bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasoactive intestinal peptide and pituitary adenylate cyclase- activating polypeptides (PACAP27) and PACAP38) protect CD4+CD8+ thymocytes from glucocorticoid-induced apoptosis
Authors: Delgado M , Garrido E , Martinez C , Leceta J , Gomariz RP
Published: October 2019

In the present study, the effects of vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptides, PACAP27 and PACAP38, in a concentration range from 10(-13) to 10(-6) mol/L were studied in vitro on the spontaneous and dexamethasone (DEX)- induced apoptosis in rat thymocytes. The results show that VIP and both PACAPs inhibit significantly and in a similar way the DNA fragmentation characteristic of glucocorticoid-induced apoptosis and increase the cell survival of thymocytes, with a maximal effect observed at 10(-8) to 10(-9) mol/L. This study showed the ability of the VIP-receptor (VIP- R) antagonist [N-Ac-Tyr1,D-Phe2]-GRF(1–29) amide to partially reverse the inhibitory effect of VIP and both PACAPs on DEX-induced apoptosis, providing evidence for a specific VIP1-R-mediated response and supporting the involvement of a single receptor for the three neuropeptides. Phenotypic analysis showed that VIP, PACAP27, and PACAP38 protect predominantly CD4+CD8+ thymocytes from glucocorticoid- induced apoptosis. These findings suggest that these neuropeptides could be involved in intrathymic T-cell maturation.
BCL-6 expression during B-cell activation
Authors: Allman D , Jain A , Dent A , Maile RR , Selvaggi T , Kehry MR , Staudt LM
Published: October 2019

Translocations involving the BCL-6 gene are common in the diffuse large cell subtype of non-Hodgkin's lymphoma. Invariably, the BCL-6 coding region is intact, but its 5′ untranslated region is replaced with sequences from the translocation partner. The present study shows that BCL-6 expression is regulated in lymphocytes during mitogenic stimulation. Resting B and T lymphocytes contain high levels of BCL-6 mRNA. Stimulation of mouse B cells with anti-IgM or IgD antibodies, bacterial lipopolysaccharide, phorbol 12-myristate 13-acetate plus ionomycin, or CD40 ligand led to a five-fold to 35-fold decrease in BCL-6 mRNA levels. Similar downregulation of BCL-6 mRNA was seen in human B cells stimulated with Staphylococcus aureus plus interleukin-2 or anti-IgM antibodies and in human T lymphocytes stimulated with phytohemagglutinin. BCL-6 mRNA levels began to decrease 8 to 16 hours after stimulation, before cells entered S phase. Although polyclonal activation of B cells in vitro invariably decreased BCL-6 MRNA expression, activated B cells from human germinal centers expressed BCL-6 mRNA at levels comparable to the levels in resting B cells. Despite these similar mRNA levels, BCL-6 protein expression was threefold to 34-fold higher in germinal center B cells than in resting B cells, suggesting that BCL-6 protein levels are controlled by translational or posttranslational mechanisms. These observations suggest that the germinal center reaction provides unique activation signals to B cells that allow for continued, high-level BCL-6 expression.
Expression of Epstein-Barr virus-gene products in Burkitt's lymphoma in Northeast Brazil
Authors: Araujo I , Foss HD , Bittencourt A , Hummel M , Demel G , Mendonca N , Herbst H , Stein H
Published: October 2019

The Epstein-Barr Virus (EBV) is consistently found in tumor cells of Burkitt's lymphoma (BL) endemic in central Africa and malaria is considered a pathogenic cofactor. In contrast, fewer than 20% of BL cases occurring in Western countries are EBV-associated. We have investigated 54 BL cases from Bahia, a tropical region of Northeast Brazil, for expression of EBV gene products by in situ hybridization and immunohistology and performed typing of the EBV by polymerase chain reaction. Ten pediatric BL cases from Germany served as controls. New cases of malaria were not observed in the period and area of our study. Small nuclear EBV encoded transcripts, EBER, were found in tumor cells of 47 of 54 Brazilian cases (87%) but in only 2 of 10 German cases (20%). Type I latency of the EBV infection with absence of EBV-encoded proteins LMP1 and EBNA2 was found in 45 of 47 of the EBER-positive Brazilian cases. In two cases, occasional LMP1-containing tumor cells were found in the neighborhood of small Schistosoma mansoni granulomas and scars. BHLF1 transcripts associated with lytic EBV infection could be detected in few cells in 3 of the 40 EBER-positive Brazilian cases investigated. EBV type A was found in the majority of Brazilian BL cases (20 of 30 A-type, 7 of 30 B-type, and 3 of 30 not amplifiable). Our results indicate that the association of Bahian BL with EBV, but not the regional prevalence of malaria, is similar to endemic African BL. In two cases, type II latency was found in association with schistosomiasis, suggesting a role of this parasitosis in the induction of an EBV expression pattern that is unusual for BL. Because chronic schistosomiasis is associated with elevated Th2 cytokine expression resulting in reduced cell-mediated cytotoxicity, it seems possible that altered local immunity is responsible for this peculiar phenotype.
Human herpesvirus 6: infection and disease following autologous and allogeneic bone marrow transplantation
Authors: Kadakia MP , Rybka WB , Stewart JA , Patton JL , Stamey FR , Elsawy M , Pellett PE , Armstrong JA
Published: October 2019

