bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous measurement of reticulocyte and red blood cell indices in healthy subjects and patients with microcytic and macrocytic anemia
Authors: d'Onofrio G , Chirillo R , Zini G , Caenaro G , Tommasi M , Micciulli G
Published: October 2019

Using the new Bayer H*3 hematology analyzer (Leverkusen, Germany), we have determined red blood cell and reticulocyte indices in 64 healthy subjects, in patients with microcytosis due to iron deficiency (58 patients) and heterozygous beta-thalassemia (40 patients), and in patients with macrocytosis (28 patients). We found in all cases that reticulocytes were larger than mature red cells by 24% to 35%, with a hemoglobin concentration 16% to 25% lower and a similar hemoglobin content. The correlation between red cell and reticulocyte indices was strikingly tight (r = .928 for volume, r = .929 for hemoglobin concentration, r = .972 for hemoglobin content) in all four groups, regardless of red blood cell size. The ratio of reticulocyte to red blood cell mean corpuscolar volume (MCV ratio) was constantly above 1. Inversion of the MCV ratio was observed only in four patients. It was always abrupt and transitory and was associated with erythropoietic changes leading to the production of red blood cells of a different volume (treatment of megaloblastic anemia, functional iron deficiency, bone marrow transplantation). In two cases of marrow transplantation, reticulocyte volume fell during the aplastic phase after conditioning chemotherapy and then rapidly increased up to values higher than before; this production of macroreticulocytes was the earliest sign of engraftment.
Interleukin-10 administration decreases survival in murine recipients of major histocompatibility complex disparate donor bone marrow grafts
Authors: Blazar BR , Taylor PA , Smith S , Vallera DA
Published: October 2019

Studies in mice and humans have indicated that the predominance of interleukin-4 (IL-4)- and IL-10-producing T-helper type 2 (Th2) cells may serve to downregulate acute graft-versus-host disease (GVHD) reactions, whereas IL-2-producing Th1 cells have been implicated in facilitating acute GVHD. We explored the possibility that the in vivo infusion of IL-10 would inhibit acute GVHD induced by fully allogeneic donor grafts. Unexpectedly, IL-10 infusions resulted in a dose- dependent increase in GVHD-induced mortality. The acceleration of lethal GVHD by IL-10 occurred in irradiated recipients of T-cell- depleted bone marrow (BM) plus 5, 15, or 25 x 10(6) splenocytes but did not influence the post-BM transplantation (post-BMT) survival rate of recipients of BM without splenocytes, suggesting that the IL-10 effects were not due to toxicity. Antimurine IL-10-neutralizing monoclonal antibody injections, administered to diminish endogenous IL-10, reduced GVHD-associated mortality and improved the clinical appearance of the recipients. For BM graft rejection studies, IL-10 was infused into sublethally irradiated recipients of anti-Thy 1.2 + C′ T-cell-depleted, fully allogeneic BM grafts. In a short-term (day 7) in vivo assay, IL- 10 infusions significantly inhibited allogeneic (but not syngeneic) BM proliferation in vivo, indicative of increased graft rejection. In long- term chimerism experiments, IL-10 infusions caused a significant increase in early post-BMT mortality caused by a profound anemia typically associated with graft rejection and aplasia. A slightly higher irradiation dose (650 cGy v 600 cGy) eliminated the anemia but did not reverse the graft rejection process associated with IL-10 administration. We conclude that the in vivo infusion of exogenous IL- 10 in recipients of fully allogeneic donor grafts results in accelerated GVHD and graft rejection in the strain combinations tested to date.
Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function
Authors: Spangrude GJ , Brooks DM , Tumas DB
Published: October 2019

Hematopoietic stem cells were isolated from normal adult mouse bone marrow based on surface antigen expression (Thy-1.1(low)Lin(neg)Ly- 6A/E+) and further selected for low retention of rhodamine 123. This population of cells (Rh-123low) could mediate radioprotection and long- term (greater than 12 months) repopulation after transplantation of as few as 25 cells. Transfer of five genetically marked Rh-123low cells in the presence of 10(5) normal bone marrow cells resulted in reconstitution of peripheral blood by greater than 10% donor cells in 64% (30 of 47) of recipient mice. Of 46 animals surviving after 24 weeks, 10 had over 50% donor-derived cells in peripheral blood. Two general patterns of long-term reconstitution were observed: one in which many donor-derived cells were observed 5 to 6 weeks after reconstitution and another in which donor-derived cells were rare initially but expanded with time. This result suggests that two classes of long-term repopulating hematopoietic stem cells exist, differing in their ability to function early in the course of transplantation. Alternatively, distinct anatomic sites of engraftment may dictate these two outcomes from a single type of cell. As an approach to measure the extent of self-renewal by the injected cells, recipients of five or 200 stem cells were killed 8 to 13 months after the transplants, and Thy- 1.1(low)Lin(neg)Ly-6A/E+ progeny of the original injected cells were isolated for a second transplant. While a numerical expansion of cells expressing the cell surface phenotype of stem cells was observed, along with activity in the colony-forming unit-spleen assay, the expanded cells were vastly inferior in radioprotection and long-term reconstitution assays when compared with cells freshly isolated from normal animals. This result demonstrates that in stem cell expansion experiments, cell surface antigen expression is not an appropriate indicator of stem cell function.
A primary T-cell immunodeficiency associated with defective transmembrane calcium influx
Authors: Le Deist F , Hivroz C , Partiseti M , Thomas C , Buc HA , Oleastro M , Belohradsky B , Choquet D , Fischer A
Published: October 2019

