bluuebunny/crossref_metadata_2025 huggingface dataset:
Human eosinophils synthesize and secrete interleukin-6, in vitro
Authors: Hamid Q , Barkans J , Meng Q , Ying S , Abrams JS , Kay AB , Moqbel R
Published: October 2019

Using the technique of in situ hybridization, we have shown that resting, unstimulated, human peripheral blood eosinophils, obtained from subjects with greater than 8% eosinophilia, transcribe and translate messenger RNA (mRNA) for interleukin-6 (IL-6). After incubation for 24 hours in culture medium alone, approximately 19% of eosinophils were positive for IL-6 mRNA. This may be a reflection of their in vivo activation, but also may suggest that the gene for this cytokine is constitutively expressed in eosinophils. After stimulation with interferon gamma (IFN gamma) (500 U/mL), the percentage of IL-6- mRNA+ cells increased to 51.3%. This was accompanied by an enhancement of intensity of the hybridization signals. The specificity of the IL-6 probe and the hybridization signals was confirmed by the use of an IL-6 sense probe and RNase pretreatment of cell preparations. Evidence for the translation of IL-6 mRNA was obtained by immunocytochemical staining. Normal and activated eosinophils gave IL-6-specific immunoreactivity with a polyclonal antihuman IL-6 antibody. A higher percentage of positive cells was detected among activated eosinophils than those treated with medium alone. Using a specific immunoenzymetric assay, we detected 190.15 +/- 18.1 and 403.32 +/- 213.6 pg/mL of IL-6 in supernatants of unstimulated and IFN gamma-treated (24 and 48 hours) eosinophils, respectively. These data indicate that eosinophils are an important cellular source of IL-6.
Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype
Authors: Arceci RJ , Stieglitz K , Bierer BE
Published: October 2019

The multidrug-resistant (MDR) phenotype is characterized in vitro by the resistance displayed by cell lines to a broad spectrum of natural product cytotoxic agents. This high level of cross-resistance is due to the increased expression of a membrane glycoprotein termed P- glycoprotein. Encoded in humans by the mdr1 gene, P-glycoprotein functions as an energy-dependent efflux pump of these cytotoxic agents. In this report, we demonstrate that the newly characterized immunosuppressant FK506 and its structural analogue, rapamycin, are capable of functioning as MDR reversal agents. FK506 and rapamycin increase both intracellular, cytotoxic drug (daunomycin) accumulation, and the cytotoxicity of chemotherapeutic agents in multidrug-resistant cells. The increase in cytotoxic drug accumulation is observed at concentrations of FK506 and rapamycin 1,000-fold greater than the concentrations required for FK506 and rapamycin to inhibit T-lymphocyte activation and similar to those shown to be effective for other MDR reversal agents such as cyclosporine A (CsA) and verapamil. The effect of FK506 or rapamycin on both intracellular accumulation and cytotoxicity of daunomycin is additive. This is supported by the ability of FK506 and rapamycin to directly compete the binding of the photoaffinity analogue 125I-iodoaryl azidoprazosin to the P- glycoprotein. The data demonstrate that FK506 and rapamycin represent a new class of structurally distinct molecules that can function as MDR reversal agents and suggest a previously unidentified, potential clinical role for these compounds.
Band 3 Memphis: a widespread polymorphism with abnormal electrophoretic mobility of erythrocyte band 3 protein caused by substitution AAG---- GAG (Lys----Glu) in codon 56
Authors: Jarolim P , Rubin HL , Zhai S , Sahr KE , Liu SC , Mueller TJ , Palek J
Published: October 2019

Band 3 Memphis (b3M) is a variant of the erythrocyte band 3 protein detected in individuals of virtually all ethnic groups and characterized by a reduced mobility of proteolytic fragments derived from the N-terminus of the cytoplasmic domain of band 3 (cdb3). We have sequenced band 3 cDNA corresponding to cdb3 in 12 heterozygotes for the b3M polymorphism including one white, one black, one Chinese, one Philippino, one Malay, and seven Melanesian subjects. In all individuals, we found a single-base substitution in codon 56 of one band 3 allele changing lysine to glutamic acid (AAG----GAG) which, in some of them, was linked with an additional mutation in cdb3. Since the change of codon 56 from AAG to GAG was the only mutation in the studied individuals found within the cDNA segment coding for the abnormally migrating fragment of cdb3, we conclude that it represents the underlying molecular basis of the b3M polymorphism. We further support this conclusion by showing that electrophoresis in the presence of 4 mol/L urea abolished the difference in migration between proteolytic products of b3M and normal band 3, and that a fusion protein prepared from cDNA coding for the b3M allele again exhibits reduced electrophoretic mobility compared with the normal fusion protein. Finally, since most of the previously cloned mouse, rat, and chicken band 3 and band 3-related proteins contain glutamic acid in the position corresponding to amino acid 56 in the human band 3, we propose that the Memphis variant is the evolutionarily older form of band 3.
Interferon-gamma increases the expression of the gene encoding the beta subunit of the granulocyte-macrophage colony-stimulating factor receptor
Authors: Hallek M , Lepisto EM , Slattery KE , Griffin JD , Ernst TJ
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activates a broad range of myeloid cells through binding to high-affinity receptors (GM-CSF-R) consisting of at least two distinct subunits, GM-CSF-R alpha and GM-CSF-R beta. The genes of these GM-CSF-R subunits have been identified recently, but little is known about the regulation of their expression. In this study, we investigated the expression of the GM-CSF- R subunit genes in normal human monocytes. Out of a panel of various cytokines and factors tested, only interferon-gamma (IFN-gamma) affected the expression of one of the GM-CSF-R subunit genes by increasing the GM-CSF-R beta mRNA expression threefold to sixfold with no effect on GM-CSF-R alpha. Maximal effects occurred 2 to 4 hours after stimulation with 500 to 5,000 U/mL IFN-gamma. Nuclear run-on assays and mRNA half-life studies showed that IFN-gamma modestly enhanced the transcription of the GM-CSF-R beta gene and stabilized the GM-CSF-R beta mRNA, with the latter mechanism predominant. Pretreatment of the monocytes with cycloheximide did not abrogate the increase of GM- CSF-R beta mRNA expression induced by IFN-gamma, indicating that de novo protein synthesis was not required for this activity. When monocytes were exposed to IFN-gamma for 6 to 24 hours, the number of GM- CSF-R per cell was increased 79% as compared with controls, whereas the receptor affinity remained unchanged. These data indicate that the GM- CSF-R expression in monocytes may be upregulated by IFN-gamma via an increased expression of the beta subunit gene, involving both transcriptional and post-transcriptional mechanisms.
Evidence that postoperative fibrinolytic shutdown is mediated by plasma factors that stimulate endothelial cell type I plasminogen activator inhibitor biosynthesis
Authors: Kassis J , Hirsh J , Podor TJ
Published: October 2019

