bluuebunny/crossref_metadata_2025 huggingface dataset:
The human TCF-1 gene encodes a nuclear DNA-binding protein uniquely expressed in normal and neoplastic T-lineage lymphocytes
Authors: Castrop J , van Wichen D , Koomans-Bitter M , van de Wetering M , de Weger R , van Dongen J , Clevers H
Published: October 2019

The TCF-1 gene encodes a putative transcription factor with affinity for a sequence motif occurring in a number of T-cell enhancers. TCF-1 mRNA was originally found to be expressed in a T cell-specific fashion within a set of human and mouse cell lines. In contrast, expression reportedly occurs in multiple nonlymphoid tissues during murine embryogenesis. We have now raised a monoclonal antibody to document expression and biochemistry of the human TCF-1 protein. As expected, the TCF-1 protein was detectable only in cell lines of T lineage. Its expression was always restricted to the nucleus. Immunohistochemistry on a panel of human tissues revealed that the TCF-1 protein was found exclusively in thymocytes and in CD3+ T cells in peripheral lymphoid tissues. Western blotting yielded a set of bands ranging from 25 kD to 55 kD, resulting from extensive alternative splicing. The TCF-1 protein was detectable in all samples of a set of 22 T-cell malignancies of various stages of maturation, but was absent from a large number of other hematologic neoplasms. These observations imply a T cell-specific function for TCF-1, a notion corroborated by recent observations on Tcf-1 knock-out mice. In addition, these results indicate that nuclear TCF-1 expression can serve as a pan-T-lineage marker in the diagnosis of lymphoid malignancies.
Immune responses to major histocompatibility complex homozygous lymphoid cells in murine F1 hybrid recipients: implications for transfusion-associated graft-versus-host disease
Authors: Fast LD , Valeri CR , Crowley JP
Published: October 2019

Graft-versus-host disease (GVHD) is currently encountered after bone marrow transplantation and transfusion. GVHD associated with transfusion (TA-GVHD) in apparently immunocompetent recipients has been recently reported with increasing frequency. A consistent finding in many of these cases is that the recipient received blood from a donor homozygous for one of the recipient's HLA haplotypes. However, the observed frequency of TA-GVHD is much lower than the estimated probability of this donor/recipient combination. The potential role of recipient immune responses in controlling TA-GVHD was investigated using an analogous murine model in which GVHD is induced by the injection of parental lymphoid cells into unirradiated F1 hybrid recipients. The effect of various immune manipulations of the recipient of GVHD induction was assessed by determining the number of donor lymphoid cells required to induce GVHD responses. Whereas depletion of recipient CD4+ cells increased the number of donor cells needed to induce GVHD, depletion of recipient CD8+ and natural killer cells resulted in fewer donor cells being needed to induce a GVHD response. These studies suggest a central role for functioning recipient CD8 and natural killer cells in the down-regulation of TA-GVHD development in recipients.
Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies
Authors: Reumaux D , Vossebeld PJ , Roos D , Verhoeven AJ
Published: October 2019

Antineutrophil cytoplasmic autoantibodies (ANCA) have been described in sera of patients with several forms of systemic vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. The two main targets of ANCA in vasculitis are proteinase 3 (PR3) and myeloperoxidase (MPO). ANCA are capable of activating neutrophils primed by tumor necrosis factor-alpha (TNF-alpha) in vitro, which may be relevant for the induction of the vascular inflammation observed in vivo. Recently, it has been suggested that engagement of Fc gamma receptor IIa (Fc gamma RIIa) on the neutrophils is involved in the activation by ANCA. In the present study, we show that activation of the neutrophil respiratory burst by anti-PR3 and anti-MPO is strongly enhanced after TNF priming and lost on removal of the Fc parts of the antibodies. Similar results were obtained when the neutrophils were activated with antibodies against known membrane antigens without major changes in the expression of the target antigens. The TNF-induced enhancement of the neutrophil activation was not observed when adherence of the cells was prevented by continuous stirring of the suspension or by the addition of CD18 antibodies before TNF exposure. Hence, our results indicate that engagement of both Fc gamma RIIa and beta 2 integrins is instrumental in neutrophil activation induced by ANCA.
Primary structure of murine red blood cell-type pyruvate kinase (PK) and molecular characterization of PK deficiency identified in the CBA strain
Authors: Kanno H , Morimoto M , Fujii H , Tsujimura T , Asai H , Noguchi T , Kitamura Y , Miwa S
Published: October 2019

