resemble thymic ones. In contrast, mouse TNFα failed to induce TNF-Rp55-deficient mice-derived Lin−c-kit+ HPCs to generate DCs with characteristic morphology, immunophenotype, and accessory function for T cells under the same culture conditions, suggesting a crucial role of TNF-Rp55 in TNFα-mediated DC differentiation from HPCs. Interestingly, human TNFα, which can bind to mouse TNF-Rp55 but not TNF-Rp75, was incapable to augment DC generation from wild-type mouse Lin−c-kit+ HPCs. Collectively, these results suggest that TNFα has a pivotal role in DC generation from murine early HPCs in collaboration with GM-CSF and SCF through the interaction of TNF-Rp55 and TNF-Rp75.
Cyclophosphamide, Doxorubicin, Vincristine, Prednisone Dose Intensification With Granulocyte Colony-Stimulating Factor Markedly Depletes Stem Cell Reserve for Autologous Bone Marrow Transplantation
Authors: Freedman Arnold , Neuberg Donna , Mauch Peter , Gribben John , Soiffer Robert , Anderson Kenneth , Robertson Michael , Fisher David C. , Schlossman Robert , Kroon Mary , Rhuda Catherine , Kuhlman Caroline , Ritz Jerome , Nadler Lee
Published: October 2019

Hematopoietic growth factors allow dose escalation of chemotherapy. This approach may potentially reduce the quality and quantity of hematopoietic stem cells. The capacity of stem cells recovered after dose intensification to support myeloablative therapy is unknown. In patients with previously untreated advanced follicular lymphoma, trilineage hematopoietic engraftment was compared in two sequential trials of induction therapy (standard dose cyclophosphamide, doxorubicin, vincristine, prednisone [CHOP] without growth factors or dose intensification CHOP supported by granulocyte colony-stimulating factor [G-CSF ]) followed by identical myeloablative therapy and autologous stem cell support. Neutrophil, platelet, and red blood cell (RBC) engraftment were compared on days 100, 180, and 360 after stem cell reinfusion. Despite similar patient characteristics including reinfusion of comparable numbers of marrow mononuclear cells, after stem cell transplantation, a highly significant prolongation of neutrophil and platelet engraftment was seen in patients who received high dose CHOP and G-CSF in comparison to standard dose CHOP. These findings suggest that dose intensified chemotherapy and G-CSF recruited stem cells into a proliferative phase and that G-CSF allowed retreatment at a time when stem cells were susceptible to damage by cytotoxic therapy. Such inadequate hematologic engraftment after myeloablative therapy might be avoided by either shortening the time that growth factor support is administered, lengthening the interval between cycles, or attempting to repetitively harvest additional stem cells either from the marrow or peripheral blood. Therefore, intensification of chemotherapy with growth factor support must be used with caution if stem cells are to be used to support myeloablative therapy.
Thrombopoietin in Patients With Congenital Thrombocytopenia and Absent Radii: Elevated Serum Levels, Normal Receptor Expression, But Defective Reactivity to Thrombopoietin
Authors: Ballmaier Matthias , Schulze Harald , Strauβ Gabriele , Cherkaoui Klara , Wittner Nicole , Lynen Stefanie , Wolters Susanne , Bogenberger Jakob , Welte Karl
Published: October 2019

The pathophysiology of thrombocytopenia in the syndrome of thrombocytopenia with absent radii (TAR) is not yet understood. We examined thrombopoietin (TPO) serum levels and the in vitro reactivity of platelets to TPO in five patients affected with TAR syndrome. We found elevated TPO serum levels in all patients tested, excluding a TPO production defect as cause for thrombocytopenia in TAR syndrome. In addition, we found similar expression of the TPO receptor c-Mpl on the surface of platelets from TAR patients (5 of 5) and a similar molecular weight of the receptor as compared with healthy controls (4 of 4). Platelet response to adenosine diphosphate or thrombin receptor agonist peptide SFLLRN (TRAP) was normal in TAR patients. However, in contrast to results with healthy controls we could show absence of in vitro reactivity of platelets from TAR patients to recombinant TPO as measured by testing TPO synergism to adenine diphosphate and TRAP in platelet activation. TPO induced tyrosine phosphorylation of platelet proteins was completely absent (3 of 4) or markedly decreased (1 of 4). Our results indicate that defective megakaryocytopoiesis/thrombocytopoiesis in TAR syndrome is not caused by a defect in TPO production but a lack of response to TPO in the signal transduction pathway of c-Mpl.
The Roles of Bcl-XL and Apopain in the Control of Erythropoiesis by Erythropoietin
Authors: Gregoli Paul A. , Bondurant Maurice C.
Published: October 2019