We investigated a T-cell activation deficiency in a 3-month-old boy with protracted diarrhea, serious cytomegalovirus pneumonia, and a family history (in a brother) of cytomegalovirus infection and toxoplasmosis. In spite of detection of normal number of peripheral lymphocytes, T cells did not proliferate after activation by anti-CD3 and anti-CD2 antibodies, although proliferation induced by antigens was detectable. We sought to determine the origin of this defect as it potentially represented a valuable tool to analyze T-cell physiology. T- cell activation by anti-CD3 antibody or phytohemagglutinin (PHA) led to reduced interleukin-2 (IL-2) production and abnormal nuclear factor- activated T cell (NF-AT; a complex regulating the IL-2 gene transcription) binding activity to a specific oligonucleotide. T-cell proliferation was restored by IL-2. Early events of T-cell activation, such as anti-CD3 antibody-induced cellular protein tyrosine phosphorylation, p59fyn and p56lck kinase activities, and phosphoinositide turnover, were found to be normal. In contrast, anti- CD3 antibody-induced Ca2+ flux was grossly abnormal. Release from endoplasmic reticulum stores was detectable as tested in the presence of anti-CD3 antibody or thapsigargin after cell membrane depolarization in a K+ rich medium, whereas extracellular entry of Ca2+ was defective. The latter abnormality was not secondary to defective K+ channel function, which was found to be normal. A similar defect was found in other hematopoietic cell lineages and in fibroblasts as evaluated by both cytometry and digital video imaging experiments at a single-cell level. This primary T-cell immunodeficiency appears, thus, to be due to defective Ca2+ entry through the plasma membrane. The same abnormality did not alter B-cell proliferation, platelet function, and polymorphonuclear neutrophil (PMN) function. Elucidation of the mechanism underlying this defect would help to understand the physiology of Ca2+ mobilization in T cells.
Functional reconstitution of the phagocyte NADPH oxidase by transfection of its multiple components in a heterologous system
Authors: de Mendez I , Leto TL
Published: October 2019

The phagocyte NADPH oxidase system, as previously defined by cell-free reconstitution, is comprised of five essential components, three of which are produced during late phagocytic differentiation--namely, two cytosolic proteins, p47- and p67-phox--and the large subunit of cytochrome b558, gp91-phox. To confirm that these are the only phagocyte-specific components necessary for oxidase activity in whole cells, the recombinant NADPH oxidase was reconstituted in a heterologous cell line. An undifferentiated multipotent leukemic cell line, K562, which expresses endogenous Rac and the small subunit of the flavocytochrome b558 (p22-phox), was cotransfected with episomal expression vectors containing cDNAs for the three other oxidase components. After 4 days of selection, the complete oxidase system was functionally reconstituted in transfected cells stimulated with phorbol myristate acetate or calcium ionophore. These easily transfected cells provide an ideal model system in which several oxidase components can be genetically manipulated and readily expressed. This system can be used to test the effects of mutations associated with any of the genes affected in chronic granulomatous disease and will facilitate studies on structure-function relationships within several oxidase components. This system will also aid in delineation of upstream regulators functioning through various signaling pathways.
Somatic mutations of the PIG-A gene found in Japanese patients with paroxysmal nocturnal hemoglobinuria
Authors: Yamada N , Miyata T , Maeda K , Kitani T , Takeda J , Kinoshita T
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder caused by deficient biosynthesis of the glycosylphosphatidylinositol (GPI) anchor. PIG-A, an X-linked gene that participates in the first step of GPI-anchor synthesis, is responsible for PNH. Abnormalities of the PIG-A gene have been demonstrated in all patients with PNH that have been studied to date. In this study, we analyzed 14 Japanese patients with PNH and identified 15 somatic mutations of PIG-A. The mutations included eight single-base changes and seven frame shift mutations. The single-base changes were two nonsense, three missense, and three splice site mutations. The frame shift mutations were four single-base deletions, two single-base insertions, and a replacement of two bases with one. They were all different, except for the same missense mutation being found in two patients. Moreover, these mutations were distributed in various regions of the gene. These results indicated that the mutations occurred at random sites and that there is no mutation hot spot in the PIG-A gene. All the mutations resulted in complete loss of function. Interestingly, the granulocytes in these patients contained variable proportions of mutant cells, suggesting that clonal expansion is not determined solely by mutations but is influenced by another factor(s).
Deletions and rearrangement of CDKN2 in lymphoid malignancy
Authors: Stranks G , Height SE , Mitchell P , Jadayel D , Yuille MA , De Lord C , Clutterbuck RD , Treleaven JG , Powles RL , Nacheva E
Published: October 2019