To clarify the molecular abnormality of pyruvate kinase (PK) deficiency identified in the mutant mice of CBA-Pk-1slc/Pk-1slc, we cloned murine red blood cell-type PK (R-PK) cDNA of those animals. The cDNA sequence spans 1827 bp, including an open reading frame that can encode 574 amino acids. Homology in the coding sequences between murine and human R-PK was 86.1% at nucleotide and 91.5% at amino acid levels. A homozygous missense mutation at nucleotide 1013 GGT-->GAT was identified in the cDNA sequence of the mutant, causing a single amino acid substitution at no. 338Gly-->Asp of the murine R-PK. Six amino acid residues, 335Val-336Ala-337Arg-338Gly-339Asp-340L eu, were encoded in exon 8 of both human and rat L (liver-type)/R-PK genes and were evolutionarily conserved in PK from bacteria through humans. 337Arg was reported to be important for substrate binding, suggesting that the amino acid change would impair substrate affinity of the PK subunit. A homozygous missense mutation at the catalytic domain has been identified in a human PK variant, PK Hong Kong (941ATT-->ACT, 314 Ile-- >Thr). Although both 1013A and 941C gave rise to an amino acid change adjacent to the active site and may interfere with substrate binding to the subunit, the degree of anemia was much more severe in the human case. The erythroid-progenitor cell number increased in the spleen of Pk-1slc/Pk-1slc mice to a level approximately 66 times higher than that in normal CBA mice, suggesting that compensatory extramedullary erythropoiesis in the spleen of the mutant mice, but not in the human variant, might account for the observed difference in the phenotype.
The flaky skin (fsn) mutation in mice: map location and description of the anemia
Authors: Beamer WG , Pelsue SC , Shultz LD , Sundberg JP , Barker JE
Published: October 2019

Flaky skin (gene symbol fsn) is an autosomal recessive mutation that causes pleiotropic effects of anemia, papulosquamous skin disorder, and gastric forestomach hyperplasia. In this report, we assign fsn to distal chromosome 17 and characterize the anemia. The decrease in hematocrit levels and red blood cell counts is significant and persists throughout life in fsn/fsn mice. There is compensatory enlargement of the heart, liver, and spleen by 8 weeks of age, whereas the thymus is less than one half normal weight. Nucleated cell counts in the peripheral blood are increased 15- to 30-fold, primarily due to an increased percentage of normoblasts. The fsn/fsn mice examined at 8 weeks of age have significantly increased reticulocyte counts and protoporphyrin levels but reduced hemoglobin concentration, suggesting possible abnormalities of hemoglobin metabolism. Erythrocyte membrane fragility is normal. Compared with normal +/? littermates, fsn/fsn mice (1) lack splenic and hepatic stores of elemental iron, (2) have the ability to transport 59Fe across the duodenal cells and into the blood, (3) have increased levels of transferrin in serum, and (4) have acute loss of urinary 59Fe. Hemolysis is indicated by increased serum bilirubin and high blood reticulocyte numbers. Collectively, the genetic, hematologic, and pathologic data indicate a severe hematologic disorder caused by homozygosity for the fsn mutation that differs from other known hematologic mutations in the mouse. The mechanism whereby fsn induces the reported pleiotropic effects has yet to be elucidated.
Analysis of beta-globin mutations shows stable mixed chimerism in patients with thalassemia after bone marrow transplantation
Authors: Kapelushnik J , Or R , Filon D , Nagler A , Cividalli G , Aker M , Naparstek E , Slavin S , Oppenheim A
Published: October 2019

Beta-thalassemia major (TM) is caused by any of approximately 150 mutations within the beta-globin gene. To establish the degree of chimerism after bone marrow transplantation (BMT), we have performed molecular analysis of beta-globin mutations in 14 patients with TM over a period of 10 years. All patients underwent T cell-depleted allogeneic BMT from HLA-identical related donors, using either in vitro T-cell depletion with CAMPATH 1M and complement or in vivo depletion using CAMPATH 1G in the bone marrow collection bag. To date, at different time periods after BMT, seven patients have some degree of chimerism; six of these patients, all blood transfusion-independent, have donor cells in the range of 70% to 95%, with stable mixed chimerism (MC). The seventh patient has less than 10% donor cells with, surprisingly, only minimal transfusion requirements. The detection of beta-globin gene point mutation, as used here, is a highly specific and sensitive marker for engraftment and MC in patients with thalassemia. In light of its specificity, the method is applicable in all cases of TM, as it is independent of sex and other non-globin-related DNA markers. The high incidence of MC found in our patients may be a consequence of the pre-BMT T-cell depletion. Because MC was associated with transfusion independence, complete eradication of residual host cells for effective treatment of TM and possibly other genetic diseases may prove not to be essential.
Unrelated donor marrow transplantation in children
Authors: Balduzzi A , Gooley T , Anasetti C , Sanders JE , Martin PJ , Petersdorf EW , Appelbaum FR , Buckner CD , Matthews D , Storb R
Published: October 2019