Erythropoietin (EP) is required by late-stage erythroid progenitor cells to prevent apoptosis. Several lines of evidence suggest that it is this action of EP that regulates erythrocyte production in vivo. To study the control of apoptosis in mouse and human erythroblasts, the expression of members of the Bcl-2 family of proteins and the expression and activation of the apoptosis-linked cysteine protease Yama/CPP32/apopain were examined. These proteins have been implicated as regulators of apoptosis in several cell models. The Bcl-2 family members analyzed were Bcl-2, Bcl-X, Bax, Bad, Bak, A1, and Mcl-1. Bcl-X expression in proerythroblasts was highly EP-dependent. Bcl-X was strongly increased during the terminal differentiation stages of human and mouse erythroblasts, reaching maximum transcript and protein levels at the time of maximum hemoglobin synthesis. This increase in Bcl-X expression led to an apparent level of approximately 50 times the level in proerythroblasts. In contrast, neither mouse nor human erythroblasts expressed Bcl-2 transcript or protein. Bax and Bad proteins remained relatively constant throughout differentiation, but diminished near the time of enucleation. Bak protein was present in early erythroblasts, but diminished progressively during differentiation. EP deprivation in both mouse and human erythroblasts led to activation of the cysteine protease, apopain, as was indicated by cleavage of the proenzyme into its proteolytically active fragments. Apopain activation was detectable within 2 hours of EP deprivation in mouse erythroblasts. These findings suggest an important role for Bcl-X in late erythroid differentiation and for apopain in apoptosis of erythroblasts caused by deprivation of EP.
Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells
Authors: Sui Xingwei , Krantz Sanford B. , Zhao Zhizhuang
Published: October 2019

Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.
Antibodies to HLA Class I α1 Domain Trigger Apoptosis of CD40-Activated Human B Lymphocytes
Authors: Genestier Laurent , Meffre Geneviève , Garrone Pierre , Pin Jean-Jacques , Liu Yong-Jun , Banchereau Jacques , Revillard Jean-Pierre
Published: October 2019

We analyzed herein whether antibodies to HLA class I α1 domain, which trigger apoptosis of activated T cells, may also control the growth/survival of human B lymphocytes. Addition of monoclonal antibody (MoAb) 90 (mouse IgG1) or YTH862 (rat IgG2b) was found to strongly inhibit the proliferation of CD40-activated total tonsil B cells as well as that of purified naive, germinal center, and memory B-cell subsets. This inhibitory effect was not prevented by addition of B-cell tropic factors, such as interleukin-2 (IL-2), IL-4, and IL-10, and was a result of induced B-cell apoptosis as shown by using a TUNEL assay and DNA electrophoresis. In contrast, engagement of another epitope of the α1 domain, as well as that of the α2 and α3 domains by specific anti-HLA class I MoAbs, failed to inhibit DNA synthesis and to induce apoptosis of CD40-activated B cells. As recently reported for acquisition of sensitivity to Fas (APO-1/CD95) -dependent apoptosis, susceptibility to MoAb90-and YTH862-induced death was restricted to CD40-activated B cells, because resting and anti–IgM-activated B cells did not undergo apoptosis after HLA class I engagement. Moreover, ligation of the B-cell receptor protected CD40-activated B cells from both HLA class I- and Fas-mediated growth inhibition and apoptosis. Taken together, these results show that engagement of the α1 domain of HLA class I induces apoptotic cell death of CD40-activated, but not of antigen-activated B cells, and would, therefore, suggest a possible role for HLA class I molecules in the control of B-cell homeostasis.
Lymphomas in Patients With Sjögren's Syndrome Are Marginal Zone B-Cell Neoplasms, Arise in Diverse Extranodal and Nodal Sites, and Are Not Associated With Viruses
Authors: Royer Bruno , Cazals-Hatem Dominique , Sibilia Jean , Agbalika Felix , Cayuela Jean-Michel , Soussi Thierry , Maloisel Frédéric , Clauvel Jean-Pierre , Brouet Jean-Claude , Mariette Xavier
Published: October 2019