Recurrent abnormalities of the short arm of chromosome 9, including translocations and interstitial deletions, have been reported in both leukemia and lymphoma. The pathologic consequences of these abnormalities remain unknown. The cyclin-dependent kinase 4 inhibitor (CDKN2) gene, which maps to 9p21, has been implicated by the finding of a high frequency of biallelic deletions in leukemic cell lines. We have determined the incidence of structural abnormalities affecting CDKN2 by DNA blot in a panel of 231 cases of leukemia and lymphoma and 66 cell lines derived from patients with lymphoid malignancies with defined cytogenetic abnormalities. Structural alterations of CDKN2 were seen in 20 (8.3%) of all fresh cases and 10 (15.1%) of all cell lines. Biallelic CDKN2 deletions were seen in 11 of 53 (21%) cases of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There was no association with any particular cytogenetic abnormality. Biallelic deletions were also found in high-grade and transformed non-Hodgkin's lymphoma (NHL) of both B- and T-cell lineages. In two cases of transformed NHL, analysis of sequential samples showed loss of CDKN2 with transformation. Neither deletions nor rearrangements of the CDKN2 gene were seen in any of the 119 leukemias of mature B or T cells analyzed. Biallelic deletions of CDKN2 were observed in 6 of 13 NHL cell lines. Three of the 6 cases had undergone transformation from low- to high-grade disease: in 2 of these cases it was possible to show that the CDKN2 deletions were present in fresh material from the patient and were therefore not an artifact of in vitro culture. Rearrangements of CDKN2 were seen in 2 cases (4%) of BCP-ALL, in 1 case of B-NHL, and in 1 Burkitt's lymphoma cell line and suggest the presence of a “hot spot” for recombination in the vicinity of the CDKN2 gene. These data indicate that the loss of CDKN2 expression may be involved in the pathogenesis of a subset of BCP-ALL, some high-grade NHL, and in the transformation of NHL from low- to high-grade disease. CDKN2 deletions and rearrangements occurred in the absence of detectable cytogenetic changes of chromosome 9p in 25 of 30 (83%) cases. Finally, of 10 cases of BCP-ALL that produced overt, transplantable leukemia in mice with severe combined immunodeficiency (SCID), seven showed biallelic CDKN2 deletions. In contrast, none of 11 cases that failed to engraft showed biallelic CDKN2 deletions. BCP-ALL cases that lack CDKN2 expression may have a particular propensity to grow in SCID mice.
A point mutation in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene in a case of acute myeloid leukemia results in the overexpression of a novel G-CSF-R isoform
Authors: Dong F , van Paassen M , van Buitenen C , Hoefsloot LH , Lowenberg B , Touw IP
Published: October 2019

A novel human granulocyte colony-stimulating factor (G-CSF) receptor isoform, designated SD, has been identified in which the distal C- terminal cytoplasmic region, previously shown to be essential for maturation signalling, is substituted by an altered C-terminus. The SD receptor has a high affinity for G-CSF and retains the membrane- proximal cytoplasmic region known to be sufficient for proliferative signalling. Nonetheless, the SD isoform lacks the ability to transduce growth signals in murine BAF3 cells and, in contrast to the wild-type G- CSF receptor, is scarcely capable of activating JAK2 kinase. Expression of SD receptor was found to be low in normal granulocytes, but was significantly increased in a patient with acute myeloid leukemia (AML). The leukemic cells of this patient harbour a point mutation in the SD splice donor site of the G-CSF receptor gene. These findings provide the first evidence that mutations in the G-CSF receptor gene can occur in certain cases of clinical de novo AML. The possible contribution of defective G-CSF receptor signalling to leukemogenesis is discussed.
The biology and treatment of acute lymphoblastic leukemia in adults
Authors: Copelan EA , McGuire EA
Published: October 2019

Despite reports to the contrary, only a small minority of adults with ALL are currently cured. Results have improved modestly with more intensive postremission chemotherapy and with tailoring of protocols in individuals with specific subsets of ALL. The use of growth factors may further improve treatment results. The performance of allogeneic BMT in first remission is clearly effective in some individuals, eg, those with Ph1-positive ALL, but it is unclear whether it is advantageous in most individuals. There are little data supporting the effectiveness of autotransplantation, as currently performed in ALL, despite its theoretical potential. Advances in understanding the biology of ALL have led to new approaches currently under basic and clinical investigation. These include serial studies of minimal residual disease by a variety of techniques to tailor treatment, the development of conjugated MoAbs to lymphoid cell antigens and immunologic and biochemical approaches to chimeric RNA and peptides generated by abnormal fusion genes. It seems likely that substantial improvement in the treatment of adult ALL awaits better characterization of the biology of this disease. However, some improvement will occur through empirical clinical research. It is critical that physicians recognize the poor results with current therapeutic approaches and enter patients into large well-designed clinical trials.
Immunosuppressive effect of human herpesvirus 6 on T-cell functions: suppression of interleukin-2 synthesis and cell proliferation [published erratum appears in Blood 1995 Jul 1;86(1):418]
Authors: Flamand L , Gosselin J , Stefanescu I , Ablashi D , Menezes J
Published: October 2019