Eighty-eight children 0.5 to 17 years of age (median, 9 years of age) received an unrelated donor marrow transplant for treatment of chronic myeloid leukemia (CML; n = 16), acute lymphoblastic leukemia (ALL) in first or second remission (n = 15) or more advanced stage (n = 28), acute myeloid leukemia (AML; n = 13), or other hematologic diseases (n = 16) between June 1985 and April 1993. All patients were conditioned with cyclophosphamide and total body irradiation and received a combination of methotrexate and cyclosporine as graft-versus-host disease (GVHD) prophylaxis. Fourty-six patients received transplants from HLA-identical donors and 42 patients received transplants from donors who were minor-mismatched at one HLA-A or B or D/DRB1 locus. The Kaplan-Meier estimates of disease-free survival and relapse were 75% and 0% for patients with CML, 47% and 20% for ALL in first or second remission, 10% and 60% for ALL in relapse or third remission, 46% and 46% for AML in first remission (n = 1) or more advanced disease (n = 12), and 29% and 69% for other diseases. HLA disparity was not significantly associated with lower disease-free survival, but the results suggest more relapses in HLA-matched recipients and there was significantly more transplant-related mortality in mismatched recipients (51% v 24%, P = .04). Most deaths were due to infections associated with acuteor chronic GVHD and occurred within the first 2 years after transplantation. Granulocyte engraftment occurred in all evaluable patients. Sixty-three percent of HLA-matched and 57% of HLA-mismatched recipients were discharged home disease-free at a median of 98 and 103 days, respectively, after transplantation (P = not significant [NS]). The incidence of grades II-IV acute GVHD was 83% in HLA-matched and 98% in HLA-mismatched recipients (P = .009). The incidence of chronic GVHD was 60% in HLA-matched and 69% in HLA- mismatched recipients (P = NS). One or multiple late adverse events such as cataracts, osteonecrosis of the hip or knee, restrictive or obstructive pulmonary disease, and hypothyroidism have occurred in 11 of 33 (33%) surviving patients. Immunosuppression was discontinued in 58% of surviving patients, including all 12 patients surviving more than 3.2 years, all of whom have a Lansky or Karnofsky score of 100%.(ABSTRACT TRUNCATED AT 400 WORDS)
Intensive therapy with peripheral blood progenitor cell transplantation in 60 patients with poor-prognosis follicular lymphoma
Authors: Bastion Y , Brice P , Haioun C , Sonet A , Salles G , Marolleau JP , Espinouse D , Reyes F , Gisselbrecht C , Coiffier B
Published: October 2019

Intensive therapy, mainly with purged autologous bone marrow transplantation (ABMT), has been proposed in recent years as consolidation treatment in young patients with follicular lymphoma. Reported experience with transplantation of peripheral blood progenitor cells (PBPC) is, so far, limited. The feasibility and the therapeutic efficacy of intensive therapy followed by unpurged autologous PBPC reinfusion were evaluated in 60 patients with poor-prognosis follicular lymphoma. Twelve patients were in first partial remission (PR), 34 were in second partial or complete remission (CR), and 14 were in subsequent progression. At the time of the procedure, 39 patients (65%) had persistent bone marrow involvement, 49 patients (82%) were in PR, and 16 patients had presented with a histologic transformation (HT). PBPC were collected after chemotherapy followed by granulocyte (G) colony-stimulating factor (CSF) or granulocyte-macrophage (GM)-CSF in 50 patients. Conditioning regimens included high-dose chemotherapy alone (14 patients); mainly the BCNU, etoposide, aracytine, melphalan [BEAM] regimen), or cyclophosphamide with or without etoposide plus total body irradiation (46 patients). The median time to reach a neutrophil count greater than 0.5 x 10(9)/L was 13 days. There were five treatment-related deaths, with four being associated with a delayed engraftment and all occurring in patients in third or subsequent progression. At a median follow-up of 21 months, 48 patients were still alive, 18 relapsed, and seven died of lymphomas progression. Estimated 2-year overall survival (OS) and failure-free survival (FFS) rates were 86% and 53%, respectively, without or plateau. Patients treated in PR1 or PR2/CR2 had a significantly longer rate of OS and FFS than those treated in subsequent progression (P = .002 and P = .001, respectively), whereas age, response to salvage treatment, presence or absence of residual bone marrow involvement, or conditioning regimen had no influence on outcome. Patients with HT tended to have a worse FFS rate (P = .04) without an OS difference. Along with an unusual rate of engraftment failure, the poor FFS observed in heavily pretreated patients suggests that intensive therapy should be performed early in the course of the disease. Given the high percentage of patients intensified in PR with residual bone marrow involvement, our results are comparable with those achieved with ABMT published to date. Prospective trials are warranted to compare this strategy with standard therapy in patients with relapsing or PR follicular lymphoma.
Opposing effects of the basic helix-loop-helix transcription factor SCL on erythroid and monocytic differentiation
Authors: Hoang T , Paradis E , Brady G , Billia F , Nakahara K , Iscove NN , Kirsch IR
Published: October 2019

The SCL gene (also called Tal-1 or TCL5) was identified because of its association with chromosomal translocations in childhood T-cell lymphoid leukemias. SCL codes for a basic helix-loop-helix (bHLH) factor that can function as a transcriptional activator or repressor. In the adult, SCL expression is restricted to hematopoietic cells and tissues, but its function in the process of lineage commitment is unknown. The present study was designed to address the role of SCL in hematopoietic cell differentiation. SCL expression was determined in primary hematopoietic cells through the screening of cDNA samples obtained by reverse transcription-polymerase chain reaction (RT-PCR) from single cells at different stages of differentiation. SCL RNA expression was highest in bipotential and committed erythroid precursors and diminished with subsequent maturation to proerythroblasts and normoblasts. In contrast, SCL mRNA was low to undetectable in precursors of granulocytes and monocytes and their maturing progeny. The same pattern of expression was observed after erythroid or monocytic differentiation of a bipotent cell line, TF-1, in that SCL mRNA levels remained elevated during erythroid differentiation and were downregulated with monocytic differentiation. Accordingly, TF-1 was chosen as a model to investigate the functional significance of this divergent pattern of SCL expression in the two lineages. Four independent clones stably transfected with an SCL expression vector exhibited enhanced spontaneous and delta-aminolevulinic acid-induced erythroid differentiation as measured by glycophorin expression and hemoglobinization, consistent with the view that SCL is a positive regulator of erythroid differentiation. Furthermore, constitutive SCL expression interfered with monocytic differentiation, as assessed by the generation of adherent cells and the expression of Fc gamma RII in response to TPA. These results suggest that the downregulation of SCL may be required for monocytic differentiation.
PAI-1-resistant t-PA: low doses prevent fibrin deposition in rabbits with increased PAI-1 activity
Authors: Krishnamurti C , Keyt B , Maglasang P , Alving BM
Published: October 2019