The occurrence of non-Hodgkin's lymphoma (NHL) is the most serious complication of Sjögren's syndrome (SS). We performed a study of 16 NHLs occurring in patients with an underlying SS. These lymphomas arose not only in salivary glands (7 cases) but also in other mucosal extranodal sites (the stomach [4 cases], the lung [3 cases], the skin [3 cases], the buccal mucosa [1 case], the thymus [1 case]) and in nodal sites (8 cases). Low-grade marginal zone lymphomas (MZL) were diagnosed in 12 of the 16 patients, 9 of mucosa-associated lymphoid tissues (MALT) type in mucosal sites and 3 exclusively nodal. The 4 other patients presented with a high-grade B-cell lymphoma that was probably a histological transformation of an underlying low-grade MZL at least in 3 of the cases involving skin, stomach, and parotid, respectively. A t(14; 18) translocation was detected in 1 of 8 lymphomas tested. We detected serum anti-p53 antibodies in 2 of the 14 studied patients. p53 protein was detected in 1 of 11 lymphomas tested. LMP protein and Eber RNAs of Epstein-Barr virus (EBV) were not detected in the 16 NHL biopsies. Using polymerase chain reaction, EBV was never detected except in 1 of 4 parotid lymphomas. No human T-lymphotropic virus 1 or human herpes virus 8 DNAs were detected in NHL biopsies. None of the patients had hepatitis C virus infection found using serological methods. Chemotherapy was usually efficient. In conclusion, lymphomas occurring in patients with an underlying SS are in most cases MZL. These lymphomas are not associated with viruses known to be present in other types of lymphomas. Some of the translocations or mutations of oncogenes or antioncogenes described in other lymphomas are detected in SS-associated lymphomas.
A Specific Stimulator of Granulocyte Colony-Stimulating Factor Accelerates Recovery From Cyclophosphamide-Induced Neutropenia in the Mouse
Authors: Fine Jay S. , Cai Xiao-Yan , Justice Luminita , Gommoll Carl P. , Hamilton Linda D. , Waters Tracey A. , Narula Satwant K. , Bober Loretta A. , Grace Michael J.
Published: October 2019

We have identified a small molecular weight compound, SCH 14988, which specifically stimulates in vitro granulocyte-colony stimulating factor (G-CSF ) production from activated human peripheral blood mononuclear cells and monocytes but not other cytokines or CSFs with hematoregulatory activity. In vivo administration of SCH 14988 to mice rendered neutropenic by cyclophosphamide treatment resulted in the accelerated recovery of the peripheral neutrophil compartment. This activity correlated with increased in vivo G-CSF levels and stimulation of marrow granulopoiesis, and was comparable to that of exogenously administered recombinant human G-CSF. No alterations to other leukocyte populations in peripheral blood, spleen, or the peritoneal cavity were observed. These findings suggest that SCH 14988 may be clinically useful to enhance neutrophil granulopoiesis, as well as to study the mechanisms involved in G-CSF gene regulation.
Wide Variety of Point Mutations in the H Gene of Bombay and Para-Bombay Individuals That Inactivate H Enzyme
Authors: Kaneko Mika , Nishihara Shoko , Shinya Naoko , Kudo Takashi , Iwasaki Hiroko , Seno Taiko , Okubo Yasuto , Narimatsu Hisashi
Published: October 2019