Human herpesvirus-6 (HHV-6), the etiologic agent of roseola, is ubiquitous, establishes latency in the host, and can infect a variety of immunocompetent cells, with CD4+ T lymphocytes being the targets in which it replicates most efficiently. The present study was undertaken to learn more about specific immunobiologic effects of HHV-6 infection on T-lymphocyte functions. Our data demonstrate that infection of peripheral blood mononuclear cells (PBMC) by HHV-6 results in suppression of T-lymphocyte functions, as evidenced by reduced interleukin-2 (IL-2) synthesis and cellular proliferation. In fact, HHV- 6-infected PBMC secreted 50% less IL-2 than mock-infected cells after mitogenic stimulation with OKT3 antibody or phytohemmaglutinin (PHA). The inhibition of IL-2 by HHV-6 was also observed in enriched T-cell cultures, suggesting a direct effect of this virus on this cell type. Messenger RNA (mRNA) analysis by reverse-transcriptase polymerase chain reaction (PCR) indicated that HHV-6 diminishes IL-2 mRNA levels in mitogen-stimulated peripheral blood T cells. These results were also confirmed by Northern blot using the leukemic T-cell line Jurkat. This inhibitory effect of HHV-6 did not require infectious virus, as the use of UV-irradiated HHV-6 produced similar results. Moreover, HHV-6- infected PBMC showed up to an 85% reduction in their mitogen-driven proliferative response, as compared with sham-infected cells. Proliferation of both CD4+ and CD8+ T cells was affected by HHV-6. Taken together, our data show that infection of T cells by HHV-6 results in immune suppression characterized by a downregulation of IL-2 mRNA and protein synthesis accompanied by diminished cellular proliferation.
A novel class of zinc finger/leucine zipper genes identified from the molecular cloning of the t(10;11) translocation in acute leukemia
Authors: Chaplin T , Ayton P , Bernard OA , Saha V , Della Valle V , Hillion J , Gregorini A , Lillington D , Berger R , Young BD
Published: October 2019

A novel class of conserved transcription factors has been identified from the molecular cloning of AF10, the gene involved in the t(10;11)(p12;q23) translocation of acute myeloid leukemias. AF10 encodes a 109-kD protein of 1,027 amino acids and contains an N- terminal zinc finger region and a C-terminal leucine zipper. These structures have been found to be conserved in sequence and position in three other proteins, AF17, BR140, and a previously unrecognized Caenorhabditis elegans gene, provisionally named CEZF. The overall structure, level of sequence conservation, and expression pattern suggest that these genes encode a new class of transcription factors, some of which are targets for chromosomal translocation in acute leukemia.
Role of the sialophorin (CD43) receptor in mediating influenza A virus- induced polymorphonuclear leukocyte dysfunction
Authors: Abramson JS , Hudnor HR
Published: October 2019

Polymorphonuclear leukocytes (PMNLs) exposed to influenza A virus (IAV) undergo activation of the respiratory burst followed by depression of cell function when subsequently exposed to particulate or soluble stimuli. The effect of IAV on PMNLs is likely to be mediated through the attachment of IAV to one or more specific receptors. Recently, IAV has been shown to bind to the sialophorin protein (CD43) receptor on PMNL plasma membranes. The present study was performed to determine if the sialophorin receptor was responsible for IAV-induced PMNL dysfunction. When PMNLs were incubated with IAV or CD43 monoclonal antibody (MoAb) for 30 minutes and then exposed to a secondary particulate (opsonized zymosan) or soluble (FMLP or phorbol 12- myristate 13-acetate) stimulus, there was significant depression of the PMNL chemiluminescence response compared with the equivalent control (P < .05). When PMNL were incubated with the CD43 MoAb and then cross- linked with a goat antimouse IgG antibody, no depression of PMNL function occurred upon secondary stimulation. Exposure of cells to IAV aggregates also eliminated the PMNL dysfunction that normally occurs due to the virus. Similar to IAV, PMNL dysfunction due to the CD43 MoAb could be overcome by priming the cells with granulocyte-macrophage colony-stimulating factor. These findings indicate that IAV-induced PMNL dysfunction is mediated, at least in part, through the sialophorin receptor.
Heterogeneous PIG-A mutations in different cell lineages in paroxysmal nocturnal hemoglobinuria
Authors: Ostendorf T , Nischan C , Schubert J , Grussenmeyer T , Scholz C , Zielinska- Skowronek M , Schmidt RE
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect of hematopoietic stem cells in which affected cells are characterized by the lack of glycosylphosphatidylinositol (GPI)-anchored proteins. The lesion in PNH lies in the defective synthesis of N-acetyl-D- glucosaminyl-phosphatidylinositol (GlcNAc-Pl), the first intermediate in GPI biosynthesis. Reintroduction of the PIG-A gene into GPI(-) patient cells reportedly complements this defect. We have analyzed here PIG-A transcripts of six PNH patients. GPI+ and GPI- cell lines from each individual were used, ie, Epstein-Barr virus-transformed B- lymphoblastoid cell lines, T-cell lines, and natural killer cell clones. Reverse transcriptase polymerase chain reaction and sequencing showed three different PIG-A splicing variants in GPI+ cell lines, in which the largest transcript contained the wild-type PIG-A coding region sequence. GPI-deficient cell lines showed abnormal splicing variants. Sequencing of PIG-A complementary DNA and genomic DNA showed heterogeneous mutations ranging from different point mutations to small deletions. Two lymphocyte cell lines (T- and B-cell lines) of one patient presented with the same mutation. For another patient, two different mutations were detected in one natural killer cell line. Therefore, different cell lineages have somatic mutations in PIG-A that lead to PNH.
Transforming growth factor beta 1 inhibits expression of the gene products for steel factor and its receptor (c-kit)
Authors: Heinrich MC , Dooley DC , Keeble WW
Published: October 2019