Postoperative fibrinolytic shutdown has been attributed to an increase in plasma levels of type I plasminogen activator inhibitor (PAI-1) activity and may contribute to postoperative venous thrombosis. The purpose of this study was to determine whether the postoperative increase in PAI-1 is contributed to by a plasma mediator(s) that stimulates PAI-1 synthesis and secretion by vascular endothelium. Plasma samples collected from patients (N = 11) before and after surgery for total hip replacement were (1) assayed for endogenous plasma PAI-1 antigen and activity, and (2) incubated with cultured human umbilical vein endothelial cells (HUVECs) and PAI-1 antigen and activity measured in the conditioned medium (CM). Eighteen hours after surgery, endogenous plasma levels of PAI-1 antigen and activity were increased by 225% (P = .003) and 190% (P = .04), respectively over the preoperative values. In addition, compared with preoperative plasma, postoperative plasma increased HUVEC secretion of PAI-1 antigen and activity by 99% (P = .001) and 66% (P = .002), respectively. This increase in HUVEC PAI-1 secretion reflects an increase in PAI-1 mRNA expression and protein biosynthesis as confirmed by metabolic radiolabeling, immunoprecipitation, and Northern blot analysis. Ultra- filtration experiments indicate that the postoperative plasma mediator(s) that stimulates HUVEC PAI-1 biosynthesis is in a molecular weight (MW) range of approximately 30 to 100 Kd. Heat treatment (56 degrees C; 30 minutes) of postoperative plasma abolished the induction of HUVEC PAI-1 production. Enzyme-linked immunosorbent assay and immunoneutralization experiments indicate that tumor necrosis factor- alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) do not contribute to the postoperative plasma effect on HUVEC PAI-1 synthesis. These observations demonstrate that postoperative patient plasma contains a factor(s) that may stimulate endothelial cell PAI-1 biosynthesis in vivo and thus mediate postoperative fibrinolytic shut- down.
Clonal evolution in a myeloid cell line transformed to interleukin-3 independent growth by retroviral transduction and expression of p210bcr/abl
Authors: Laneuville P , Sun G , Timm M , Vekemans M
Published: October 2019

Current evidence suggests that the expression of the tyrosine kinase p210bcr/abl in chronic myelogenous leukemia (CML) may directly induce the initial phase of granulocytic hyperplasia. However, the dysregulation of additional genes appears to be required for transition to the acute leukemic phase, as inferred by the appearance of recurrent secondary cytogenetic abnormalities in the majority of patients. To determine whether the expression of p210bcr/abl alone is responsible for this genetic instability, we introduced and expressed the bcr/abl gene from a retroviral vector in a clone of the interleukin-3 (IL-3) dependent myeloblastic 32D C13(G) cell line. Clonal and polyclonal cells transformed to IL-3 independent growth were observed for a period extending up to 6 months for changes in the expression of p210bcr/abl, cell proliferation, inhibition by prostaglandin E1 (PGE1), forskolin, and cyclic adenosine monophosphate (cAMP) analogues, regulation of the cell cycle, and karyotype. Whereas the properties of control vector infected 32D C13(G)′ cells remained stable over time, cells expressing p210bcr/abl were phenotypically unstable. In cells expressing p210bcr/abl, we observed selective modulation of p210bcr/abl mRNA and protein expression, evolution from partial to full abrogation of IL-3 dependence, reduced serum requirements, increased cell proliferation, decreased inhibition by PGE1 and cAMP analogues, and the appearance of new structural and numerical chromosomal abnormalities with successive cell passages. These results indicate that expression of p210bcr/abl can directly predispose 32D C13(G)′ cells to genetic instability, promotes the emergence of clones with an increased proliferative advantage, and may represent an in vitro model suitable for the study of mechanisms underlying progression to the acute leukemic phase in CML.
Involvement of tumor necrosis factor (TNF) receptors p55 and p75 in TNF responses of acute myeloid leukemia blasts in vitro
Authors: Delwel R , van Buitenen C , Lowenberg B , Touw I
Published: October 2019