The H genes, encoding an α1,2fucosyltransferase, which defines blood groups with the H structure, of four Bombay and 13 para-Bombay Japanese individuals were analyzed for mutations. Four Bombay individuals were homologous for the same null H allele, which is inactivated by a single nonsense mutation at position 695 from G to A (G695A), resulting in termination of H gene translation. The allele inactivated by the G695A was designated h1. The other 13 para-Bombay individuals possessed a trace amount of H antigens on erythrocytes regardless of their secretor status. Sequence analysis of their H genes showed four additional inactivated H gene alleles, h2, h3, h4, and h5. The h2 allele possesed a single base deletion at position 990 G (990-del). The h3 and h4 alleles possessed a single missense mutation, T721C, which changes Tyr 241 to His, and G442T, which changes Asp148 to Tyr, respectively. The h5 allele possessed two missense mutations, T460C (Tyr154 to His) and G1042A (Glu348 to Lys). The h2, h3, h4, and h5 enzymes directed by these alleles were not fully inactivated by the deletion and the missense mutations expressing some residual enzyme activity resulting in synthesis of H antigen on erythrocytes. Thirteen para-Bombay individuals whose erythrocytes retained a trace amount of H antigen were determined to be heterozygous or homozygous for at least one of h2, h3, h4, or h5 alleles. This clarified that the levels (null to trace amount) of H antigen expression on erythrocytes of Bombay and para-Bombay individuals are determined solely by H enzyme activity. These mutations found in the Japanese H alleles differ from a nonsense mutation found in the Indonesian population. To determine the roles of the H, Se, and Le genes in the expression of H antigen in secretions and Lewis blood group antigen on erythrocytes, the Lewis and secretor genes were also examined in these Bombay and para-Bombay individuals. The Lewis blood group phenotype, Le(α- b+), was determined by the combinatorial activity of two fucosyltransferases, the Lewis enzyme and the secretor enzyme, and the secretor status was solely determined by the secretor enzyme activity, not by H enzyme activity. Bombay individuals were confirmed to be homozygous for the inactivated H and Se genes. As expected from the very low frequency of Bombay and para-Bombay individuals in the population, ie, approximately one in two or 300,000, the H gene mutations were found to be very variable, unlike the cases of the point mutations in the other glycosyltransferase genes; the ABO genes, the Lewis gene, and the secretor gene.
Essential Role of the Thymus to Reconstitute Naive (CD45RA+) T-Helper Cells After Human Allogeneic Bone Marrow Transplantation
Authors: Heitger Andreas , Neu Nikolaus , Kern Hannelore , Panzer-Grümayer Eva-Renate , Greinix Hildegard , Nachbaur David , Niederwieser Dietger , Fink Franz Martin
Published: October 2019

To contribute to the understanding of the role of the thymus in humans in the reconstitution of naive (CD45RA+) T cells after bone marrow transplantation (BMT), we compared T-cell regeneration in a unique situation, namely a thymectomized cancer patient (15 years old), with that of thymus-bearing patients after allogeneic BMT. These cases shared features of transplantation (total body irradiation, HLA-matched donors, and graft-versus-host disease prophylaxis with cyclosporine A) and all had an uncomplicated posttransplantation course. As shown by fluorescence-activated cell sorting analyses, the thymectomized host failed to reconstitute CD45RA+ T-helper cells even 24 months after BMT (11% CD45RA+ of CD4+ cells). In this patient, preferentially CD45RO+ cells contributed to the recovery of CD4+ cells (206 of 261/μL at 6 months and 463 of 558/μL at 24 months after BMT, CD45RA+ of CD4+ cells), whereas CD45RA+ cells remained low (<60/μL). In contrast, nine thymus-bearing hosts (5 children and 4 adults) examined between 6 and 24 months after BMT effectively reconstituted CD4+/CD45RA+ cells according to their normal age-related range (≥28% in adults and ≥50% in children). Five of these were analyzed sequentially at 6 and 9 months after BMT. Within this period, CD45RA+ cells increasingly contributed to the recovery of CD4+ cells (median, +21%), even when total CD4+ cells decreased. With respect to T-cytotoxic/suppressor cells, the thymectomized host retained the capacity to recover CD45RA+ cells (137 of 333/μL at 6 months and 596 of 1,046/μL at 24 months after BMT, CD45RA+ of CD8+ cells), a proportion similar to that seen in thymus-bearing hosts. These findings suggest that a thymus-independent pathway exists to regenerate CD45RA+ T-cytotoxic/suppressor cells, but residual thymus is essential to reconstitute naive (CD45RA+) T-helper cells after BMT in humans.
Myelodysplastic Syndrome and Acute Myelogenous Leukemia as a Late Clonal Complication in Children With Acquired Aplastic Anemia
Authors: Ohara Akira , Kojima Seiji , Hamajima Nobuyuki , Tsuchida Masahiro , Imashuku Shinsaku , Ohta Shigeru , Sasaki Hideki , Okamura Jun , Sugita Kanji , Kigasawa Hisato , Kiriyama Yukio , Akatsuka Junichi , Tsukimoto Ichiro
Published: October 2019