The present study compared the activities of recombinant tissue-type plasminogen activator (t-PA) and a plasminogen activator inhibitor-1 (PAI-1)-resistant variant t-PA (Kt-PA, KHRR 296–299 AAAA) in preventing renal fibrin deposition in rabbits with elevated PAI-1 activity. In this model, all rabbits were infused with endotoxin (10 micrograms/kg), followed by initiation of thrombin (130 U/kg) 4 hours later, when plasma PAI-1 activity was greater than 200 arbitrary units (AU)/mL (baseline, < 3 AU/mL). Thirty minutes after completion of the 1-hour thrombin infusion, rabbits were killed and the kidneys fixed and stained for identification of fibrin deposition. Rabbits received one of the following treatments initiated 30 minutes before thrombin and continued during the 1-hour thrombin infusion: (1) saline (n = 7); (2) low-dose t-PA (17 micrograms/kg, n = 4); (3) higher-dose t-PA (170 micrograms/kg, n = 4); or (4) low-dose Kt-PA (17 micrograms/kg, n = 6). Fibrin deposition occurred in 86% and 100% of the rabbits receiving saline or low-dose t-PA, respectively. Fibrin deposition did not occur in any of the rabbits receiving low-dose Kt-PA or higher-dose t-PA. Low-dose Kt-PA and higher dose t-PA also caused a reduction in fibrin deposition when infused after thrombin administration had been completed. The data provide in vivo evidence that Kt-PA is more effective than t-PA in preventing fibrin deposition in an animal model that combines thrombogenic stimulation with increased PAI-1 activity.
Protein kinase C regulates tyrosine phosphorylation of pp125FAK in platelets adherent to fibrinogen
Authors: Haimovich B , Kaneshiki N , Ji P
Published: October 2019

Platelet adhesion to immobilized fibrinogen stimulates the induction of tyrosine phosphorylation of multiple proteins. However, platelet spreading and tyrosine phosphorylation of three proteins, the focal adhesion kinase pp125FAK and proteins of 101 and 105 kD (pp101 and pp105), require a second adenosine diphosphate (ADP)-dependent costimulatory event. In this study we show that protein kinase C (PKC) inhibitors prevented the induction of tyrosine phosphorylation of pp125FAK, pp101 and pp105, and abolished spreading. These inhibitory effects were not observed after treatment of the platelets with the intracellular Ca2+ chelator BAPTA-AM. This suggested that in platelets, PKC regulates spreading and related protein tyrosine phosphorylation. In addition, the inhibitory effects of apyrase, an ADP scavenger, on spreading and tyrosine phosphorylation of pp125FAK, pp101, and pp105, were not observed in the presence of phorbol 12-myristate 13-acetate (PMA). These data implied that in fibrinogen-adherent platelets integrin ligation and an agonist receptor occupancy are required for the functional association of PKC and the alpha IIb beta 3-mediated signaling pathways. Taken together these results show that PKC plays a central role in the transduction of intracellular signals downstream from alpha IIb beta 3 that regulate spreading and pp125FAK phosphorylation.
Differential regulation of vascular cell adhesion molecule-1 gene transcription by tumor necrosis factor alpha and interleukin-1 alpha in dermal microvascular endothelial cells
Authors: Gille J , Swerlick RA , Lawley TJ , Caughman SW
Published: October 2019

As part of the inflammatory response, the localization of leukocytes depends to an important degree on cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells (EC). We have previously shown that VCAM-1 expression is induced on human umbilical vein EC (HUVEC) by both tumor necrosis factor alpha (TNF- alpha) and interleukin-1 alpha (IL-1 alpha), whereas on human dermal microvascular EC (HDMEC) only TNF alpha results in VCAM-1 expression. To explore molecular mechanisms responsible for these contrasting patterns of VCAM-1 induction in HUVEC versus HDMEC, we performed transcriptional activation studies with VCAM-1-based reporter constructs and in vitro binding assays using two adjacent NF-kappa B binding sequences of the VCAM-1 promoter as a DNA probe. Previous studies have established that these NF-kappa B motifs are required for cytokine-induced VCAM-1 transcription, and may further mediate cell- specific VCAM-1 gene activation by cytokines. The findings reported here demonstrate a significant HDMEC-specific attenuation of VCAM-1 gene transcription in response to IL-1 alpha, but not TNF alpha. An upstream VCAM-1 gene regulatory region distinct from the NF-kappa B sites appears to function as an IL-1 alpha-mediated transcriptional repressor within HDMEC. This repressor region conveys IL-1 alpha- dependent, but not TNF alpha-dependent, inhibition of transcription driven by a heterologous cytokine response element and promoter.
REL proto-oncogene is frequently amplified in extranodal diffuse large cell lymphoma
Authors: Houldsworth J , Mathew S , Rao PH , Dyomina K , Louie DC , Parsa N , Offit K , Chaganti RS
Published: October 2019