Human herpesvirus 6 activity (HHV-6) was studied in 15 allogeneic and 11 autologous marrow transplantation patients. After transplantation, HHV-6 was isolated from the peripheral blood mononuclear cells of 12 of 26 patients (6 allogeneic and 6 autologous). All isolates were variant B. Eleven of 26 and 12 of 19 patients showed salivary shedding of HHV-6 DNA before and after transplantation, respectively. The antibody titer increased in 7 of 26 patients. Thus, 23 of 26 patients showed evidence of active HHV-6 infection either by virus isolation, salivary shedding, or increases in antibody titers. The fraction of saliva specimens positive in 19 patients was negatively associated with their antibody titers (P= .005). The proportion of cultures positive increased after transplantation (P = .007). Sinusitis was associated with HHV-6 isolation in autologous recipients (P= .002). In allogeneic patients, active human cytomegalovirus infection was associated with HHV-6 isolation (P = .04). No association was observed between HHV-6 infection and GVHD, pneumonia, delay in engraftment, or marrow suppression. Of the 120 clinical events analyzed in 26 patients, HHV-6 was defined as a probable cause of 16 events in 9 patients based on the propinquity of HHV-6 activity and the clinical event plus the absence of other identified causes of the event.
Individual stem cell quality in leukapheresis products is related to the number of mobilized stem cells
Authors: Breems DA , van Hennik PB , Kusadasi N , Boudewijn A , Cornelissen JJ , Sonneveld P , Ploemacher RE
Published: October 2019

Peripheral blood stem cells (PBSC) are used for stem cell support in patients after intensive chemotherapy and generally permit faster hematopoietic recovery than bone marrow. The development of different protocols for chemotherapy conditioning, mobilization, and ex vivo manipulation of PBSC may potentially lead to loss of primitive hematopoietic stem cells or reduction of their quality. To characterize the frequency of different stem cell subsets and their quality per mobilized PBSC, we have studied 47 leukapheresis products (LPs) of 21 cancer patients using stroma-dependent long-term culture (LTC) and limiting dilution-type cobblestone area forming cell (CAFC) assays. A large variation in CAFC week-type frequencies between the LPs was observed. The frequencies of CAFC week 2 as a tentative indicator of progenitor cells and transiently repopulating hematopoietic stem cells ranged from 0.89 to 205 per 10(5) mobilized nucleated cells and the frequencies of more primitive CAFC week 6 varied between 0.37 and 48. The average total colony-forming cell (CFC) production per CAFC at week 6 varied between 1.2 and 730, as determined in parallel LTC. In contrast to LPs, bone marrow samples generated 4.2 to 48 CFC per CAFC at week 6. Notably, a poor stem cell quality was consistently found in LPs that contained less than 5,000 CAFC week 6 per kilogram of body weight. Frequency analyses of CFCs, CAFC subtypes, and immunophenotypic subsets showed a good level of mutual correlation, suggesting identical mobilization kinetics of different stem cell subsets. The premobilization chemotherapy intensity was directly correlated with both decreasing frequency and quality of the CAFC week 6 in LPs. The frequency of CFCs, immunophenotypic subsets, and CAFC subsets transplanted and the transplant quality as determined in LTC assays was related to the neutrophil and platelet recovery time after PBSC transplantation. Although the number of progenitor cells transplanted and the in vitro transplant quality showed the best correlation with early hematopoietic recovery, the data did not permit determination of which stem cell subsets are predominantly responsible for early posttransplantation recovery. As a result, frequency and quality analysis of stem cell subsets may be a useful tool to monitor and calibrate the efficacy of novel mobilization regimens and ex vivo manipulation of PBSC.
Distinctive expression pattern of the BCL-6 protein in nodular lymphocyte predominance Hodgkin's disease
Authors: Falini B , Bigerna B , Pasqualucci L , Fizzotti M , Martelli MF , Pileri S , Pinto A , Carbone A , Venturi S , Pacini R , Cattoretti G , Pescarmona E , Lo Coco F , Pelicci PG , Anagnastopoulos I , Dalla-Favera R , Flenghi L
Published: October 2019