Tumor necrosis factor (TNF)-alpha and TNF-beta have multiple effects on human acute myeloid leukemia (AML) cells in vitro, including (1) synergistic stimulation of proliferation with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and upregulation of interleukin-3 (IL-3) and GM-CSF receptors; (2) inhibition of granulocyte-CSF (G-CSF)-induced growth and rapid downmodulation of G-CSF receptors; and (3) induction of autocrine growth. Recently, two distinct TNF receptors (TNF-Rs), TNF-R(p55) and TNF-R(p75), have been identified. In this study, we show that both receptor types may be expressed by AML blasts. It has been investigated whether the different effects of TNF on AML blasts can be explained by differential activation of the distinct TNF-R structures. For this purpose, we used the monoclonal antibodies HTR-1 and HTR-9, specifically recognizing TNF-R(p55), and UTR-1, specific for TNF- R(p75). TNF-(alpha and -beta) mediated synergistic activation with IL- 3/GM-CSF, upregulation of IL-3/GM-CSF receptors, inhibition of G-CSF- induced growth, and rapid downmodulation of G-CSF receptors exclusively result from activation of TNF-R(p55). In certain cases in which TNF- alpha, rather than TNF-beta, induces AML growth through an autocrine mechanism, both TNF-R(p55) and (p75) are involved. These data indicate that the variety of TNF responses observed in AML can only be partially explained by differential activation of the TNF-R(p55) and (p75) structures, and that TNF-R(p55) on AML blasts can transduce both positive (synergism with IL-3/GM-CSF) and negative regulatory signals (inhibition of G-CSF-induced proliferation) following TNF activation.
Heterogeneous Epstein-Barr virus infection patterns in peripheral T- cell lymphoma of angioimmunoblastic lymphadenopathy type
Authors: Anagnostopoulos I , Hummel M , Finn T , Tiemann M , Korbjuhn P , Dimmler C , Gatter K , Dallenbach F , Parwaresch MR , Stein H
Published: October 2019

In this study, 32 cases of T-cell lymphoma of angioimmunoblastic lymphadenopathy type (AILD-TCL) were investigated for their association with Epstein-Barr virus (EBV). For this purpose, three different approaches were applied: polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small nuclear RNAs (EBER), and immunohistology for EBV-encoded latent membrane protein (LMP). PCR and EBER-ISH produced almost identical results, showing that all but one case of AILD-TCL contained EBV genomes. Three distinctive patterns of EBV infection were observed after immunophenotypical characterization of EBER-positive cells: (1) in 26% of the cases, B and T cells were infected, the majority of which were B cells of immunoblastic morphology located in the remnants of lymphoid follicles; (2) in 42% of the cases, the vast majority of infected cells were neoplastic T cells diffusely distributed in the lymph nodes, but infected B cells were also present; and (3) in 32% of the cases, there were only a few infected small lymphoid cells. Detectable LMP was frequent in cases exhibiting patterns 1 and 2. These findings suggest that in AILD-TCL patients, B cells and especially T cells are highly susceptible to a persistent EBV infection, which often leads to a growth advantage of the infected cells. Thus EBV, in conjunction with genetic abnormalities and selective defects of the immune system, might be involved in the pathogenesis of AILD-TCL.
Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients
Authors: Billadeau D , Quam L , Thomas W , Kay N , Greipp P , Kyle R , Oken MM , Van Ness B
Published: October 2019

One of the distinguishing features of multiple myeloma (MM) is the proliferation of plasma cells that home to the bone marrow (BM). However, there still remains some uncertainty concerning the presence of related malignant cells in the peripheral blood of myeloma patients. Using consensus oligonucleotide primers, we amplified the third complementary determining region (CDR3) of rearranged immunoglobulin heavy chain alleles from MM marrow samples by polymerase chain reaction (PCR). From the sequences of the products, we derived allele-specific oligonucleotides (ASO), and these were used in subsequent amplification reactions to detect malignant clones in the peripheral blood of myeloma patients. This method is highly specific and sensitive to 1 malignant cell in the background of 10(5) normal cells. Using this method we detected circulating malignant cells in 13 of 14 previously untreated MM patients. Furthermore, by applying ASO-PCR to artificial titrations of initial BM DNA sample into normal peripheral blood lymphocyte (PBL) DNA we were able to generate standard curves and quantitate the amount of tumor in the patient PBL. We observed a wide variation in the amount of circulating tumor between patients. In addition, we found that the incidence of circulating tumor cells was independent of BM tumor burden and stage of disease. The detection and quantitation of circulating tumor cells in the PBL of MM patients may offer an alternative assessment of the disease and may be an important consideration in the use of peripheral stem cells in bone marrow transplantation.
Acute graft-versus-host disease: analysis of risk factors after allogeneic marrow transplantation and prophylaxis with cyclosporine and methotrexate [see comments]
Authors: Nash RA , Pepe MS , Storb R , Longton G , Pettinger M , Anasetti C , Appelbaum FR , Bowden RA , Deeg HJ , Doney K
Published: October 2019