The improved outcome of acquired aplastic anemia (AA) has revealed later complications, such as myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). We retrospectively analyzed 167 children with severe acquired AA. Eleven of 50 children treated with cyclosporin (CSA) and recombinant human granulocyte colony-stimulating factor (rhG-CSF ) developed MDS/AML; 8 of these were within 36 months of the diagnosis of AA, much earlier than previous reports. Six of the 11 children received rhG-CSF exceeding 10 μg/kg/d, and 9 received rhG-CSF therapy for over 1 year. Ten children showed monosomy 7 at diagnosis of MDS. All of the 11 children were administered both CSA and rhG-CSF. There was no development of MDS/AML among 41 children treated with either CSA or rhG-CSF or among 48 children who underwent bone marrow transplantation. A well-controlled clinical trial is warranted to determine whether therapeutic modalities affect the development of MDS/AML in children with severe acquired AA.
A Comparison of Murine and Human Factor XI
Authors: Gailani David , Sun Mao-Fu , Sun Yuehui
Published: October 2019

Factor XI is a plasma glycoprotein that is required for contact activation initiated fibrin formation in vitro and for normal hemostasis in vivo. In preparation for developing a mouse model of factor XI deficiency to facilitate investigations into this protease's contributions to coagulation, we cloned the complementary DNA for murine factor XI, expressed the protein in a mammalian expression system, and compared its properties with human recombinant factor XI. The 2.8-kb murine cDNA codes for a protein of 624 amino acids with 78% homology to human factor XI. Both recombinant murine and human factor XI are 160 kD homodimers comprised of two 80 kD polypeptides connected by disulfide bonds. Murine factor XI shortens the clotting time of human factor XI deficient plasma in an activated partial thromboplastin time assay, with a specific activity 50% to 70% that of the human protein. In a purified system, murine factor XI is activated by human factor XIIa and thrombin in the presence of dextran sulfate. Murine factor XI differs from human factor XI in that it undergoes autoactivation slowly in the presence of dextran sulfate. This is due primarily to murine factor XIa preferentially cleaving a site on zymogen factor XI within the light chain, rather than the activation site between Arg371 and Val372. Northern blots of polyadenylated messenger RNA show that murine factor XI message is expressed, as expected, primarily in the liver. In contrast, messenger RNA for human factor XI was identified in liver, pancreas, and kidney. The studies show that murine and human factor XI have similar structural and enzymatic properties. However, there may be variations in tissue specific expression and subtle differences in enzyme activity across species.
The Acquisition of Host-Derived Major Histocompatibility Complex Class II Glycoproteins by Human Immunodeficiency Virus Type 1 Accelerates the Process of Virus Entry and Infection in Human T-Lymphoid Cells
Authors: Cantin Réjean , Fortin Jean-François , Lamontagne Ginette , Tremblay Michel
Published: October 2019

Infection by human immunodeficiency virus type 1 (HIV-1) results in a progressive depletion of CD4+ T lymphocytes, leading to fatal immunodeficiency. The mechanisms causing the marked loss of CD4+ T lymphocytes are incompletely understood. However, several lines of evidence indicate that direct cytopathology mediated by HIV-1 is a key element in such CD4+ T-cell depletion. In this study, we investigated whether the previously reported incorporation of host-derived major histocompatibility class II glycoproteins (MHC-II) on HIV-1 can alter its replicative capacity. To achieve this goal, virus stocks were produced in parental MHC-II–expressing RAJI cells and in MHC-II–negative RAJI mutants (RM3), both of which have been stably transfected with human CD4 cDNA to allow productive infection with HIV-1. An enhancement of the rate/efficiency of virus entry was seen after infection with normalized amounts of virions carrying host-derived MHC-II on their surface as compared with inoculation with virions devoid of cellular MHC-II. Data from time-course and infectivity experiments showed that the kinetics of infection were more rapid for virions bearing host-derived MHC-II glycoproteins than for MHC-II–free HIV-1 particles. These results suggest that virally embedded cellular MHC-II glycoproteins are functional and can have a positive effect on early events in the virus replicative cycle. Therefore, we show that the acquisition of cellular MHC-II glycoproteins by HIV-1 can modify its biologic properties and might, consequently, influence the pathogenesis of this retroviral disease.
FcγRIIIa-158V/F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcγRIIIa, Independently of the FcγRIIIa-48L/R/H Phenotype
Authors: Koene Harry R. , Kleijer Marion , Algra Johan , Roos Dirk , E.G.Kr. von dem Borne Albert , de Haas Masja
Published: October 2019