Transforming growth factor beta 1 (TGF-beta 1), a product of marrow stromal cells, inhibits the proliferation and differentiation of hematopoietic progenitor cells within the hematopoietic microenvironment. Steel factor (SF), also a product of marrow stromal cells, is an essential positive regulator of hematopoiesis in vivo. TGF- beta 1 has been shown to repress human and murine leukemic cell and murine lin-bone marrow mononuclear cell expression of the receptor for SF (c-kit). We speculated that TGF-beta 1 might exert its inhibitory effect on hematopoiesis in part by decreasing SF/c-kit interactions. Therefore, we tested the hypothesis that TGF-beta 1 inhibits both stromal cell expression of SF and hematopoietic progenitor cell expression of c-kit. We measured stromal cell expression of SF protein and hematopoietic progenitor cell expression of membrane-bound c-kit before and after exposure to recombinant human TGF-beta 1. Both stromal cell expression of SF protein and hematopoietic progenitor cell expression of c-kit protein were inhibited 50% to 80% by TGF-beta 1. Using Northern blot and ribonuclease protection assays, we determined that TGF-beta 1 repressed stromal cell SF mRNA, but did not alter SF transcript stability. TGF-beta 1 was also found to repress c-kit mRNA in human leukemic myeloblasts as well as in normal lin-hematopoietic progenitor cells. In contrast with its effect on SF mRNA, TGF-beta 1 accelerated the degradation of c-kit mRNA. We conclude that TGF-beta 1 inhibits stromal cell production of SF by repression of SF gene transcription and represses hematopoietic progenitor cell expression of c-kit by decreasing the stability of c-kit transcripts. Taking into account the importance of SF and c-kit in maintaining steady-state hematopoiesis in vivo, the dual effect of TGF-beta 1 on both SF and c-kit gene expression is likely to be one of the major mechanisms by which TGF-beta 1 inhibits hematopoiesis in vivo.
A new congenital dysmegakaryopoietic thrombocytopenia (Paris-Trousseau) associated with giant platelet alpha-granules and chromosome 11 deletion at 11q23 [see comments]
Authors: Breton-Gorius J , Favier R , Guichard J , Cherif D , Berger R , Debili N , Vainchenker W , Douay L
Published: October 2019

This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha-granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.
G protein subunit G alpha 16 expression is restricted to progenitor B cells during human B-cell differentiation
Authors: Mapara MY , Bommert K , Bargou RC , Leng C , Beck C , Ludwig WD , Gierschik P , Dorken B
Published: October 2019

Recently G alpha 16, a new guanosine triphosphate (GTP) binding protein alpha subunit has been described to be specifically expressed in human hematopoietic cells. Expression of G alpha 16 was observed in human cell lines of myelomonocytic and T-lymphocytic origin, but not in human B-cell lines Raji and IM9. We studied the expression of G alpha 16 in human B cells corresponding to different stages of B-cell differentiation by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The human Burkitt's lymphoma cell lines Raji, Ramos, BJAB, the lymphoblastoid cell line SKW6.4, and the plasmocytoma cell line U266 were devoid of G alpha 16. In contrast, G alpha 16 was detected in the human progenitor B cell lines Reh and Nalm-6. Using the mu+, k-cell line BLIN-1 (pre-B cell phenotype) and its derived subclone 1E8 (surface mu+, k+; B-cell phenotype) G alpha 16 expression was found to disappear on transition from pre-B to B-cell differentiation stage. The analysis of a broad panel of human neoplastic B lymphocytes ranging from progenitor B-acute lymphatic leukemia (pre-pre-B-ALL), common acute leukemias (cALL), pre-B-ALL, mature B-ALL to low grade B-cell lymphoma (chronic lymphocytic leukemia of B-cell type, leukemic centrocytic non-Hodgkins lymphoma [NHL], hairy cell leukemia) showed that G alpha 16 expression is limited to progenitor and pre-B-ALL cells. Therefore, we conclude that within B-cell differentiation, G alpha 16 is expressed solely during early B cell ontogeny and downregulated during differentiation. Thus, G alpha 16 might be an important regulator involved in signaling processes in progenitor B cells.
Naturally occurring human anti-band 3 autoantibodies accelerate clearance of erythrocytes in guinea pigs
Authors: Giger U , Sticher B , Naef R , Burger R , Lutz HU
Published: October 2019

A variety of naturally occurring autoantibodies (NOAs) have been found in sera of animals and humans. Although their specific homeostatic role in the clearance of altered or senescent cells has been proposed and in vitro studies support such functions, in vivo evidence has been lacking. We studied the effect of affinity-purified human anti-band 3 NOA on the survival of untreated and diamide-treated erythrocytes in normal and complement C3-deficient guinea pigs. In vitro exposure to diamide, an oxidative agent, severely reduced the erythrocyte deformability and increased the amount of high-molecular-weight forms of band 3 protein and band 3-hemoglobin adducts in erythrocyte membranes, thereby markedly shortening the survival of these cells in vivo. Human anti-band 3 NOA bound in a dose-dependent manner to erythrocytes, and binding increased with exposure to diamide. In normal guinea pigs anti-band 3 NOA significantly accelerated the clearance of erythrocytes that were mildly damaged by iodine surface labeling and of those that were further oxidized by diamide. However, the anti-band 3 effect was transient and small. In contrast, anti-band 3 NOA did not significantly alter erythrocyte survival in functionally C3-deficient guinea pigs, thereby supporting the C3b requirement for anti-band 3 NOA activity. On the other hand, a pretreatment of animals with purified human band 3 protein slowed down the clearance of erythrocytes incubated with IgG depleted of anti-band 3 NOA. These results provide the first in vivo evidence of a role for anti-band 3 NOA in the clearance of erythrocytes.
Blood-derived autografts collected during granulocyte colony- stimulating factor-enhanced recovery are enriched with early Thy-1+ hematopoietic progenitor cells
Authors: Haas R , Mohle R , Pforsich M , Fruehauf S , Witt B , Goldschmidt H , Hunstein W
Published: October 2019