The BCL-6 gene encoding a nuclear-located Kruppel-type zinc finger protein is rearranged in about 30% diffuse large B-cell lymphomas and is expressed predominantly in normal germinal center B cells and related lymphomas. These findings suggest that BCL-6 may play a role in regulating differentiation of normal germinal center B cells and that its deregulated expression caused by rearrangements may contribute to lymphomagenesis. This prompted us to investigate the expression of the BCL-6 protein in Hodgkin's disease (HD), focusing on the nodular lymphocyte predominance subtype (NLPHD), which differs from classical HD by virtue of the B-cell nature of the malignant cell population (so- called L&H cells) and its relationship with germinal centers. Forty-one HD samples (19 NLPHD, 12 nodular sclerosis, and 10 mixed cellularity) were immunostained with the monoclonal antibodies PG-B6 and PG-B6p that react with a fixative-sensitive and a formalin-resistant epitope on the aminoterminal region of the BCL-6 gene product, respectively. Strong nuclear positivity for the BCL-6 protein was detected in tumor (L&H) cells in all cases of NLPHD. In contrast, BCL-6 was expressed only in a small percentage of Hodgkin and Reed-Sternberg cells in about 30% of classical HD cases. Notably, the nuclei of reactive CD3+/CD4+ T cells nearby to and rosetting around L&H cells in NLPHD were also strongly BCL-6+, but lacked CD40 ligand (CD40L) expression. This staining pattern clearly differed from that of classical HD, whose cellular background was made up of CD3+/CD4+ T cells showing the BCL-6-/CD40L+ phenotype. These results further support the concept that NLPHD is an histogenetically distinct, B-cell-derived subtype of HD and suggest a role for BCL-6 in its development.
Targeted gene transfer to human hematopoietic progenitor cell lines through the c-kit receptor
Authors: Schwarzenberger P , Spence SE , Gooya JM , Michiel D , Curiel DT , Ruscetti FW , Keller JR
Published: October 2019

In this report, we describe a novel gene therapy approach for hematopoietic stem/progenitor cells using a specific receptor-mediated gene transfection procedure to target c-kit+ cell lines. The vector consists of plasmid DNA containing a luciferase reporter gene that is condensed by electrostatic forces with polylysine (PL) covalently linked to streptavidin (binds biotinylated ligand) and PL covalently linked to adenovirus (AD; to achieve endosomal lysis) with the final addition of biotinylated steel factor (SLF-biotin). Targeted transfection of growth factor-dependent hematopoietic progenitor cell lines that express c-kit showed specific luciferase gene expression over cell lines that did not express c-kit. This effect was dependent on the dose of SLF-biotin and was competed by excess SLF or with monoclonal antibodies that recognize c-kit and block the binding of SLF to its receptor. Maximum transfection efficiency (> 90%) requires a 2- hour incubation period of the vector with the cells, and maximum gene expression occurred 30 hours later. Removal of the endosomalytic agent, AD, from the vector resulted in the loss of gene expression. Vector targeting was versatile and could be changed by the addition of other biotinylated ligands. In principle, this vector should be broadly applicable to deliver genes to hematopoietic stem/progenitor cells in vitro and in vivo.
Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture
Authors: Young JC , Varma A , DiGiusto D , Backer MP
Published: October 2019

Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells. Ex vivo expansion of this population is proposed as a means of providing accelerated short-term, as well as long-term, engraftment after myeloablative therapy. Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibiting quiescent behavior during culture. CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells on a murine stromal layer in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking of the number of cell divisions. Progenitor cells present after a 2-week expansion period were sorted into CD34+/Lin-/dyebright and CD34+/Lin- /dyedim fractions and then cultured in a 4-week single-cell proliferation assay to characterize the proliferative capacity of each group. Fifty-nine percent of progenitors remaining dyebright after bulk culture (four or fewer cell divisions) were observed to proliferate in single cell culture, and produced an average of 1,780 cells per plated cell. In contrast, only 26% of dyedim (more than four divisions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell. Similar behaviors were observed after a second consecutive cycle of bulk culture, indicating that quiescent cells with high proliferative capacity existed in culture for at least 4 weeks. Single CD34+/Lin-/dyebright progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and these progeny included multilineage colony forming cells. These data demonstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent behavior. The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy.
Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats
Authors: Murakami K , Okajima K , Uchiba M , Johno M , Nakagaki T , Okabe H , Takatsuki K
Published: October 2019

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS- induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.
An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes
Authors: Baricordi OR , Ferrari D , Melchiorri L , Chiozzi P , Hanau S , Chiari E , Rubini M , Di Virgilio F
Published: October 2019

We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation.
Serum neural cell adhesion molecule differentiates multiple myeloma from paraproteinemias due to other causes
Authors: Ong F , Kaiser U , Seelen PJ , Hermans J , Wijermans PW , de Kieviet W , Jaques G , Kluin-Nelemans JC
Published: October 2019