We analyzed a genetic polymorphism of Fcγ receptor IIIa (CD16) that is present on position 158 (Phe or Val) in the membrane-proximal, IgG-binding domain. With a polymerase chain reaction–based allele-specific restriction analysis assay we genotyped 87 donors and found gene frequencies of 0.57 and 0.43 for FcγRIIIA-158F and −158V, respectively. A clear linkage was observed between the FcγRIIIA-158F and −48L genotypes on the one hand and the FcγRIIIA-158V and −48H or −48R genotypes on the other hand (χ2 test; P < .001). To determine the functional consequences of this FcγRIIIa-158V/F polymorphism, we performed IgG binding experiments with natural killer (NK) cells from genotyped donors. All donors were also typed for the recently described triallelic FcγRIIIa-48L/R/H polymorphism. NK cells were treated with lactic acid to remove cell-associated IgG. FcγRIIIaNK158F bound significantly less IgG1, IgG3, and IgG4 than did FcγRIIIaNK-158V, irrespective of the FcγRIIIa-48 phenotype. Moreover, freshly isolated NK cells from FcγRIIIa-158VV individuals carried significantly more cytophilic IgG than did NK cells from FcγRIIIa-158FF individuals. In addition, CD16 monoclonal antibody (MoAb) MEM154 bound more strongly to FcγRIIIa-158V, compared with -158F, again independently of the FcγRIIIa-48 phenotype. The binding of MoAb B73.1 was not influenced by the FcγRIIIa-158V/F polymorphism, but proved to depend solely on the amino acid present at position 48 of FcγRIIIa. In conclusion, the previously reported differences in IgG binding among the three FcγRIIIa-48L/R/H isoforms are a consequence of the linked, biallelic FcγRIIIa-158V/F polymorphism at amino-acid position 158.
Detectable Molecular Residual Disease at the Beginning of Maintenance Therapy Indicates Poor Outcome in Children With T-Cell Acute Lymphoblastic Leukemia
Authors: Dibenedetto Salvatore P. , Lo Nigro Luca , Pine Mayer Sharon , Rovera Giovanni , Schilirò Gino
Published: October 2019

The aims of this study were twofold: (1) to assess the marrow of patients with T-lineage acute lymphoblastic leukemia (T-ALL) for the presence of molecular residual disease (MRD) at different times after diagnosis and determine its value as a prognostic indicator; and (2) to compare the sensitivity, rapidity, and reliability of two methods for routine clinical detection of rearranged T-cell receptor (TCR). Marrow aspirates from 23 patients with T-ALL diagnosed consecutively from 1982 to 1994 at the Division of Pediatric Hematology and Oncology, University of Catania, Italy, were obtained at diagnosis, at the end of induction therapy (6 to 7 weeks after diagnosis), at consolidation and/or reinforced reinduction (12 to 15 weeks after diagnosis), at the beginning of maintenance therapy (34 to 40 weeks after diagnosis), and at the end of therapy (96 to 104 weeks after diagnosis). DNA from the patients' marrow was screened using the polymerase chain reaction (PCR) for the four most common TCR δ rearrangements in T-ALL (Vδ1Jδ1, Vδ2Jδ1, Vδ3Jδ1, and Dδ2Jδ1) and, when negative, further tested for the presence of other possible TCR δ and TCR γ rearrangements. After identification of junctional rearrangements involving V, D, and J segments by DNA sequencing, clone-specific oligonucleotide probes 5′ end-labeled either with fluorescein or with [γ-32P]ATP were used for heminested PCR or dot hybridization of PCR products of marrows from patients in clinical remission. For 17 patients with samples that were informative at the molecular level, the estimated relapse-free survival (RFS) at 5 years was 48.6% (±12%). The sensitivity and specificity for detection of MRD relating to the outcome were 100% and 88.9% for the heminested fluorescence PCR and 71.4% and 88.9% for Southern/dot blot hybridization, respectively. Predictive negative and positive values were 100% and 90.7% for heminested fluorescence PCR, respectively. The probability of RFS based on evidence of MRD as detected by heminested fluorescence PCR at the time of initiation of maintenance therapy was 100% and 0% for MRD-negative and MRD-positive patients, respectively. Thus, the presence of MRD at the beginning of maintenance therapy is a strong predictor of poor outcome, and the molecular detection of MRD at that time might represent the basis for a therapeutic decision about such patients. By contrast, the absence of MRD at any time after initiation of treatment strongly correlates with a favorable outcome. The heminested fluorescence PCR appears to be more accurate and more rapid than other previously used methods for the detection of residual leukemia.
Disparate Affinities of Antifolates for Folylpolyglutamate Synthetase From Human Leukemia Cells
Authors: Longo Giuseppe S.A. , Gorlick Richard , Tong William P. , Ercikan Emine , Bertino Joseph R.
Published: October 2019