Comparative genomic hybridization (CGH) analysis of DNA extracted from a diffuse lymphoma with a large cell component (DLLC) that displayed double minute chromosomes upon conventional karyotypic analysis indicated overt amplification of DNA sequences derived from the 2p13–15 region. Southern blot analysis of this tumor DNA with a cDNA probe for the proto-oncogene REL, previously mapped to 2p14–15, indicated a greater than 35-fold amplification of REL. To determine the incidence of REL amplification and possible clinical or histologic association with DLLC, a panel of 111 tumor DNAs from DLLC specimens was screened for REL amplification by Southern blot analysis. A copy number of > or = 4 was noted in 26 cases (23%). Southern blot analysis of these 26 tumor DNAs with a cDNA probe for TGFA, mapped to 2p13, indicated lack of coamplification except in one case. Another member of the Rel/NF- kappa B family of transcriptional activators, RELA/p65 mapped to 11q13, was amplified in five cases as determined by Southern blot analysis using a cDNA probe. Nineteen of the 26 DLLC (73%) with REL amplification were primary extranodal lymphomas. As a group, the tumors with REL amplification demonstrated an increased frequency of chromosomal aberrations previously associated with tumor progression, suggesting an oncogenic effect of amplified REL in B-lymphoid cells that already contained a transforming genetic lesion. Thus, REL amplification is a frequent event in DLLC, and probably constitutes a progression-associated marker of primary extranodal lymphomas. This study shows the usefulness of the CGH technique in identifying chromosomal regions overrepresented in tumors that can point to amplified genes and may be correlated with clinical features of the disease.
Tumor-specific rearrangements of the immunoglobulin heavy-chain gene in B-cell non-Hodgkin's lymphoma detected by in situ hybridization
Authors: Ueda Y , Matsuda F , Misawa S , Taniwaki M
Published: October 2019

We have recently described the potential use of fluorescence in situ hybridization (FISH) to detect tumor-specific rearrangements of the immunoglobulin heavy-chain (IgH) gene in interphase nuclei. Using yeast artificial chromosome (YAC) clone Y6 containing variable region (VH) gene and bacteriophage clones Ig gamma, we analyzed 70 patients with B- cell non-Hodgkin's lymphoma (NHL) and compared the results with those obtained by the conventional G-banding method. Tumor-specific rearrangements of the IgH gene equivalent to 14q32 translocations were defined as separate signals of VH and Ig gamma genes or those of Ig gamma genes and referred to split signals. Twenty-nine patients (41.4%) showed split signals. Among these, 13 did not show 14q32 translocations by G-banding: three with other chromosomal abnormalities, one with normal karyotype, and nine with no analyzable metaphases. The partner sites of 14q32 translocations were identified in 17 patients by FISH: t(3;14)(q27;q32) including a complex variant was observed in nine patients, t(14;18)(q32;q21) in four, t(8;14)(q24;q32) in three, t(14;19)(q32;q13) in one, and t(11;14)(q13;q32) in one. Six of nine patients with t(3;14) or its variant and one of three with t(8;14) were diagnosed as having respective translocations only by FISH. Translocation t(3;14) was found most commonly, and was correlated histologically with diffuse lymphoma with large-cell components. These results indicate that interphase FISH with IgH gene probes promises to be a rapid and reliable method for use in the diagnosis of B-cell NHL.
Marginal zone B-cell lymphomas of different sites share similar cytogenetic and morphologic features [see comments]
Authors: Dierlamm J , Pittaluga S , Wlodarska I , Stul M , Thomas J , Boogaerts M , Michaux L , Driessen A , Mecucci C , Cassiman JJ
Published: October 2019

Clinical, histologic, cytogenetic, and molecular genetic data of 31 patients with extranodal, nodal, and splenic marginal zone B-cell lymphoma (MZBCL) are presented. Despite these variable clinical manifestations, a similar spectrum of morphologic features as well as distinctive immunophenotypic findings were noted. In all cases, a monotypic B-cell proliferation consistently negative for CD5, CD10, and CD23 was found expanding the marginal zone of the B follicle with and without colonization of the follicle centers. Clonal chromosomal abnormalities were detected in 23 of the 31 patients. Recurrent aberrations included whole or partial trisomy 3 (18 cases), trisomy 18 (9 cases), and structural rearrangements of chromosome 1 with breakpoints in 1q21 (9 cases) or 1p34 (6 cases), all of which were seen in extranodal, nodal, as well as splenic MZBCL. Abnormalities of the additional chromosome 3, such as +del(3)(p13),+i(3)(q10), or structural changes involving the distal part of the long arm, were evident in 9 of the 18 cases. All recurrent abnormalities were found in MZBCL more frequently than in other histologic entities of B-cell non-Hodgkin's lymphoma (B-NHL). None of the known lymphoma-associated chromosomal changes or rearrangements of the BCL1, BCL2, BCL3, BCL6, and CMYC genes were detected. We conclude that MZBCL represent a distinct entity of B- NHL with characteristic morphologic and immunophenotypic features and particular chromosomal abnormalities, and that a close histogenetic relationship between extranodal, nodal, and splenic MZBCL is likely, although the clinical presentation may vary.
Distinct temporal patterns of defensin mRNA regulation during drug- induced differentiation of human myeloid leukemia cells
Authors: Herwig S , Su Q , Zhang W , Ma Y , Tempst P
Published: October 2019