Serum neural cell adhesion molecule (NCAM) has been described as a prognostic marker in multiple myeloma (MM). Both C-reactive protein (CRP) and beta 2-microglobulin (beta 2M) are established prognostic markers in MM. We tested the diagnostic value of these markers in 212 serum samples of patients with paraproteinemia registered prospectively in a population-based registry. Sixty patients had MM and 152 had other monoclonal gammopathies (hematologic diseases [48], paraneoplastic disease [35], autoimmune disease [15], and monoclonal gammopathy of undetermined significance [56]). CRP and beta 2M had wide and overlapping ranges in all diagnostic categories. However, serum neural cell adhesion molecule (NCAM) was low (< 20 U/mL) in all but 4 of 152 nonmyeloma cases and high (> or = 20 U/mL) in 31 (52%) of the 60 MM cases. Two patients with non-Hodgkin's lymphoma, 1 with chronic lymphatic leukemia, and 1 with autoimmune disease had serum NCAM values between 20 and 30 U/mL. In a discriminant analysis in which serum NCAM, CRP, beta 2M, paraprotein type and concentration, hemoglobin, leukocyte and thrombocyte counts, creatinine, corrected calcium, lactate dehydrogenase, and alkaline phosphatase were included, paraprotein type and concentration and serum NCAM turned out to be the best combination of parameters predicting whether a patient had MM, with 89% of cases being correctly classified. Even without bone marrow and x-ray examinations, serum NCAM, in combination with paraprotein type and concentration, can differentiate between MM and nonmyeloma patients.
Adhesion of sickle neutrophils and erythrocytes to fibronectin
Authors: Kasschau MR , Barabino GA , Bridges KR , Golan DE
Published: October 2019

The pathophysiology of vaso-occlusive crisis in sickle cell disease involves interactions among blood cells, plasma proteins, and vessel wall components. The initial goal of this work was to quantify the adhesion of sickle red blood cells (RBCs) to fibronectin immobilized on glass under both static and dynamic shear stress conditions. High-power microscopic inspection of static assay plates showed striking numbers of adherent neutrophils as well as RBCs. Sickle neutrophils and RBCs were significantly more adherent to fibronectin than the corresponding normal cells in static adhesion assays. Adhesion of both sickle neutrophils and sickle RBCs in dynamic adhesion assays was promoted by a period of static incubation preceding initiation of shear stress conditions. Adherent neutrophils remained attached at shear stresses up to 51 dyne/cm2; most adherent RBCs were attached at shear stresses up to 13 dyne/cm2, but detached at a shear stress of 20 dyne/cm2. Sickle neutrophil adhesion was enhanced significantly by autologous plasma. Elevated levels of plasma interleukin-6 (IL-6; but not IL-1 or IL-8) were found in 6 of 9 sickle cell disease samples examined, and elevated levels of tumor necrosis factor were found in 2 of 9 samples. Plasma IL- 6 levels correlated positively with both the number of sickle neutrophils adherent to fibronectin and the ability of sickle plasma to enhance adhesion of normal neutrophils to fibronectin. These data suggest possible roles for neutrophil activation and for fibronectin in mediating sickle neutrophil and RBC adhesion.
Lethal murine graft-versus-host disease induced by donor gamma/delta expressing T cells with specificity for host nonclassical major histocompatibility complex class Ib antigens
Authors: Blazar BR , Taylor PA , Panoskaltsis-Mortari A , Barrett TA , Bluestone JA , Vallera DA
Published: October 2019

Although T-cell receptor (TCR) alpha/beta expressing cells have a well- known role in graft-versus-host disease (GVHD) generation, the role of TCR gamma/delta expressing cells in this process has remained unclear. To elucidate the potential function of TCR gamma/delta cells in GVHD, we have used transgenic (Tg) H-2d mice (termed G8) that express gamma/delta heterodimers on a high proportion of peripheral T cells. In vitro, G8 Tg gamma/delta T cells proliferate to and kill C57BL/6 (B6) (H-2b) which express gene products (T10b and T22b) from the nonclassical major histocompatibility complex (MHC) class Ib H-2T region. The infusion of G8 Tg (H-2Td) TCR gamma/delta cells into lethally irradiated [900 cGy total body irradiation (TBI)] B6 (H-2b) mice resulted in the generation of lethal GVHD characterized histologically by destruction of the spleen, liver, lung, and colon. Lethal GVHD was prevented by the injection of anti-TCR gamma/delta monoclonal antibodies. Immunohistochemical analysis of B6 recipients post-bone marrow transplantation (BMT) confirmed that G8 Tg TCR gamma/delta cells infiltrated GVHD target tissues (skin, liver, colon, and lung) and were absent in recipients treated with anti-TCR gamma/delta monoclonal antibodies (MoAbs) but not anti-CD4 plus anti- CD8 MoAbs. In contrast, injection of TCR gamma/delta+ cells into irradiated (900 cGy TBI) B6.A-TIaa BoyEg mice that do not express either T10b or T22b did not induce lethal GVHD. Similarly, in a different GVHD system in which sublethal irradiation without bone marrow (BM) rescue was used, B6 but not B6.A-TIaa/BoyEg mice were found to be susceptible to TCR gamma delta+ cell mediated GVHD-induced lethality characterized by an aplasia syndrome. These results demonstrate that TCR gamma/delta cells have the capacity to cause acute lethal GVHD in mice and suggest that nonclassical MHC class Ib gene products expressed on GVHD target organs are responsible for G8 Tg TCR gamma/delta+ cell mediated lethality.
Analysis of the Bruton's tyrosine kinase gene promoter reveals critical PU.1 and SP1 sites
Authors: Himmelmann A , Thevenin C , Harrison K , Kehrl JH
Published: October 2019