Previous studies of risk factors for acute graft-versus-host disease (GVHD) involved patients receiving predominantly single-agent prophylaxis. Therefore, a retrospective analysis was performed on 446 patients, from a single institution, who received transplants of marrow from HLA-identical siblings and the combination of cyclosporine (CSP) and methotrexate (MTX) to determine risk factors for acute GVHD associated with this more effective form of GVHD prophylaxis. The incidences of Grades II-IV and Grades III-IV (severe) acute GVHD were 35% and 16%, respectively. Increased clinical grades of acute GVHD in patients without advanced malignant disease were associated with a decreased survival. In a multivariate Cox regression analysis, risk factors associated with the onset of Grades II-IV acute GVHD were sex mismatch and donor parity (P = .001), increased dose of total body irradiation (TBI) (P = .001), and reduction to less than 80% of the scheduled dose of MTX (P = .02) or CSP (P = .02). The multivariate analysis indicated a relative risk of 1.37 for acute GVHD in a group defined as having advanced malignant disease at transplant; however, this difference failed to reach conventional levels of statistical significance (P = .07). Reduction of MTX and CSP occurred in up to 36% and 44% of patients, respectively, primarily because of renal or hepatic dysfunction. The periods of increased risk for the onset of acute GVHD were up to 1 week after a reduction of MTX and 2 weeks after a reduction in CSP. When only patients who developed Grades II-IV acute GVHD were considered, the more severe acute GVHD of Grades III-IV was associated with increased patient age of 40 years or greater (P = .05) and dose reductions of CSP (P = .008). Serologic status of patient and donor for cytomegalovirus (CMV), HLA antigens in the A and B loci, and isolation in a laminar air flow room during marrow transplantation, all previously identified as risk factors for acute GVHD, were not confirmed as risk factors in this study population. The toxicity of MTX and CSP and the development of acute GVHD from inadequate immunosuppression because of dose reduction warrants further trials with potentially less toxic immunosuppressive agents. Risk factors for acute GVHD should be considered in clinical management and in the design of clinical trials.
Anti-CD3 + interleukin-2 stimulation of marrow and blood: comparison of proliferation and cytotoxicity
Authors: Anderson PM , Ochoa AC , Ramsay NK , Hasz D , Weisdorf D
Published: October 2019

The proliferation and in vitro cytolytic activity of interleukin-2 (IL- 2)-activated and anti-CD3 + IL-2-stimulated marrow mononuclear cells (MMC) and peripheral blood mononuclear cells (PBMC) were studied. Samples from 8 normal donors, 15 patients with acute lymphoblastic leukemia (ALL), and 7 patients with non-Hodgkin's lymphoma (NHL) in remission were cultured in IL-2 (100 U/mL) or IL-2 (100 U/mL) plus anti- CD3 (10 ng/mL). MMC as well as PBMC samples demonstrated significant synergy between IL-2 and anti-CD3 in the promotion of proliferation as measured by 3H thymidine incorporation on day 5 (P less than .001) or fold increase in cell number on day 14. Cryopreserved marrow specimens had equally rapid proliferation as fresh MMC when cultured in the presence of anti-CD3 + IL-2. Anti-CD3 concentrations of 3, 11, 33, and 100 ng/mL augmented proliferation similarly in the presence of IL-2 (0.1 to 100 U/mL). Mean fold increases in cell number of both marrow- and blood-derived cultures after 14 days were significantly higher for anti-CD3 + IL-2-stimulated cultures compared with cultures stimulated with IL-2 only (50- to 200-fold increase in cell number; P = .01). Comparison of remission MMC and PBMC from ALL and NHL patients with normal controls showed equivalent growth rates of activated cultures at 7, 14, and 21 days. Marrow purging with immunotoxin anti-CD19 pokeweed antiviral protein plus 4HC had no significant effect on proliferation of anti-CD3 + IL-2-stimulated MMC cultures in patients with ALL. Cytolytic activity of IL-2- and IL-2 + anti-CD3-activated PBMC and MMC cultures was assessed in 51Cr release assays using K562 (natural killer ([NK]-sensitive), Daudi (Burkitt's lymphoma-, NK-resistant), and Nalm-6 (ALL-, lymphokine-activated killer [LAK]-resistant) cell lines and cryopreserved ALL blasts. Cytolytic activity on a per-cell basis (percent cytotoxicity at an effector:target ratio of 30:1) was similar in IL-2-activated PBMC- and MMC-derived cultures from ALL patients. MMC activated with anti-CD3 plus IL-2 killed Daudi significantly less well than IL-2-activated cultures on days 12 and 19 (P = .03); no significant differences were observed in lysis of LAK-resistant Nalm-6 or cryopreserved ALL blast targets. Dose response of anti-CD3 augmentation of Daudi and Nalm-6 killing was different in IL-2- and IL- 2 + anti-CD3-stimulated cultures.
Differentiation and erythropoietin receptor gene expression in human erythroid progenitor cells [see comments]
Authors: Wickrema A , Krantz SB , Winkelmann JC , Bondurant MC
Published: October 2019

Partially purified human burst-forming unit-erythroid (BFU-E) cells from peripheral blood were cultured for 6 to 8 days to obtain colony- forming unit-erythroid (CFU-E) cells. When these BFU-E-derived CFU-E were further purified and recultured in liquid suspension cultures with erythropoietin (EPO), they matured and differentiated into reticulocytes in vitro. A maximum rate of hemoglobin synthesis was observed at day 10 of cumulative culture time by measuring 59Fe incorporation into heme. Withdrawal of EPO from erythroblast cultures at various times during development showed that between day 10 and day 11 (when the majority of the cells are in the polychromatic erythroblast stage), these cells became independent of EPO. The timing of the disappearance of the EPO requirement in these cells coincided with the marked decline in proliferation. Measurement of EPO receptor messenger RNA (mRNA) levels by Northern analysis showed that there is a slight decline during the day 8 to day 10 time period, followed by a rapid decline between days 10 and 14. Binding of 125I-EPO to erythroblasts also showed a steady decline of the cell surface binding during maturation and terminal differentiation. The half-life of the human EPO receptor was 90 minutes in the presence of the transcriptional inhibitor actinomycin D and the half-life measured at two different times during the 8- to 14-day culture period remained constant. These results indicate that human EPO receptor mRNA must be transcribed continuously to maintain the levels seen by Northern analysis. The human cell system described here is well suited for the study of a wide variety of biochemical events during late erythroid differentiation.
Role of tumor necrosis factor-alpha and its specific 55-Kd and 75-Kd receptors in patients with lymphoproliferative disease of granular lymphocytes
Authors: Zambello R , Trentin L , Bulian P , Cassatella M , Raimondi R , Chisesi T , Agostini C , Semenzato G
Published: October 2019