Defensins are microbicidal peptides and the principal constituents of neutrophil primary granules. They are presumed to play a prominent role in innate host defenses. We examined defensin mRNA levels during drug-induced differentiation of the promyelocytic leukemia cell line, HL-60. Transcription was restricted to promyelocyte, myelocyte, and very early metamyelocyte stages of the granulocytic pathway. Complete downregulation occurred during late granulocytic maturation or early during phorbol ester-promoted differentiation along the monocyte/macrophage lineage. Retinoic acid (RA) was the strongest inducer of defensin mRNA accumulation, even at doses too low to effect morphologic changes; the initial (first 48 hours), gradual increase resulted from transcriptional activation and was enhanced by granulocyte colony-stimulating factor. In contrast, addition of hybrid polar compounds led to a transient, drug-specific downregulation within the same time period, apparently by means of selectively induced, biphasic degradation of transcripts. Subsequent increase in transcript levels was faster and more pronounced with hexamethylene bisacetamide, relative to dimethyl sulfoxide (DMSO). DMSO-promoted effects were strikingly different in serum-free medium or in the presence of the tyrosine kinase inhibitor, genistein. Under these conditions, and although differentiation was unaffected, early defensin mRNA downregulation was final. The effect did not occur with RA and expression of other myeloid-specific genes was also unchanged. Addition of selected cytokines caused a similar “dip,” only at earlier times and uncoupled from differentiation. Tumor necrosis factor-alpha markedly induced defensin levels after 2 days in previously untreated HL-60 cells, but inhibited expression in RA-differentiated cells. These results begin to detail a complex regulation of defensin mRNA synthesis with both spatial and temporal control elements, and a unique modulation by chemical agents, cytokines, and serum-factors.
Human cytomegalovirus increases constitutive production of interleukin- 6 and leukemia inhibitory factor by bone marrow stromal cells
Authors: Lagneaux L , Delforge A , Snoeck R , Bosmans E , Moreau JF , Taupin JL , De Clercq E , Stryckmans P , Bron D
Published: October 2019

Human cytomegalovirus (CMV) infection is often associated with myelosuppression and acute inflammatory reaction in immunocompromised patients. We have previously documented that CMV exposure of bone marrow (BM) stromal cells reduces the capacity of these cells to support hematopoiesis because of a decreased production of colony- stimulating factors. This study examines the potential role of CMV on constitutive and lipopolysaccharide (LPS)-stimulated production of cytokines involved in inflammatory reaction, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) by BM stromal cells. The release of IL- 6 was already detectable 2 hours post CMV-infection (2.5-fold increase in production) and the cumulative production of IL-6 after 5 days of infection was 23 +/- 1.2 ng/mL (ninefold increase in production). CMV was also able to induce a time-dependent production of LIF that was maximal 8 hours after CMV infection (2.5-fold increase in production). Concomitantly, there was no detectable release of granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) by CMV-infected stromal cells. The similar IL-6 and LIF production in the presence of polymyxin B ruled out the possibility that this increase could be caused by contamination of the viral stock by endotoxin. In addition, ultraviolet-inactivated virus behaved similarly to live virus and caused the release of IL-6 and LIF. However, heat-inactivated CMV was unable to induce IL-6 and LIF secretion by BM stromal cells. The production of IL-6 and LIF was also evaluated after stimulation by LPS. After 5 days of CMV exposure, the LPS-stimulated production of IL-6 and LIF was significantly lower than uninfected controls. This LPS-induced release of cytokine production was found to be dependent of viral replication. The experiments have shown that CMV is a potent inducer of IL-6 and LIF with differential effect on constitutive and LPS- stimulated cytokine production by stromal cells; we suggest that CMV induction of IL-6 and LIF during the first hours of infection could play a role in CMV-induced inflammatory reaction. Moreover, our results show that human CMV can disturb the balanced cytokine network involved in the regulation of hematopoiesis.
The synthesis and localization of alternatively spliced fibronectin EIIIB in resting and thrombin-treated megakaryocytes
Authors: Schick PK , Wojenski CM , Bennett VD , Ivanova T
Published: October 2019

There are several species of alternatively spliced fibronectin (FN). One of these, FN EIIIB, is primarily present in embryonic and in proliferating and migrating cells and is believed to be important for cell maturation. We have studied the synthesis, localization, and secretion of this FN isoform in isolated guinea pig megakaryocytes, nonmegakaryocytic bone marrow cells, and platelets. There was 7.5 times more general FN in megakaryocytes than in nonmegakaryocytic cells based on the analysis of equivalent amounts of protein. FN EIIIB was detected by Western blotting in megakaryocytes but not in nonmegakaryocytic cells present in bone marrow. Neither megakaryocytes nor platelets secreted FN EIIIB, while megakaryocytes secreted 25.3% +/- 4.6% general FN and platelets secreted about 61% general FN in response to thrombin. Analysis of immunostained cells by confocal microscopy revealed that FN EIIIB had been redistributed to the surface of megakaryocytes in response to thrombin. Synthesis was studied by metabolic labeling, and megakaryocytes were shown to synthesize FN and FN EIIIB. Thus, megakaryocytes and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. The expression of FN EIIIB on the megakaryocyte surface may influence migration and maturation.
Six different cytokines that share GP130 as a receptor subunit, induce serum amyloid A and potentiate the induction of interleukin-6 and the activation of the hypothalamus-pituitary-adrenal axis by interleukin-1
Authors: Benigni F , Fantuzzi G , Sacco S , Sironi M , Pozzi P , Dinarello CA , Sipe JD , Poli V , Cappelletti M , Paonessa G , Pennica D , Panayotatos N , Ghezzi P
Published: October 2019

Ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6) potentiate the elevation of serum corticosterone induced by suboptimal doses of interleukin-1 (IL-1). CNTF also potentiates IL-1-induced serum IL-6. Here, we report that four other cytokines (leukemia inhibitory factor [LIF], oncostatin M [OSM], interleukin-11 and cardiotrophin-1) also potentiated the elevation of serum corticosterone and IL-6 levels induced by IL-1. Furthermore, all the six cytokines studied induced the acute-phase protein serum amyloid A when administered alone. Because these cytokines differ both in structure and in function, but share gp130 as a subunit of their receptors, these results indicate that signaling through gp130 mediates potentiation of IL-1 activities. The potentiation of IL-1-induced serum corticosterone levels is not a consequence of the increased serum IL-6 observed after IL-1 administration. In fact, in IL-6 deficient mice, IL-1 increased serum corticosterone to a level comparable to that observed in wild-type mice. Thus, either endogenous IL-6 does not mediate IL-1-induced corticosterone increase, or its role may be fulfilled by other cytokines. To the extent that gp130-dependent cytokines may serve this role, they may be important feedback regulators of inflammation through the activation of the hypothalamus-pituitary-adrenal axis and the potentiation of acute-phase protein synthesis.
Transforming growth factor-beta1: differential effects on multiple myeloma versus normal B cells
Authors: Urashima M , Ogata A , Chauhan D , Hatziyanni M , Vidriales MB , Dedera DA , Schlossman RL , Anderson KC
Published: October 2019

Interleukin-6 (IL-6), a product of bone marrow stromal cells (BMSCs), is a growth factor for multiple myeloma (MM) cells. Transforming growth factor-beta1 (TGF-beta1) is also produced by BMSCs and can regulate IL- 6 secretion by several tissues, including BMSCs. The present study was designed to characterize in vitro tumor growth regulation by TGF-beta1 in MM. Sorted CD38+CD45RA- MM cells secreted significantly more TGF- beta1 (8.2 +/- 2.0 ng/mL) than peripheral blood mononuclear cells (P < .001), splenic B cells (P < .001), and CD40 ligand (CD40L) pretreated B cells (P < .05). TGF-beta1 secretion by MM-BMMCs (3.8 +/- 0.9 ng/mL) was significantly greater than by N-BMMCs (1.2 +/- 0.1 ng/mL, P < .001). MM-BMSCs also secreted significantly more TGF-beta1 (6.6 +/- 2.5 ng/mL, n = 11) than N-BMSCs (4.4 +/- 0.6 ng/mL, P < .02, n = 10) and N- BMSC lines (3.9 +/- 0.2 ng/mL, P < .02, n = 6). TGF-beta1 secretion was correlated with IL-6 secretion in MM-BMSCs. Anti-TGF-beta1 monoclonal antibody both blocked IL-6 secretion by BMSCs and inhibited the increments in IL-6 secretion by BMSCs induced by MM cell adhesion. Moreover, exogenous TGF-beta1 upregulated IL-6 secretion by MM-BMSCs, normal BMSCs, and CD38+ CD45RA- MM cells, as well as tumor cell proliferation. This is in contrast to the inhibitory effect of TGF- beta1 on proliferation and Ig secretion of normal splenic B cells. Finally, retinoblastoma proteins (pRB) are constitutively phosphorylated in MM cells; TGF-beta1 either did not alter or increased pRB phosphorylation. pRB are dephosphorylated in splenic B cells and phosphorylated in CD40L triggered B cells in contrast to its effects on MM cells, TGF-beta1 decreased phosphorylation of pRB in CD40L treated B cells. These results suggest that TGF-beta1 is produced in MM by both tumor cells and BMSCs, with related tumore cell growth. Moreover, MM cell growth may be enhanced by resistance of tumor cells to the inhibitory effects of TGF-beta1 on normal B-cell proliferation and Ig secretion.
Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase
Authors: Jelinek J , Fairbairn LJ , Dexter TM , Rafferty JA , Stocking C , Ostertag W , Margison GP
Published: October 2019

A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony- forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.
Circulating clonal lymphocytes in myeloma constitute a minor subpopulation of B cells [see comments]
Authors: Chen BJ , Epstein J
Published: October 2019