It was the objective of the study to characterize CD34+ hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) in a group of 24 cancer patients. After cytotoxic chemotherapy, R-metHu granulocyte colony-stimulating factor (R-metHuG-CSF; filgrastim, 300 micrograms daily, subcutaneously) was given to shorten the time of neutropenia as well as to increase the rebound of peripheral blood progenitor cells (PBPC) for harvesting. The proportion of CD34+ cells in the leukapheresis products (LPs) was 1.4-fold greater than in BM samples that were obtained at the same day (LP: median, 1.4% v BM: median, 1.0%, P < .01). Two- and three-color immunofluorescence showed that blood-derived CD34+ cells comprised a greater proportion of a particular early progenitor cell than CD34+ cells of bone marrow. Blood-derived progenitor cells tended to have a higher mean fluorescence intensity of CD34 and expressed significantly lower levels of HLA-DR (mean fluorescence intensity of HLA-DR: 442.6 +/- 44.9 [LP] v 661.5 +/- 64.6 [BM], mean +/- SEM, P < .01). Furthermore, the blood-derived CD34+ cells comprised a 1.7-fold greater proportion of Thy-1+ cells (LP: median, 24.4% v BM: median, 14.4%, P < .001) and expressed significantly less c-kit (LP: median, 20.5% v BM: median, 31.0%, P < .01). Three-color analysis showed that high levels of Thy-1 expression were restricted to CD34+/HLA-DRdim or CD34+/HLA-DR-cells confirming the early developmental stage of this progenitor cell subset. The proportion of CD34+/CD45RA(bright) cells representing late colony-forming unit granulocyte-macrophage (CFU-GM) was smaller in LPs compared with BM (P < .05). For an examination of BM CD34+ cells before the mobilization chemotherapy, samples of 16 patients were available. The mean proportion of c-kit expressing CD34+ cells in the bone marrow during G-CSF-stimulated reconstitution decreased 1.8-fold compared with baseline values. There was no difference in the proportion of BM-derived CD34+/Thy-1+ cells and CD34+/CD45RA+ cells between steady-state hematopoiesis and G-CSF-supported recovery. Our data suggest that during G-CSF-enhanced recovery, CD34+ cells in the PB are enriched with more primitive progenitor cells to evenly replenish the BM after the chemotherapy-related cytotoxic damage.
Constitutive in vivo cytokine and hematopoietic growth factor gene expression in the bone marrow and peripheral blood of healthy individuals
Authors: Cluitmans FH , Esendam BH , Landegent JE , Willemze R , Falkenburg JH
Published: October 2019

We investigated hematopoietic growth factor (HGF) and cytokine gene expression in the bone marrow (BM) and peripheral blood (PB) of healthy individuals as a starting point for delineating the physiologic role of cytokines in steady state hematopoiesis. BM biopsy specimens and PB samples from 7 healthy individuals were analyzed by polymerase chain reaction amplification of reverse-transcribed RNA using gene-specific primer sets. Consistent gene expression in the BM of all 7 individuals was detected for macrophage colony-stimulating factor (CSF), stem cell factor, interleukin-6 (IL-6), IL-7, erythroid-potentiating factor, erythroid-differentiating factor, and insulinlike growth factor 1, all cytokines with reported direct stimulatory effects on in vitro hematopoiesis. Of these, erythroid-potentiating factor and erythroid-differentiating factor appeared to be the only stimulating factors that were also expressed in the PB. Among the cytokines with inhibitory effects on in vitro hematopoiesis IL-4, tumor necrosis factor-alpha (TNF-alpha), TNF-beta, transforming growth factor-beta, and macrophage inflammatory protein-1 alpha were expressed in the BM of the 7 individuals. Except for TNF-alpha, the latter cytokines were also expressed in the PB. Consistent expression in the BM and PB of all tested individuals was also observed for IL-1 beta, IL-1 receptor antagonist, and IL-1 beta converting enzyme, which are all members of the IL-1 family with a possible indirect effect on hematopoiesis. Remarkably, no expression of granulocyte CSF, granulocyte-macrophage CSF, and IL-3 was found in the BM or PB of all investigated individuals (n = 15). This was also the case for IL-1 alpha, IL-2, IL-5, IL-9, IL-12, IL-13, leukemia-inhibiting factor, interferon-gamma, and inhibin. Weak IL-8 and IL-10 expression was found in the BM and/or PB of a minority of investigated individuals. These findings provide insight into which cytokines or HGFs potentially are involved in the autocrine or paracrine regulation of in vivo steady state hematopoiesis. The absence of expression of granulocyte CSF, granulocyte-macrophage CSF, and IL-3 in the BM of healthy individuals implicates that it is highly unlikely that these HGFs are involved in the autocrine or paracrine regulation of constitutive hematopoiesis.
Interleukin-3 and interleukin-7 are alternative growth factors for the same B-cell precursors in the mouse
Authors: Winkler TH , Melchers F , Rolink AG
Published: October 2019