The role of tumor necrosis factor-alpha (TNF-alpha) in the development of in vitro proliferative and cytotoxic abilities of granular lymphocytes (GL) in patients with lymphoproliferative disease of GL (LDGL) has been investigated. To this aim, taking advantage of the recent generation of specific monoclonal antibodies (MoAbs) reacting with the p55 and p75 TNF receptors (TNF-R) (htr-9 and utr-1 MoAb, respectively), we evaluated the expression and the functional role of each TNF-R in freshly isolated highly purified GL from a series of 10 LDGL patients (six CD3+ T-lineage GL and four CD3- natural killer [NK]- lineage GL). The expression of TNF-alpha transcripts and the release of TNF-alpha in the culture medium at resting conditions and following cell activation were also studied. Our data indicate that at resting conditions both CD3+ and CD3- GL express only the p75 TNF-R. Accordingly, a specific inhibition of phycoerythrin (PE)-conjugated TNF- alpha binding was demonstrated by the anti-p75 TNF-R utr-1 MoAb, but not by the anti-p55 htr-9 MoAb. Following activation with interleukin-2 (IL-2), anti-CD3, or anti-CD16 MoAbs, an increased expression of the p75 TNF-R and a slight induction of the p55 TNF-R was observed. Weak expression of specific TNF-alpha transcripts was detected at resting conditions and on unstimulated cells, whereas both IL-2 or anti-CD3 MoAb induced TNF-alpha mRNA. Under these in vitro conditions, detectable amounts of this cytokine were demonstrated in the culture supernatant of GL. The cytotoxic and proliferating activities mediated by IL-2 or anti-CD3 MoAb were dampened by anti-TNF-alpha antibody, suggesting a role for endogenous TNF-alpha in these functions. Both utr- 1 and htr-9 MoAbs showed a moderate inhibition of proliferative activity, whereas cytotoxicity was not reduced. Taken together, our results suggest that TNF-alpha plays a role in the mechanisms leading to CD3+ and CD3- GL in vitro activation in patients with LDGL.
Glutathione depletion in chronic lymphocytic leukemia B lymphocytes
Authors: Silber R , Farber CM , Papadopoulos E , Nevrla D , Liebes L , Bruck M , Brown R , Canellakis ZN
Published: October 2019

Glutathione (GSH) content may be the major determinant of a cell's sensitivity to cytotoxic alkylating agents. In the present study, the GSH concentration was determined in lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia (CLL). Comparable levels were found in both types of cells. Incubation for 20 hours led to a decrease in GSH to 51% of baseline values in CLL B cells. Under the same conditions, normal B- or T-lymphocyte GSH content remained constant. GSH depletion was shown to be a characteristic of the B-CLL B lymphocyte. It was not found in the T cells of patients with B-CLL or in cells from patients with T-CLL. Chlorambucil (CLB) contributes to the decrease in GSH in B-CLL lymphocytes; after incubation with the drug, lower levels of GSH were found than in the normal B or T lymphocytes, B-CLL T cells, or T-CLL (CD4 or CD8) cells. GSH depletion of CLL B lymphocytes may be related to the greater therapeutic efficacy of CLB in B-CLL than in T-CLL.
Detection of bcl-2 protein and bcl-2 messenger RNA in normal and neoplastic lymphoid tissues by immunohistochemistry and in situ hybridization
Authors: Kondo E , Nakamura S , Onoue H , Matsuo Y , Yoshino T , Aoki H , Hayashi K , Takahashi K , Minowada J , Nomura S
Published: October 2019

bcl-2 protein has been detected in surgical specimens and cultured permanent cell lines of non-Hodgkin's lymphomas and leukemias using enzyme immunohistochemistry and immunofluorescence with anti-bcl-2 monoclonal antibodies. Of 40 surgical specimens, bcl-2 protein was expressed in 50% of B-cell and 41% of T-cell lymphomas, both with and without the bcl-2 gene rearrangement. In investigations of 38 hematopoietic cell lines, bcl-2 protein was detected not only in lymphoid cell lines but also in myeloid cell lines. In situ hybridization and immunohistochemical analysis of reactive lymph nodes showed that lymphocytes in mantle zones and paracortical areas expressed bcl-2 protein consistent with the messenger RNA distribution and that germinal center cells showed abundant bcl-2 transcript, despite the absence of detectable bcl-2 protein. These results suggest that bcl-2 protein is broadly expressed in various hematopoietic neoplasms not restricted in t(14; 18) lymphomas and that germinal center cells may be involved in some arrest of bcl-2 protein expression at the posttranscriptional level.
Interferon-alpha downregulates the abnormal intracytoplasmic free calcium concentration of tumor cells in hairy cell leukemia
Authors: Genot E , Bismuth G , Degos L , Sigaux F , Wietzerbin J
Published: October 2019

Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiation. One important feature of the disease is its exquisite sensitivity to interferon-alpha (IFN-alpha) therapy. Because we showed earlier that the CD20 molecule is consistently hyperphosphorylated in hairy cells and because previous studies showed that CD20 is involved in regulating intracytoplasmic free calcium concentrations ([Ca2+]i) in normal B lymphocytes, we measured [Ca2+]i in tumor cell samples from patients with HCL and studied the effect of IFN-alpha on this parameter. Using the Ca(2+)-sensitive fluorophore fura-2, we observed that hairy cells display a slightly but consistently higher [Ca2+]i than normal 48-hour-activated B cells or other leukemic cells. Furthermore, both in vitro preincubation of cell samples with IFN-alpha and in vivo administration of this cytokine reduced the [Ca2+]i in hairy cells. This effect was observed together with a decrease in transmembrane Ca2+ influx. However, preincubation with IFN-gamma had no effect. The in vivo correlation between the diminution of CD20 phosphorylation and [Ca2+]i in tumor cell samples from patients at the beginning of IFN-alpha therapy suggests that these two parameters are connected.
Genomic hypomethylation in human chronic lymphocytic leukemia
Authors: Wahlfors J , Hiltunen H , Heinonen K , Hamalainen E , Alhonen L , Janne J
Published: October 2019