Previous work showed that acute myelocytic leukemia blasts accumulate less long chain polyglutamates of methotrexate (MTX) than acute lymphocytic leukemia blasts when incubated with this radiolabeled antifolate. This difference likely explains the increased sensitivity of lymphoid leukemias to short-term exposure of MTX as compared with myeloid leukemias. In this study, we examined the basis for differences between long chain MTX polyglutamate accumulation between different leukemia cell types using both leukemia cell lines and blasts freshly isolated from blood of leukemic patients. The major difference found between leukemia cells that accumulate long chain polyglutamates and those that do not were differences in Km values for the enzyme folylpolyglutamate synthetase. Km values did not change with partial purification of this enzyme, indicating that interfering substances in crude lysates were not responsible for this difference. We postulate that there may be differences in the properties of this enzyme related to tissue specific expression. In contrast to MTX, both Tomudex (Zeneca Pharmaceuticals, Wilmington, DE) and 1843U89, potent inhibitors of thymidylate synthetase, have low Kms for folylpolyglutamate synthetase, and polyglutamate forms of these inhibitors are accumulated to the same degree in both myeloid and lymphoid acute leukemia cells, paralleling the equivalent cytotoxicity found between myeloid and lymphoid leukemia cell lines. Based on these results, we believe a clinical trial of Tomudex in patients with acute myeloid leukemia is warranted.
Outbreak of Hepatitis C Virus Infection in Patients With Hematologic Disorders Treated With Intravenous Immunoglobulins: Different Prognosis According to the Immune Status
Authors: Rossi Giuseppe , Tucci Alessandra , Cariani Elisabetta , Ravaggi Antonella , Rossini Angelo , Radaeli Enrico
Published: October 2019

The influence of immunodeficiency on the course of hepatitis C virus (HCV) infection is still debated, although a worsening effect has been suggested. We compared the characteristics of hepatitis C in two groups of hematologic patients with different levels of immunocompetence who acquired the same virus strain after treatment with contaminated intravenous immunoglobulins (IVIG). Indications for IVIG therapy were idiopathic thrombocytopenic purpura (ITP) in six patients and hypogammaglobulinemia in 7 patients with various hematologic disorders, who were defined immunodeficient (ID). Infection rate was 100%. Five ID patients never developed HCV antibodies despite serum HCV-RNA positivity. The same HCV genotype was shown in 10 patients tested. Moreover, E1-E2 gene partial nucleotide sequencing, performed in four patients, showed identical or closely related amino acid sequences, thus strongly supporting the hypothesis of a common source of infection. Clinical acute infection did not differ significantly between the two groups, but subsequent liver failure developed in five of the seven ID patients and in none of the ITP patients (P = .04). Liver biopsy, performed in three cases, documented HCV as the only cause of liver damage. Six ID patients died, with liver disease being the primary cause of death in four cases and a contributory cause in two cases. Their median survival after IVIG was 12 months, significantly worse than that of ITP patients (P = .0028). We conclude that immunodeficiency markedly worsens the course of IVIG-acquired HCV infection in hematologic patients.
Hepatitis C and G Virus Infection and Liver Dysfunction After Allogeneic Bone Marrow Transplantation: Results From a Prospective Study
Authors: Rodriguez-Iñigo Elena , Tomás José-Francisco , Gómez-Garcı́a de Soria Valle , Bartolomé Javier , Pinilla Inmaculada , Amaro Marı́a-José , Carreño Vicente , Fernández-Rañada José-Marı́a
Published: October 2019