Clones and lines of precursor (pre) B cells can be established by limiting dilutions of unseparated cell suspensions of fetal liver or bone marrow on stromal cells in the presence of interleukin (IL)-7. When IL-3 is used instead of IL-7, cultures are regularly overgrown by different precursor cells of the myeloid lineage, as well as by adherent cells that inhibit pre-B-cell expansion. However, in the presence of either IL-7 or IL-3, clones of pre-B cells can be established on stroma cells at frequencies near one in one when the cultures are initiated with cell sorter purified CD45RO (B220)+/c-kit+ fetal liver or bone marrow derived pre-B cells. Clones grown on stromal cells in the presence of IL-7 can be regrown in IL-3, and vice versa. Pre-B cells that proliferate on stromal cells in the presence of IL-7 or IL-3 have the same phenotype, ie, are B220+ c-kit+, CD43+, and surrogate light chain+. Removal of the growth factors (IL-7, respectively IL-3) from the cultures results in differentiation to surface immunoglobulin (slg) positive, c-kit-, CD43-, surrogate light chain-B cells, a fraction of which is lipopolysaccharide (LPS) responsive as shown by IgM secretion. These results show that IL-7 and IL-3 stimulate largely overlapping populations of precursor B cells from bone marrow to proliferate for long periods of time in the presence of stromal cells. Thus, IL-7 and IL-3 are alternative growth factors for the same pre-BI cell.
Identification and characterization of a soluble c-kit receptor produced by human hematopoietic cell lines
Authors: Turner AM , Bennett LG , Lin NL , Wypych J , Bartley TD , Hunt RW , Atkins HL , Langley KE , Parker V , Martin F
Published: October 2019

Stem cell factor (SCF) triggers cell growth by binding to cell surface c-kit receptors. Soluble forms of several cytokine receptors have been described and may play a role in the modulation of cytokine activity in vivo. For these reasons, we investigated whether human hematopoietic cells produce soluble c-kit receptors. The human leukemia cell lines OCIM1 and MO7e display approximately 80,000 and approximately 35,000 high-affinity cell surface c-kit receptors, respectively. Soluble c-kit receptors were detected by enzyme immunoassay in OCIM1 and MO7e culture supernatants. We determined the molecular weight and binding affinity of soluble c-kit receptor produced by OCIM1 cells, soluble c-kit receptor purified from human serum, and recombinant soluble c-kit receptor expressed in CHO cells. The three soluble c-kit receptors each have a molecular weight of 98 kD. Quantitative binding experiments with 125I-SCF indicate that the soluble c-kit receptors obtained from human serum or OCIM1 cells have binding affinities for SCF of approximately 200 to 300 pmol/L, in contrast to the recombinant form, which has a binding affinity of approximately 1.5 nmol/L. All three forms of the soluble c-kit receptor were able to compete with c-kit receptors on OCIM1 cells for 125I-SCF binding. Thus human hematopoietic cells can produce a soluble form of the c-kit receptor that retains high-affinity SCF binding activity. We speculate that the soluble c-kit receptor may bind SCF and function as a receptor antagonist in vivo.
Localization of the inducible enhancer in the mouse interleukin-5 gene that is responsive to T-cell receptor stimulation
Authors: Bourke PF , van Leeuwen BH , Campbell HD , Young IG
Published: October 2019

Transcriptional regulation of the interleukin-5 (IL-5) gene in T lymphocytes appears to be of central importance in the control of the eosinophilia characteristic of allergic responses and certain parasite infections. Previous studies of IL-5 gene regulation have been hampered by the lack of a transfection assay, which detects the antigen-responsive enhancer in the IL-5 promoter. Here we show that stable transfection of the Th2 clone D10.G4.1 and the T lymphoma EL4.23 with chloramphenicol acetyltransferase reporter gene constructs carrying the region to -3859 gives inducible expression with the known regulatory characteristics of the endogenous IL-5 gene. To facilitate detailed analysis of the promoter region, 3.9 kb of DNA sequence immediately up stream of the start of transcription was determined and the minimum upstream region required for inducible expression was further localized, by stable transfection studies in EL4.23 cells, to the region up to -1016. A CTF/NF1 site in the upstream enhancer at -940 to - 928 was shown to be required for regulated inducible expression. Mutation of this sequence motif abolished inducibility and also prevented binding of the sequence to a nuclear protein(s). A TCATTT-containing element in the proximal promoter region was also demonstrated to be essential for inducible expression of the IL-5 gene, similar to the role of this conserved element in the transcriptional regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 genes.
Detection of myc translocations in lymphoma cells by fluorescence in situ hybridization with yeast artificial chromosomes
Authors: Veronese ML , Ohta M , Finan J , Nowell PC , Croce CM
Published: October 2019