DNA samples from patients with chronic lymphocytic (CLL), chronic myelocytic (CML), acute myelocytic (AML), and acute lymphocytic leukemia (ALL), as well as samples from patients with multiple myeloma (MM) and healthy volunteers, were analyzed for their genomic methylation status using Hpa II and Msp I digestions followed by a simple gel electrophoresis and ethidium bromide staining. A densitometric method was developed to measure more accurately the extent of methylation in genomic DNA samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis of hydrolyzed DNA. Southern analysis with ornithine decarboxylase (ODC) gene probe was also employed, and the levels of ODC mRNA were determined with the aid of polymerase chain reaction (PCR). The results indicated that a general genomic hypomethylation was present in almost all of the samples obtained from patients with B-cell CLL. This hypomethylation was most striking among the patients who were freshly diagnosed and among untreated chronic patients. CML appeared to be a heterogenous group, comprising patients with normal methylation status in addition to patients with slight hypomethylation. Patients with ALL, AML, or MM did not show any signs of DNA methylation changes in comparison to healthy volunteers. Although all analyzed samples from patients with B-CLL showed hypomethylation of ODC sequences, little correlation existed between the mRNA levels and the extent of hypomethylation of ODC gene.
Allogeneic leukocytes but not therapeutic blood elements induce reactivation and dissemination of latent human immunodeficiency virus type 1 infection: implications for transfusion support of infected patients
Authors: Busch MP , Lee TH , Heitman J
Published: October 2019

Various immunologic stimuli and heterologous viral regulatory elements have been shown to increase susceptibility to, and replication of, human immunodeficiency virus type 1 (HIV-1) in lymphocytes and monocytes in vitro. Transfusion of allogeneic blood components from heterologous donors constitutes a profound immunologic stimulus to the recipient, in addition to being a potential route of transmission of lymphotropic viral infections. To investigate the hypothesis that transfusions, and particularly those containing leukocytes, activate HIV-1 replication in infected recipient cells, we cocultured peripheral blood mononuclear cells (PBMC) from three anti-HIV-1-positive individuals with allogeneic donor PBMC, as well as partially purified populations of donor lymphocytes, monocytes, granulocytes, platelets, and red blood cells (RBC) and allogeneic cell-free plasma. Allogeneic PBMC induced a dose-related activation of HIV-1 expression in in vivo infected cells, followed by dissemination of HIV-1 to previously uninfected patient cells. Activation of HIV-1 replication was observed with donor lymphocytes, monocytes, and granulocytes, whereas no effect was seen with leukocyte-depleted RBC, platelets, or plasma (ie, therapeutic blood constituents). Allogeneic donor PBMC were also shown to upregulate HIV-1 expression in a “latently” infected cell line, and to increase susceptibility of heterologous donor PBMC to acute HIV-1 infection. Studies should be performed to evaluate whether transfusions of leukocyte-containing blood components accelerate HIV-1 dissemination and disease progression in vivo. If so, HIV-1-infected patients should be transfused as infrequently as possible and leukocyte-depleted (filtered) blood components should be used to avoid this complication.
The generation of human natural killer cells from CD34+/DR- primitive progenitors in long-term bone marrow culture
Authors: Miller JS , Verfaillie C , McGlave P
Published: October 2019

We have adapted the stroma-dependent long-term bone marrow culture (LTBMC) system to study the development of human natural killer cells (NK) from the CD34+/HLA-DR- (CD34+/DR-) BM mononuclear cell (BMMNC) population. The CD34+/DR- population does not express any known antigens associated with myeloid or lymphoid lineage and has been shown by us and others to contain primitive hematopoietic progenitors capable of both self-renewal and differentiation to myeloid lineage. CD34+/DR- cells obtained from normal human BM by fluorescence-activated cell sorting were plated on allogeneic, irradiated BM stromal layers. After 5 weeks of culture in the presence of media containing recombinant interleukin-2 and human serum, 147- +/- 21-fold expansion of cells with the morphologic appearance of large granular lymphocytes was observed. Cultured cells (84.8% +/- 1.5%) expressed the characteristic CD56+/CD3- phenotype of NK. A proportion of CD56+/CD3- cells expressed other markers of lymphoid lineage that have been associated with mature NK, including CD2 (7.8% +/- 1.2%), CD7 (19.5% +/- 2.8), CD8 (3.1% +/- 1.0%), and CD16 (4.5% +/- 1.3%). The cultured cells did not express other antigens associated with T-lymphocyte (CD3, CD5, T-cell receptor [TCR] alpha/beta and TCR gamma/delta), B-lymphocyte (CD19), myeloid (MY8, CD33, and CD71), or monocytoid (CD14 and CD15) lineage and did not express the CD34 antigen associated with hematopoietic progenitors present on the starting population. This NK population was cytotoxic against both K562 (E:T 20:1; 79% +/- 1.9%) and Raji (E:T 20:1; 38% +/- 5.7%) target cell lines. The NK progenitor frequency in the CD34+/DR- cell population determined by limiting dilution of CD34+DR- on stromal layers followed by a functional chromium release assay against K562 targets was 1:169 +/- 50 CD34+/DR- cells. The data suggest that human LTBMC developed to study myeloid differentiation can be modified to study the origin and development of the NK and possibly other lymphoid lineages. Modified cultures show that cells with morphologic, phenotypic, and functional characteristics of NK can be derived from a population of BMMNC with the phenotype of primitive hematopoietic progenitors and without phenotypic evidence of lymphoid- or myeloid- lineage commitment. Further studies will address the cell of origin and the ontogeny of human NK and other lymphoid lineages.
Response of human platelets to activating monoclonal antibodies: importance of Fc gamma RII (CD32) phenotype and level of expression
Authors: Tomiyama Y , Kunicki TJ , Zipf TF , Ford SB , Aster RH
Published: October 2019