The gene defective in X-linked agammaglobulinemia (XLA) encodes a novel protein kinase termed Bruton's tyrosine kinase (Btk). Whereas the XLA phenotype is confined to abnormalities of B-cell development and function, Btk is expressed not only in B-lymphocyte lineage but also in myeloid lineage cells. The first 450 basepairs of the Btk promoter fused to a luciferase gene displayed a similar cell-type specificity. Critical binding sites for the transcription factors PU.1 and Sp1 were identified in the proximal portion of the Btk promoter upstream of a cluster of transcriptional start sites. Mutation of either the PU.1 or Sp1 site markedly reduced the activity of a Btk promoter-luciferase reporter construct in transfection experiments. In addition, PU.1 directly transactivated the Btk promoter, and deletion of the PU.1 binding site abolished this effect. This study implicates PU.1 and Sp1 as major regulators of Btk expression and provides a foundation for further study of the regulation of this gene in XLA patients that lack Btk mRNA.
Epstein-Barr virus genome in non-Hodgkin's lymphomas occurring in immunocompetent patients: highest prevalence in nonlymphoblastic T-cell lymphoma and correlation with a poor prognosis. Danish Lymphoma Study Group, LYFO
Authors: d'Amore F , Johansen P , Houmand A , Weisenburger DD , Mortensen LS
Published: October 2019

A series of 520 cases of non-Hodgkin's lymphoma (NHL; 374 of B-cell, 130 of T-cell, 5 of non-B/non-T-cell, and 11 of undetermined phenotype) was analyzed for the presence of Epstein-Barr virus (EBV) using RNA in situ hybridization (RISH). The aims of the study were to assess the frequency of EBV-encoded small nuclear RNAs 1 and 2 (EBER), abundant immediate early RNAs (BHLF), and latent membrane protein-1 (LMP-1) in cases covering the entire histologic spectrum of NHL, and to analyze whether EBV status had prognostic relevance with regard to patient survival. EBER positivity was found in 25 of 374 (7%) B-NHL and 40 of 130 (31%) T-NHL (P < .00005) cases, but in only 1 of 16 cases with non- B/non-T-cell or undetermined phenotype. Among T-NHL cases, EBER positivity was confined to angioimmunoblastic, lymphadenopathy-like lymphoma (11 of 13 cases, 85%), Lennert's lymphoma (five of seven cases, 71%), and pleomorphic T-NHL (24 of 67 cases, 36%). Mycosis fungoides, lymphoblastic, and CD30-positive anaplastic large T-cell NHL cases were consistently EBV-negative. Double-labeling by RISH and immunophenotyping demonstrated the presence of EBV in neoplastic T cells, but no CD21 expression was found in the EBER-positive T-NHL cases. LMP-1 was expressed in 12 of 40 (30%) EBER-positive T-NHL and 5 of 25 (20%) EBER-positive B-NHL cases. For both T- and B-NHL, no correlation was found for EBER positivity and age, sex, clinical stage, or serum level of lactate dehydrogenase (LDH) at diagnosis. However, in T-NHL but not B-NHL, EBER positivity correlated with the presence of constitutional symptoms and a poor performance score (PS < 1; scale, 0 to 4). EBER status did not have any prognostic significance in B-NHL, but it had a negative prognostic impact in high-grade T-NHL (7-year survival of EBER-negative v EBER-positive cases: 33% v 14%; P = .01). A multivariate analysis including all B- and T-NHL of intermediate-/high- grade histology showed that EBER positivity in T-NHL was one of the three most significant factors recognized by the final prognostic model, only surpassed by PS greater than 1 and age greater than 67 years, and more powerful than B symptoms, an elevated LDH, or disseminated disease (clinical stage greater than II). We conclude that patients with EBV-positive T-NHL have a very poor clinical outcome, that EBV status should be considered as additional useful information in the classification of T-NHL, and that EBV-positive T-NHL should be treated as a separate entity in the future.
Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B
Authors: Chauhan D , Uchiyama H , Akbarali Y , Urashima M , Yamamoto K , Libermann TA , Anderson KC
Published: October 2019

Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells (BMSCs) not only localizes MM cells in the marrow microenvironment, but also triggers interleukin-6 (IL-6) secretion by BMSCs and related MM cell proliferation. In the present study, we characterized the regulation of IL-6 gene expression in BMSCs during MM cell adhesion. Adhesion of ARH-77, HS-Sultan, IM-9, and U266 MM cell lines to BMSCs and BMSC lines (LP 101 and AA 101) triggered 5-through 15-fold and 2- through 4-fold increases in IL-6 secretion, respectively. IL-6 mRNA transcripts were undetectable by Northern blotting in IM-9 MM cells or LP 101 BMSCs cultured alone; however, adherence of IM-9 cells to LP 101 cells induced a transient increase in IL-6 transcripts at 6 hours, followed by peak IL-6 secretion at 24 hours. To confirm increased IL-6 transcription and characterize its regulation, LP101 BMSCs were transiently transfected with full length and deletion fragments of the IL-6 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transient transfection of LP101 BMSCs with plasmid containing an intact NF-kappa B site showed a 6.8 +/- 0.4-fold increase in CAT activity triggered by IM-9 MM cell adhesion (n = 3, P < .05). Transfection of LP 101 cells with plasmid containing a single base pair deletion from the NF-kapp B binding motif abolished the MM adhesion- induced increase in CAT activity, whereas transfection with plasmid containing three copies of synthetic NF-kappa B sequence resulted in an 8.1 +/- 0.7-fold increase in CAT activity related to MM adhesion (n = 3, P < .05). These data suggest that the NF-kappa B site is one of the essential regulatory elements for MM cell adhesion-induced IL-6 transcription in BMSCs. Electrophoretic mobility shift assays confirmed the involvement of NF-kappa B activation in regulating MM adhesion- induced IL-6 transcription in BMSCs. Further characterization of the upstream events in the signalling cascade regulating IL-6 may not only delineate mechanisms of IL-6 regulation during paracrine MM cell growth, but also provide new therapeutic strategies based on interruption of IL-6 mediated tumor cell growth.
BCR-ABL does not prevent apoptotic death induced by human natural killer or lymphokine-activated killer cells
Authors: Roger R , Issaad C , Pallardy M , Leglise MC , Turhan AG , Bertoglio J , Breard J
Published: October 2019

The erythromyeloid cell line, K562, the most sensitive target in human natural killer (NK) cell mediated cytotoxicity, is derived from a chronic myeloid leukemia (CML) patient and expresses the characteristic reciprocal translocation t(9;22). The resulting BCR-ABL fusion protein has been shown to mediate the unusual resistance of K562, and other BCR- ABL expressing lines, to apoptosis induced by a variety of agents (irradiation, UV light, cytotoxic drugs). Here we show that human NK and lymphokine-activated killer (LAK) cells, when tested at low effector to target ratio, can readily induce apoptotic death in K562 cells. This was accompanied with classical DNA oligonucleosomal fragmentation, an unexpected finding given the reported lack of such fragmentation when apoptosis is induced in K562 by chemical agents, after downregulation of BCR-ABL. Apoptosis was assessed by several means: morphological studies, 125I-DNA versus 51Cr release, DNA agarose gel electrophoresis, and results were always concordant, with a delayed kinetics for DNA oligonucleosomal fragmentation. Similar data were obtained with a pluripotent human hematopoietic cell line, UT-7, infected with a defective amphotropic p210 BCR-ABL retrovirus. The BCR- ABL expressing subclone UT-7/9, while being no longer sensitive to cytotoxic drugs or to tumor necrosis factor, a lytic mediator to which UT-7 cells are sensitive, underwent apoptotic death when exposed to LAK effector cells to the same degree as the parental UT-7 line. With these targets, DNA oligonucleosomal fragmentation occurred concomitantly with isotope release. Results obtained with several inhibitors of exocytosis strongly suggest that cytotoxic granules mediate NK and LAK cell- induced apoptotic death. In conclusion, NK and LAK cell-induced apoptotic signals, unlike those activated by chemotherapeutic agents, are unaffected by the antiapoptotic action of BCR-ABL. This unique property may support the observed curative effect of allogeneic bone marrow transplantation in CML.
The human homologue of rat NG2, a chondroitin sulfate proteoglycan, is not expressed on the cell surface of normal hematopoietic cells but is expressed by acute myeloid leukemia blasts from poor-prognosis patients with abnormalities of chromosome band 11q23
Authors: Smith FO , Rauch C , Williams DE , March CJ , Arthur D , Hilden J , Lampkin BC , Buckley JD , Buckley CV , Woods WG , Dinndorf PA , Sorensen P , Kersey J , Hammond D , Bernstein ID
Published: October 2019

In our efforts to produce monoclonal antibodies that recognize cell- surface antigens expressed by hematopoietic precursor and stromal cells, we generated a monoclonal antibody, 7.1, which recognizes a 220- to 240-kD cell-surface protein whose N-terminal amino acid sequence is identical to the rat NG2 chondroitin sulfate proteoglycan molecule. This chondroitin sulfate proteoglycan, previously reported to be expressed by human melanoma cells, was not found to be expressed by normal hematopoietic cells, nor was it expressed on the cell surface of cell lines of hematopoietic origin including cell lines with 11q23 abnormalities. It was found on the cell surface of acute myeloid leukemia (AML) blasts and cell lines derived from nonhematopoietic tissues. Samples of leukemic marrow from 166 children with AML enrolled on Childrens Cancer Group protocol 213 were evaluated for cell-surface expression of this proteoglycan molecule. In 18 of 166 (11%) patient samples, greater than 25% of leukemic blasts expressed the NG2 molecule. These 18 patients had a poorer outcome with respect to survival (P = .002) and event-free survival (P = .035) with an actuarial survival at 4 years of 16.7%. Blast cell expression of the NG2 molecule was strongly associated with French-American-British M5 morphology (P < .0001) and abnormalities in chromosome band 11q23, site of the MLL gene. These results show that the NG2 molecule is expressed by malignant hematopoietic cells that have abnormalities in chromosome band 11q23, suggesting that antibody 7.1 may be useful in the rapid identification of this group of poor-prognosis patients.
Human homologue of the rat chondroitin sulfate proteoglycan, NG2, detected by monoclonal antibody 7.1, identifies childhood acute lymphoblastic leukemias with t(4;11)(q21;q23) or t(11;19)(q23;p13) and MLL gene rearrangements
Authors: Behm FG , Smith FO , Raimondi SC , Pui CH , Bernstein ID
Published: October 2019