Acute and chronic liver dysfunction is common after allogeneic bone marrow transplantation (BMT). Although toxicity, graft versus-host disease (GVHD), and viral infections are the major causes, etiologic diagnosis is difficult and often remains unknown. We conducted a prospective study to establish the role of the infection with both the hepatitis C virus (HCV) and the recently discovered hepatitis G virus (HGV) in liver dysfunction after BMT. From January 1994 to December 1995, 59 patients who had undergone an allogeneic BMT at our institution were enrolled in the study. HGV-RNA was identified in serum by nested polymerase chain reaction (PCR), and HCV was studied by the presence of second generation enzyme-linked immunosorbent assay (ELISA)-antibodies and HCV-RNA by nested PCR. HGV-RNA was detected in 25 patients (42%) (before BMT in 18 and after BMT in 7). HCV-RNA was present in 12 patients (20%) (before BMT in 11 and after BMT in one). The presence of HCV-RNA and HGV-RNA was clearly associated with a previous history of blood transfusions. No significant association was found between viral infection and acute liver toxicity. Some degree of liver dysfunction was present 6 months after BMT in 25 of 40 evaluable patients (62%). Long-term liver dysfunction was more common among patients infected with HCV alone (3 of 4) or with both HCV and HGV (3 of 3) than in those infected with either HGV alone (eight of 13) or with no virus infection (10 of 20). We found a high prevalence of HGV infection in our BMT population. However, no role for HGV in liver disease could be established in this study, and the relationship between HGV infection and liver dysfunction requires further clarification.
Activation of p38 MAP Kinase Pathway by Erythropoietin and Interleukin-3
Authors: Nagata Yuka , Moriguchi Tetsuo , Nishida Eisuke , Todokoro Kazuo
Published: October 2019

Activation of p38 MAP kinase (p38) as well as JNK/SAPK has been described as being induced by a variety of environmental stresses such as osmotic shock, ultraviolet radiation, and heat shock, or the proinflammatory cytokines tumor necrosis factor-α and interleukin-1 (IL-3). We found that the hematopoietic cytokines erythropoietin (Epo) and IL-3, which regulate growth and differentiation of erythroids and hematopoietic progenitors, respectively, also activate a p38 cascade. Immunoblot analyses and in vitro kinase assay clearly showed that Epo and IL-3 rapidly and transiently phosphorylated and activated p38 in Epo– or IL-3–dependent mouse hematopoietic progenitor cells. p38 can generally be activated by the upstream kinase MKK3 or MKK6. However, in vitro kinase assays in the immunoprecipitates with anti-MKK6 antibody and anti-phosphorylated MKK3/MKK6 antibody showed that activation of neither MKK3 nor MKK6 was detected after Epo or IL-3 stimulation, while osmotic shock clearly induced activation of both MKK3/MKK6 and p38. Together with previous observations, these results suggest that both p38 and JNK cascades play an important role not only in stress and proinflammatory cytokine responses but also in hematopoietic cytokine actions.
Lipoprotein(a) Is a Potent Chemoattractant for Human Peripheral Monocytes
Authors: Syrovets Tatjana , Thillet Joëlle , Chapman M. John , Simmet Thomas
Published: October 2019

We have previously reported that the serine protease plasmin triggers chemotaxis in human peripheral monocytes, but not in polymorphonuclear leukocyte. We now show that the structurally related lipoprotein(a) (Lp[a]) as well as recombinant apolipoprotein(a) (apo[a]) trigger chemotactic responses in human monocytes equipotent to that observed with the standard chemoattractant FMLP. The chemotactic effects of Lp(a) and FMLP were additive. Low density lipoprotein (LDL) did not elicit any significant chemotactic response nor did it interfere with that triggered by Lp(a). As assessed by checkerboard analysis, Lp(a)-mediated monocyte locomotion was a true chemotaxis. Both plasminogen as well as catalytically inactivated plasmin inhibited monocyte migration elicited by Lp(a), suggesting binding of Lp(a) to plasminogen binding sites. Lp(a)-mediated signaling proceeds through a pertussis toxin-sensitive guanosine triphosphate (GTP)-binding protein and activation of protein kinase C as implicated by the effects of 1-O-hexadecyl-2-O-methyl-rac-glycerol and chelerythrine. Lp(a) induced generation of guanosine 3′,5′-cyclic monophosphate (cGMP), apparently crucial for the Lp(a)-mediated chemotaxis, because an inhibitor of soluble guanylyl cyclase, LY83583, reduced both the Lp(a)-induced cGMP formation as well as the monocyte migration. The latter effect of LY83583 was antagonized by the stable cGMP analog 8-pCPT-cGMP. The data indicate that Lp(a) triggers chemotaxis in human monocytes by way of a cGMP-dependent mechanism. Our findings may have important implications for the atherogenesis associated with elevated levels of Lp(a).