Certain monoclonal antibodies (MoAbs) specific for platelet membrane glycoproteins are known to be capable of activating platelets, and it is generally thought that platelets from normal subjects are equally susceptible to stimulation by such MoAbs. We found that platelets from 20 normal donors varied significantly in their sensitivity to three IgG1 murine MoAbs specific for membrane glycoproteins CD9, GPIV (CD36), and the GPIIb/IIIa complex (CD41), respectively. The response of platelets to these MoAbs was blocked by prior addition of MoAb IV.3 specific for the Fc gamma RII receptor, indicating that activation was Fc receptor mediated. Platelets that responded poorly to these MoAbs failed to bind the MoAb 41H.16, specific for the “responder” form of Fc gamma RII, but platelets that responded well reacted with this MoAb. The average number of Fc gamma RII receptors on platelets from “responders” and “non-responders” was approximately the same. However, the number of Fc gamma RII receptors expressed influenced sensitivity of a subgroup of “responder” platelets to the anti-CD41 MoAb. These platelets were judged on the basis of MoAb binding studies to be heterozygous for the two alleles of Fc gamma RIIA. In contrast to their varying sensitivity to IgG1 MoAbs, members of the platelet panel responded equally well to 50H.19, an IgG2a MoAb specific for CD9, and these responses could not be blocked by MoAb IV.3 in the presence of plasma. This appears to be because of dual actions of 50H.19 on platelets: one FcR-dependent and the other complement-dependent. Our findings confirm previous reports that certain IgG1 MoAbs activate platelets through binding of their Fc domains to Fc gamma RII receptors and demonstrate that this response is influenced both by Fc gamma RII phenotype and (in the case of the anti-CD41 MoAb) by the number of Fc gamma RII receptors expressed. The failure of nonresponding platelets to bind detectable amounts of MoAb 41H.16, which is thought to recognize all Fc gamma RII receptors except for one allele of the Fc gamma RIIA gene, is consistent with the possibility that Fc gamma RIIA gene products, but not Fc gamma RIIB or Fc gamma RIIC gene products, are expressed on platelets.
Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation
Authors: Bakkus MH , Heirman C , Van Riet I , Van Camp B , Thielemans K
Published: October 2019

To investigate whether somatic hypermutation occurs in multiple myeloma (MM) Ig VH region genes, we have cloned and sequenced the expressed VH genes from five cases of MM. The sequences were obtained after polymerase chain reaction (PCR) on total RNA isolated from the bone marrow, using 5′ VH family-specific leader and 3′ C gamma- or C alpha- specific primers. MM-specific CDR3 oligonucleotides were produced to isolate VH genes expressed by the malignant plasma cells. In all five cases, the productive Ig gene used the VH3 family. Extensive sequence analysis of multiple independent M13 clones showed no intraclonal variation with no evidence for ongoing somatic hypermutation in MM VH region genes. We were able to identify possible germline counterparts of the expressed VH genes in two cases. Comparison of these genes shows that the MM VH region genes have somatic mutations characteristic for an antigen-driven process. In the other three cases, no close homology could be found with published VH3 sequences. These findings implicate that, in MM, clonal proliferation takes place in a cell type that has already passed through the phase of somatic hypermutation.
Exaggerated cation leak from oxygenated sickle red blood cells during deformation: evidence for a unique leak pathway
Authors: Sugihara T , Hebbel RP
Published: October 2019

An abnormal susceptibility of the sickle red blood cell (RBC) membrane to deformation could compromise its permeability barrier function and contribute to the exuberant cation leakiness occurring during the sickling phenomenon. We examined this hypothesis by subjecting RBCs at ambient oxygen tension to elliptical deformation, applying shear stress in a viscous medium under physiologic conditions. Compared with normal and high-reticulocyte control RBCs, sickle RBCs manifest an exaggerated K leak response to deformation. This leak is fully reversible, is both Cl and Ca independent, and at pHe 7.4 is fully balanced so that Kefflux equals Nainflux. This abnormal susceptibility is also evident in that the K leak in response to deformation occurs at an applied shear stress of only 141 dyne/cm2 for sickle RBCs, as compared to 204 dyne/cm2 for normal RBCs. Fresh sickle RBC membranes contain elevated amounts of lipid hydroperoxide, the presence of which is believed to provide the biochemical basis for enhanced deformation susceptibility. When examined at pHe 6.8, oxygenated sickle RBCs acquire an additional, unbalanced (Kefflux > Nainflux) component to the K leak increment specifically ascribable to deformation. Studies with inhibitors suggest that this additional component is not caused by a known leak pathway (eg, either K:Cl cotransport or the Gardos channel). This abnormal susceptibility of the sickle membrane to development of cation leakiness during deformation probably contributes to the exuberant cation leak taking place during RBC sickling.
Enhancement of T-cell-depleted bone marrow allografts in the absence of graft-versus-host disease is mediated by CD8+ CD4- and not by CD8- CD4+ thymocytes
Authors: Lapidot T , Faktorowich Y , Lubin I , Reisner Y
Published: October 2019