Monoclonal antibody 7.1, which recognizes the chondroitin sulfate proteoglycan molecule NG2, was used to screen prospectively blast cells from 104 consecutive children at initial presentation with acute lymphoblastic leukemia (ALL). Reactivity with this antibody was found in 9 cases (8.6%), of whom 5 had a t(4;11)(q21;q23) and 4 had a t(11;19)(p13;q23). None of the NG2- cases had either translocation. Southern blot analysis disclosed MLL gene rearrangement in only the 9 cases with 7.1 reactivity plus the t(4;11)(q21;q23) or t(11;19)(q23;p13) translocation. MLL gene rearrangements were not detected in 89 patient leukemic samples that did not express NG2, including 7 patients with del(11)(q23) or inv(11)(p13q23). As expected from the association with t(4;11) and t(11;19), NG2+ cases were significantly more likely to be infants, to have hyperleukocytosis and central nervous system involvement, to be CD10-, and to express myeloid- associated antigens CD15 and CD65. Despite short follow-up duration, 3 of the NG2+ cases have relapsed while the other 101 patients remain in remission. Thus, blast cell surface expression of NG2 is useful for identifying patients with ALL having t(4;11) or t(11;19) translocations that are associated with poor prognosis, especially in the infant age group.
Clinical relevance of BCL-2 overexpression in childhood acute lymphoblastic leukemia
Authors: Coustan-Smith E , Kitanaka A , Pui CH , McNinch L , Evans WE , Raimondi SC , Behm FG , Arico M , Campana D
Published: October 2019

Enforced BCL-2 gene expression in leukemic cell lines suppresses apoptosis and confers resistance to anticancer drugs, but the clinical significance of increased BCL-2 protein levels in acute lymphoblastic leukemia (ALL) is unknown. Among 52 children with newly diagnosed ALL, BCL-2 expression in leukemic lymphoblasts ranged widely, from 4,464 to 59,753 molecules of equivalent soluble fluorochrome per cell (MESF), as determined by flow cytometry. The mean (+/- SD) level of MESF in 43 cases of B-lineage ALL (19,410 +/- 11,834) was higher than that detected in CD10+ B-lymphoid progenitors from normal bone marrow (450 +/- 314; P < .001), and CD19+ peripheral blood B lymphocytes (7,617 +/- 1,731; P = .02). Levels of BCL-2 in T-ALL cases (17,909 +/- 18,691) were also generally higher than those found in normal CD1a+ thymocytes (1,762 +/- 670), or in peripheral blood T lymphocytes (9,687 +/- 3,019). Although higher levels of BCL-2 corresponded to higher leukemic cell recoveries after culture in serum-free medium, they did not correlate with higher cell recoveries after culture on stromal layers, or with in vitro resistance to vincristine, dexamethasone, 6- thioguanine, cytarabine, teniposide, daunorubicin or methotrexate. BCL- 2 protein levels did not correlate with presenting clinical features. Unexpectedly, however, lower-than-median MESF values were significantly associated with the presence of chromosomal translocations (P = .010). Notably, all six cases with the Philadelphia chromosome, a known high- risk feature, had low levels of BCL-2 expression (P = .022). Higher levels of BCL-2 were not associated with poorer responses to therapy among 33 uniformly treated patients, and were not observed in three patients studied at relapse. In conclusion, increased BCL-2 expression in childhood ALL appears to enhance the ability of lymphoblasts to survive without essential trophic factors, and is inversely related to the presence of chromosomal translocations. However, it does not reflect increased disease aggressiveness or resistance to chemotherapy.
Combination therapy of erythropoietin, hydroxyurea, and clotrimazole in a beta thalassemic mouse: a model for human therapy
Authors: de Franceschi L , Rouyer-Fessard P , Alper SL , Jouault H , Brugnara C , Beuzard Y
Published: October 2019

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.
Hydroxyurea increases hemoglobin F levels and improves the effectiveness of erythropoiesis in beta-thalassemia/hemoglobin E disease
Authors: Fucharoen S , Siritanaratkul N , Winichagoon P , Chowthaworn J , Siriboon W , Muangsup W , Chaicharoen S , Poolsup N , Chindavijak B , Pootrakul P , Piankijagum A , Schechter AN , Rodgers GP
Published: October 2019