The mononuclear cells in the blood of myeloma patients have been reported to contain a high proportion of phenotypically abnormal myeloma B lymphocytes. These cells have been proposed to constitute the drug-resistant proliferative myeloma cell compartment. To determine the extent of B lymphocyte involvement, the proportion of clonotypic cells among the CD19-expressing cells from myeloma patients was estimated by quantitative polymerase chain reaction analysis of the third complementarity determining region (CDR3). The results indicate that the B lymphocytes constitute, on average, 6% of blood mononuclear cells, and that only a minor fraction of these are clonally related to the myeloma cells. While the small number of circulating clonal cells is not incompatible with their proposed role as a reservoir of proliferating myeloma progenitors, the majority of the B cells appear not to be clonally related to the myeloma cells.
Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18
Authors: Diacovo TG , Roth SJ , Buccola JM , Bainton DF , Springer TA
Published: October 2019

Platelets bound to thrombogenic surfaces have been shown to support activation-dependent firm adhesion of neutrophils in flow following selectin-mediated tethering and rolling. The specific receptor(s) responsible for mediating adhesion-strengthening interactions between neutrophils and platelets has not previously been identified. Furthermore, the ability of adherent platelets to support the migration of bound neutrophils has not been tested. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte binding in vascular shear flow and emigration at thrombogenic sites. Our results demonstrate that the beta 2-integrin Mac-1 (CD11b/CD18) is required for both firm attachment to and transmigration of neutrophils across surface-adherent platelets. In flow assays, neutrophils from patients with leukocyte adhesion deficiency-1 (LAD-I), which lack beta 2-integrin receptors, formed P-selectin-mediated rolling interactions, but were unable to develop firm adhesion to activated platelets, in contrast to healthy neutrophils, which developed firm adhesion within 5 to 30 seconds after initiation of rolling. Furthermore, the adhesion-strengthening interaction observed for healthy neutrophils could be specifically inhibited by monoclonal antibodies (mAbs) to Mac-1, but not to lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) or intercellular adhesion molecule-2 (ICAM-2; CD102). Further evidence for a beta 2-integrin-dependent neutrophil/platelet interaction is demonstrated by the complete inhibition of interleukin (IL)-8-induced neutrophil transmigration across platelets bound to fibronectin-coated polycarbonate filters by mAbs to Mac-1. Thus, Mac-1 is required for firm adhesion of neutrophils to activated, adherent platelets and may play an important role in promoting neutrophil accumulation on and migration across platelets deposited at sites of vascular injury.
Prospective evaluation of the clinical usefulness of an antigen- specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias
Authors: Brighton TA , Evans S , Castaldi PA , Chesterman CN , Chong BH
Published: October 2019

The diagnosis of idiopathic immune thrombocytopenia remains a clinical diagnosis based on the exclusion of other causes of immune and nonimmune thrombocytopenia. Measurement of platelet-associated Ig (PAIg), while sensitive, is nonspecific for the diagnosis of immune thrombocytopenia. Published experience of antigen capture assays (including monoclonal antibody immobilization of platelet antigens or MAIPA) suggest a high sensitivity and specificity (70% to 80%) in selected groups of patients. In a prospective evaluation of 158 patients with thrombocytopenia from all causes, we report a sensitivity of 51% and specificity of 80% for direct MAIPA assays. MAIPA was considerably better in discriminating immune from nonimmune thrombocytopenia than two assays of PAIgG. Antiplatelet antibodies detected by MAIPA were more frequently directed against the glycoprotein (GP) IIb/IIIa than the GP Ib/IX complex. Our experience suggests that MAIPA assays are useful in the laboratory assessment of thrombocytopenia, should be performed before therapy, and that some patients with ‘nonimmune’ thrombocytopenia may have genuine antiplatelet antibodies.
T cells from patients with Hodgkin's disease have a defective T-cell receptor zeta chain expression that is reversible by T-cell stimulation with CD3 and CD28
Authors: Renner C , Ohnesorge S , Held G , Bauer S , Jung W , Pfitzenmeier JP , Pfreundschuh M
Published: October 2019

To investigate the mechanisms underlying the deficiency of T lymphocytes from patients with Hodgkin's disease, we investigated the expression of the T-cell receptor (TCR) zeta chain in patients with Hodgkin's disease. By flow cytometry using an anti-zeta chain monoclonal antibody, peripheral blood T lymphocytes from patients with untreated Hodgkin's disease were shown to express decreased levels of the TCR zeta chain. After stimulation by combined CD3 and CD28 cross- linking, T cells from Hodgkin's disease patients upregulated zeta chain protein expression to normal values within 48 hours and achieved a cytolytic potential and levels of interleukin (IL)-2 secretion that were not different from T cells obtained from healthy controls. These results show that downregulation of the TCR zeta chain in Hodgkin's T lymphocytes is a reversible event. Costimulation of CD3 and CD28 is a novel approach for overcoming the T-cell deficiency in Hodgkin's disease and might be exploited clinically. As upregulation of the zeta chain can also be achieved using bispecific monoclonal antibodies (BI- MoAbs) with specificity for tumor antigens and CD3 and CD28, respectively, an immunotherapy with CD3/CD30 and CD28/CD30 Bi-MoAbs may overcome and should therefore, not be jeopardized by the inherent T- cell deficiency in patients with Hodgkin's disease.
Laminin promotes differentiation of NB4 promyelocytic leukemia cells with all-trans retinoic acid
Authors: Becker PS , Li Z , Potselueva T , Madri JA , Newburger PE , Berliner N
Published: October 2019

The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all- trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.