Transplantation of T-cell-depleted C57BL/6-Nu/Nu (“nude”) bone marrow (BM) into C3H/HeJ recipients, conditioned with 8 Gy total body irradiation plus chemotherapy with the myeloablative drug dimethyl myleran, resulted in poor hematopoietic reconstitution 14 days posttransplant, compared with transplantation with T-cell-depleted BM from normal C57BL/6 donors. Hematopoietic reconstitution of “nude” BM could be improved by the addition of (C57BL/6xC3H/HeJ)F1 thymocytes void of graft-versus-host activity. Enhancement of BM allografting by thymocytes is sensitive to low radiation doses (> or = 5.0 Gy) and can be achieved by transplanting the BM 24 hours before the administration of thymocytes. Fractionation of F1 thymocytes by differential agglutination with peanut agglutinin (PNA) and by fluorescence activated cell sorting showed that this hematopoietic enhancing activity is enriched in the unagglutinated (PNA-) thymocyte fraction and is mediated by PNA- CD8+ and not by PNA- CD4+ thymocytes.
Optimization of busulfan dosage in children undergoing bone marrow transplantation: a pharmacokinetic study of dose escalation
Authors: Yeager AM , Wagner JE Jr , Graham ML , Jones RJ , Santos GW , Grochow LB
Published: October 2019

Busulfan (BU) is a widely used myeloablative and antineoplastic agent in clinical bone marrow transplantation (BMT). The lower incidence of BU-associated toxicities and lower therapeutic effectiveness in young children given BU doses based on body weight (ie, 16 mg/kg) is associated with altered pharmacokinetics of BU; the area under the curve (AUC) of BU concentration versus time is significantly less in these patients than those observed in older children and adults. To optimize BU dosage in young BMT recipients, we developed a dosage regimen based on body surface area (BSA) and determined BU pharmacokinetics and BU-associated toxicities. Seven children (median age, 3.9 years, range, 1.1 to 5.7) undergoing allogeneic or autologous BMT for leukemia received 40 mg/m2/dose BU every 6 hours for 16 doses; BU concentrations were measured in the plasma, and AUCs were determined for each patient after the first and 13th doses. Expressed as a function of body weight, the median BU dosage was 26.4 mg/kg (range, 24.3 to 28.2), a 60% increase over the BU dosage based on body weight. Four patients developed mucositis, and one of them also developed nonfatal hepatic veno-occlusive disease (VOD). No patients receiving 40 mg/m2 BU developed neurotoxicity (eg, seizures) or interstitial pneumonitis. Prompt and sustained engraftment was observed in the allogeneic BMT recipients, and late graft failure was not seen. The mean BU AUCs were 1,105 mumol/L.min (range, 790 to 2,080) after the first dose and 1,022 mumol/L.min (range, 632 to 1,860) after the 13th dose of BU, comparable to the AUCs in adults given 16 mg/kg of BU. These studies suggest that, in young children, BSA-based dosing of BU (40 mg/m2) provides drug exposures (AUCs) closer to adult values with acceptable toxicities and may improve therapeutic effects.
Differentiation-dependent expression of sialyl stage-specific embryonic antigen-1 and I-antigens on human lymphoid cells and its implications for carbohydrate-mediated adhesion to vascular endothelium
Authors: Ohmori K , Takada A , Yoneda T , Buma Y , Hirashima K , Tsuyuoka K , Hasegawa A , Kannagi R
Published: October 2019

Expression of two developmentally regulated carbohydrate antigens, the sialyl stage-specific embryonic antigen-1 (SSEA-1) and I-antigens, in human lymphocytes and lymphocytic leukemia cells was investigated using specific monoclonal antibodies. Sialyl SSEA-1 was expressed only on natural killer (NK) cells, and was essentially absent on resting mature T and B cells among normal peripheral lymphocytes. On the other hand, the I-antigen was strongly expressed on virtually all mature B cells, moderately expressed on most mature T cells, but not expressed on NK cells in normal donors. Expression of the two antigens on normal T and B cells was reversible; in vitro stimulation of normal lymphocytes with concanavalin A (Con A) resulted in the loss of I-antigen and appearance of sialyl SSEA-1 on CD3+ T blasts, whereas stimulation with pokeweed mitogen led to loss of I-antigen expression and appearance of sialyl SSEA-1 antigen on CD19+ B blasts. Among lymphocytic leukemia cells, sialyl SSEA-1 was detected primarily on leukemia cells having immature properties such as most common acute lymphocytic leukemia (cALL) blasts, while the I-antigen was frequently expressed on malignant cells having relatively mature properties, such as those found in adult T- cell leukemia or chronic lymphocytic leukemia, and only occasionally on cALL blasts. Among normal peripheral lymphocytes, the sialyl SSEA-1+I- antigen- NK cells selectively underwent E-selectin (ELAM-1, endothelial- leukocyte adhesion molecule-1)-dependent adhesion to endothelial cells, while the I-antigen+sialyl SSEA-1- mature T and B cells did not, in line with the recent finding that sialyl SSEA-1 serves as a specific ligand for E-selectin. Con A blasts, which are sialyl SSEA-1+I-antigen- , also exhibited significant E-selectin-dependent adhesion to endothelial cells. These results indicate that expression of the sialyl SSEA-1 and I-antigens varies alternately depending on the differentiation/activation status of the lymphocytes, and that this at least partly regulates the behavior of lymphocytes at the vessel wall.