Hydroxyurea (HU) is one of several agents that have been shown to enhance hemoglobin (Hb) F levels in patients with sickle cell disease and may be useful as a therapy for beta-globinopathies. However, limited information exists on the effects of HU in patients with thalassemia. Accordingly, we examined the hematologic effects of orally administered HU in 13 patients with beta-thalassemia/Hb E, including four patients who had been splenectomized. These patients were treated with escalating doses (final range, 10 to 20 mg/kg/d) for 5 months and were observed in the outpatient hematology clinic every 2 to 4 weeks. Complete blood counts including reticulocyte counts, amounts of Hb E and Hb F, G gamma:A gamma and alpha:non-alpha globin biosynthetic ratios were evaluated before and during treatment. Almost all patients responded with an average increase of 33% in Hb F levels, from a mean (+/- SD) of 42% +/- 11% to 56% +/- 8% (P < .0001), and a reciprocal decline in the percentage of Hb E from 59% +/- 9% to 49% +/- 8% (P < .001). Reticulocytosis was decreased from a mean (+/- SD) of 18.0% +/- 15.6% to 11.7% +/- 9.1% (P < .05); there was also a slight (10%) but statistically significant increase in hemoglobin levels and an improved balance in alpha:non-alpha globin chains ratios. The side effects were minimal in most patients, although these patients tended to tolerate a lower dose of HU before significant myelosuppression than has been our previous experience in sickle cell disease. One splenectomized patient died of sepsis during the trial. We conclude that increased Hb F production in beta-thalassemia/Hb E patients, with an improvement in the alpha:non-alpha globin ratios and, probably, the effectiveness of erythropoiesis, can be achieved using HU. Longer trials of HU in this population, including at other doses and in combination with other agents, appear warranted.
Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor gamma chain
Authors: Ohbo K , Suda T , Hashiyama M , Mantani A , Ikebe M , Miyakawa K , Moriyama M , Nakamura M , Katsuki M , Takahashi K , Yamamura K , Sugamura K
Published: October 2019

The interleukin-2 (IL-2) receptor gamma chain is indispensable for IL-2- , IL-4-, IL-7-, IL-9-, and IL-15-mediated signaling. Mutations of the human gamma chain cause the X-linked severe combined immunodeficiency (XSCID), showing that T and natural killer cells absolutely require the gamma chain for their development in humans. To elucidate the roles of the gamma chain in hematopoiesis, we have generated mice, by gene targeting, that express a form of the gamma chain lacking the cytoplasmic region. Male mice carrying the truncated gamma-chain mutant, which mimics mutations in patients with XSCID, showed a decrease in the number of lymphocytes and an increase in monocytes; the number of T cells was profoundly reduced and no natural killer cells were detected, which is similar to the characteristic of human XSCID. Unlike human XSCID, the levels of B cells were also reduced. In spite of the severe decrease in CD45R+/sIgM+ B cells, the level of IgM in serum of the 8-week-old mutant mice was higher than that of control littermates. Interestingly, the stem cell population with surface phenotypes of CD34, c-kit, and Sca-1 was significantly increased. Furthermore, the colony-forming assay showed that the mutant mice had 15-fold higher numbers of hematopoietic progenitor cells in the spleen as compared with that of controls. These results indicate that functional loss of the gamma chain causes significant effects on the immunological system in mice.
Phorbol ester enhances integrin alpha IIb beta 3-dependent adhesion of human erythroleukemic cells to activation-dependent monoclonal antibodies
Authors: Boudignon-Proudhon C , Patel PM , Parise LV
Published: October 2019

Following platelet stimulation by agonists, integrin-alpha IIb beta 3 (or glycoprotein IIb-IIIa) is converted to an activated state that can bind soluble fibrinogen and mediate platelet aggregation. However, little is known about modulation of alpha IIb beta 3 in cell lines. In the present study, we show that agonist stimulation modulates alpha IIb beta 3-dependent adhesive properties of a human erythroleukemic (HEL) cell line. Brief treatment with phorbol 12-myristate 13-acetate (PMA) caused a significant increase in HEL cell adhesion to monoclonal antibodies (MoAbs) specific for activated alpha IIb beta 3 (PAC1 or pl- 55). This adhesion was inhibited by blocking MoAbs or peptides specific for alpha IIb beta 3, but not by anti-Fc gamma receptor-specific MoAb. Similarly, PMA enhanced HEL cell adhesion to immobilized fibrinogen by 10-fold. However, the activation-dependent ligands in solution (ie, PAC1, pl-55, or fibrinogen) did not inhibit the enhanced HEL cell adhesion to immobilized MoAbs PAC1 or pl-55 after PMA treatment. Thus, PMA may increase alpha IIb beta 3-dependent adhesion to immobilized activation-dependent antibodies and fibrinogen by increasing the local concentration of alpha IIb beta 3 to participate in low-affinity interactions, resulting in an increased avidity, changing the affinity state of alpha IIb beta 3, or both.