Translocations involving chromosome 8 at band q24 and one of the Ig loci on chromosomes 14q32, 22q11, and 2p11 are the hallmark of Burkitt's lymphoma (BL). It has been previously observed that the exact localization of the breakpoints at chromosome 8q24 can vary significantly from patient to patient, scattering over a distance of more than 300 kb upstream of c-myc and about 300 kb downstream of c-myc. To generate probes for fluorescence in situ hybridization (FISH) that detect most c-myc translocations, we screened a yeast artificial chromosome (YAC) library from normal human lymphocytes by colony hybridization, using three markers surrounding the c-myc gene as probes. We obtained 10 YAC clones ranging in size between 500 and 200 kb. Two nonchimeric clones were used for FISH on several BL cell lines and patient samples with different breakpoints at 8q24. Our results show that the YAC clones detected translocations scattered along approximately 200 kb in both metaphase chromosomes and interphase nuclei. The sensitivity, rapidity, and feasibility in nondividing cells render FISH an important diagnostic tool. Furthermore, the use of large DNA fragments such as YACs greatly simplifies the detection of translocations with widely scattered breakpoints such as these seen in BL.
Allogeneic bone marrow transplantation for chronic myeloid leukemia in first chronic phase: a randomized trial of busulfan-cytoxan versus cytoxan-total body irradiation as preparative regimen: a report from the French Society of Bone Marrow Graft (SFGM)
Authors: Devergie A , Blaise D , Attal M , Tigaud JD , Jouet JP , Vernant JP , Bordigoni P , Ifrah N , Dauriac C , Cahn JY
Published: October 2019

From March 1988 to March 1991, 19 French bone marrow transplant (BMT) centers participated in a prospective randomized trial comparing two conditioning regimens for patients with chronic myeloid leukemia transplanted in first chronic phase with an HLA identical sibling donor. A total of 120 consecutive patients were randomized to receive either 120 mg/kg of cyclophosphamide followed by total body irradiation (CY-TBI; n = 55) or 16 mg/kg of busulfan followed by 120 mg/kg of CY (BU-CY; n = 65). Two different TBI regimens were used. Thirteen patients received a 10-Gy single-dose TBI (SDTBI), and 42 received a fractionated TBI (FTBI). Median time between diagnosis and BMT was 315 days. Overall 5-year actuarial survival was 62.9% (65.8% +/- 12.5% for CY-TBI and 60.6 +/- 11.7% for BU-CY; P = .5), and overall disease-free survival was 55% (51% +/- 14% for CY-TBI and 59.1% +/- 11.8% for BU-CY; P = .75). All patients conditioned with CY-TBI experienced sustained engraftment; in contrast, 4 of 65 patients conditioned with BU-CY rejected the graft (P = .18). There was no significant statistical difference between the two groups regarding transplant-related mortality (29% for CY-TBI and 38% for BU-CY; P = .44). So far, with a median follow up of 42 months, 11 patients have relapsed; 9 relapses occurred after CY-TBI, mostly after FTBI (8 of 9) and 2 after BU-CY (P = .02). The actuarial risk of relapse was 4.4% +/- 6.7% after BU-CY, 11.1% +/- 20.8% after SDTBI, and 31.3% +/- 18.1% after FTBI (P = .039). In addition, independently of the conditioning regimen, the increase of posttransplant immunosuppression in 16 patients with an anti- interleukin-2 receptor monoclonal antibody (MoAb) in addition to a short course of methotrexate and cyclosporine was shown to increase the actuarial risk of relapse (57% +/- 30% with MoAb v 9% +/- 7.3% without MoAb; P = .001). We conclude that BU is an acceptable alternative to TBI for patients with chronic myeloid leukemia in first chronic phase receiving BMT from HLA identical sibling donors. Both BU-CY and CY-TBI regimens gave similar transplant-related mortality, and the antileukemic efficiency of BU-CY regimen was either similar or even higher than that of CY-TBI.
CD8+ T lymphocytes in the lung of acquired immunodeficiency syndrome patients harbor human immunodeficiency virus type 1
Authors: Semenzato G , Agostini C , Ometto L , Zambello R , Trentin L , Chieco-Bianchi L , De Rossi A
Published: October 2019

Human immunodeficiency virus-1 (HIV-1) infection of CD8+ lymphocytes has been described in several in vitro culture systems, but whether CD8+ cells are a target and also serve as a reservoir for infection in vivo as yet is unknown. We addressed this issue in patients with acquired immunodeficiency syndrome (AIDS)-related lower respiratory tract chronic inflammation, which is characterized by a massive influx of CD8+ HIV-1-specific cytotoxic T lymphocytes (CTL). Proviral load in lung T lymphocytes and their subpopulations was evaluated by using the DNA-polymerase chain reaction (PCR) technique on cells retrieved by bronchoalveolar lavage. To avoid the possibility that the presence of HIV-1 DNA could be caused by contaminating CD4+ cells, serial dilutions of highly purified CD8+ cells were also analyzed by PCR. Our findings showed that lung CD8+ cells harbor and express HIV-1. To explore the possible mechanisms leading to pulmonary CD8+ lymphocyte infection, we evaluated CD4 gene expression on highly purified CD8+ cells by means of reverse transcriptase PCR. Despite the lack of membrane CD4 reactivity, we could show that CD8+ cells may express CD4 RNA. Coinfection of lung CD8+ cells harboring proviral HIV-1 sequences by viral agents capable of inducing CD4 expression (ie, HHV-6) was not detected. Our data indicate that not only CD4+ T lymphocytes and macrophages, but also CD8+ cells, may represent a target and/or a reservoir for HIV-1 in vivo, and suggest that lung CD8+ lymphocytes could derive from precursors equipped with enough CD4 molecules to become HIV-1 permissive. Aside from the cell-to-cell contact between activated HIV-1 specific CTL and relevant targets, the infection of precursors could represent an additional mechanism accounting for the infection of pulmonary CD8+ cells and their functional impairment.