bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of myelopoiesis in a patient with congenital neutropenia: biology and nature of response to recombinant human granulocyte- macrophage colony-stimulating factor
Authors: Vadhan-Raj S , Jeha SS , Buescher S , LeMaistre A , Yee G , Lu L , Lloreta J , Hoots WK , Hittelman WN , Gutterman JU
Published: October 2019

To stimulate granulopoiesis, we gave recombinant human granulocyte- macrophage colony-stimulating factor (GM-CSF; 120 microgram/m2/d) to a patient with congenital neutropenia. The treatment resulted in marked increases in white blood cell counts (maximum, 17,400/microL), consisting mainly of eosinophils (maximum, 13,050/microL) and monocytes (maximum, 1305/microL), rather than neutrophils (maximum, 798/microL). Circulating phagocytes (97% eosinophils) derived after GM-CSF treatment were less effective in chemotaxis, slower but equally effective in phagocytosis, and more effective in H2O2 production compared with normal control neutrophils, but comparable in chemotaxis and H2O2 production to control eosinophils. Before GM-CSF treatment, the bone marrow showed a maturation defect in the neutrophilic series that persisted after treatment despite marked increases in mature cells of other lineages. In vitro agar culture of bone marrow cells before GM- CSF treatment showed a normal number of granulocyte colonies; however, maturation was limited to the metamyelocyte stage. Although the absolute number and cycling rates of myeloid colony forming cells (predominantly eosinophils) increased after treatment, the maturation defect in the neutrophilic series persisted. The finding that GM-CSF induced stimulation of proliferation, which was coupled with maturation in the eosinophilic and monocytic but not the neutrophilic components, suggests that this patient had an intrinsic cellular or humoral defect in neutrophil maturation.
Interactions of C1(-)-inhibitors from normal persons and patients with type II hereditary angioneurotic edema with purified activated Hageman factor (factor XIIa)
Authors: Donaldson VH , Mitchell BH , Everson B , Ratnoff OD
Published: October 2019

Activated high molecular weight Hageman factor (75 Kd) and Hageman factor carboxy-terminal fragments both formed complexes with purified C1(-)-inhibitor, but the Hageman factor fragments appeared to have a higher affinity for the C1(-)-inhibitor than activated Hageman factor. Therefore, the clot-promoting activity of activated Hageman factor might be relatively unimpaired if Hageman factor fragments are also present. Normal C1(-)-inhibitor was cleaved by Hageman factor fragments. Clot-promoting activity was not generated in Hageman factor by exposure to Hageman factor fragments, nor was Hageman factor cleaved by Hageman factor fragments. When Hageman factor was cleaved by streptokinase-activated plasminogen, a 40 Kd fragment was released. In contrast to their interactions with other proteinases, which are blocked by normal C1(-)-inhibitor, Type II C1(-)-inhibitors from plasmas of affected members of eight different kindred with this form of hereditary angioneurotic edema all inhibited the specific coagulant activity of activated Hageman factor to some degree. They did not all form complexes with activated Hageman factor that were stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Accessory function of interleukin-1 and interleukin-6: preferential costimulation of T4 positive lymphocytes
Authors: Tosato G , Miller J , Marti G , Pike SE
Published: October 2019

Two monocyte-derived cytokines, interleukin-1 (IL-1) and interleukin-6 (IL-6), have been reported to costimulate monocyte-depleted T cell populations in the presence of mitogen, and this effect has been attributed to an accessory function of these molecules. We have now examined further the accessory function potential of IL-1 plus IL-6, and examined how these cytokines promote T cell growth with mitogen. Together, IL-1 and IL-6 additively and, to a small degree, synergistically promote the proliferation of highly purified human peripheral blood T cells with phytohemagglutinin (PHA). However, maximum costimulation by IL-1 plus IL-6 over a wide range of concentrations is significantly smaller than that induced by optimal numbers of monocytes. Also, in contrast to monocytes that costimulate equally effectively T4 positive and T8 positive cells, IL-1 plus IL-6 costimulate T4 positive lymphocytes in marked preference to T8 positive cells. IL-1 plus IL-6 induces IL-2 secretion in T cell cultures costimulated with PHA, and an antibody to the IL-2 receptor, anti-Tac, markedly inhibits PHA-activated T cells costimulated by IL-1 plus IL-6. In addition, IL-1 plus IL-6 enhances the expression of surface IL-2 receptors. Because the costimulatory effect of IL-1 plus IL-6 is quantitatively smaller than that of monocytes, and it is preferentially directed toward T4 positive as opposed to T8 positive T cells, IL-1 plus IL-6, together, appear to represent a selective set of monocyte- derived accessory signals.
Anti-CD3 monoclonal antibody-mediated cytotoxicity occurs through an interleukin-2-independent pathway in CD3+ large granular lymphocytes
Authors: Loughran TP Jr , Aprile JA , Ruscetti FW
Published: October 2019

The mechanism of induction of cytotoxicity produced by anti-CD3 monoclonal antibody (MoAb) was studied in four patients with CD3+ large granular lymphocyte (LGL) leukemia. Anti-CD3 MoAb treatment resulted in increased target cell binding and increased granule formation. After activation, leukemic LGL remained Tac-, with the exception of a patient with CD4+ LGL leukemia. Radiolabeled interleukin-2 (IL-2) binding studies demonstrated that treatment with anti-CD3 MoAb resulted in upregulation of the number of p75 intermediate affinity IL-2 receptor sites per cell. Northern blot hybridization analysis showed expression of gamma-interferon gene transcripts 24 to 48 hours after activation. There was no evidence for expression of IL-2 messenger RNA or secretion of IL-2 after activation. Anti-CD3 MoAb and IL-2 provide different signals for activation of CD3+ LGL. Induction of cytotoxicity produced by anti-CD3 MoAb in leukemic CD3+ LGL is not associated with IL-2 production.
Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells
Authors: Ulrich MJ , Ley TJ
Published: October 2019

We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated “A gamma-neo” transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.
Open-wedge testicular biopsy in childhood acute lymphoblastic leukemia after two years of maintenance therapy: diagnostic accuracy and influence on outcome--a report from Children's Cancer Study Group
Authors: Nachman J , Palmer NF , Sather HN , Bleyer WA , Coccia PF , Lukens JN , Siegel SE , Hammond GD
Published: October 2019

Bilateral testicular biopsies were performed on 708 males with acute lymphoblastic leukemia completing 24 to 30 months of maintenance chemotherapy in continuous remission. The 73 patients (10.3%) with occult testicular leukemia (TL) had a significantly increased risk of subsequent relapse (P = .0001) and death (P less than .0001) when compared with patients with negative biopsies. Protocol-specified therapy for occult TL included reinduction therapy with concurrent bilateral testicular radiation, and 2 years of maintenance therapy. Four-year event-free survival for patients with negative biopsies was 78.2% +/- 4% versus 65% +/- 14% for patients with occult TL who received protocol-specified therapy (P = .05). This study suggests that (1) occult TL occurs in 10% of males completing 2 years of maintenance therapy; (2) occult TL significantly increases risk for subsequent relapse and death; (3) treatment results for occult TL and isolated overt off therapy TL (no previous biopsy) are similar; and (4) given current therapy, documentation of occult TL after 2 years of therapy does not improve disease-free survival.
Clinical implications of increased plasma levels of CD8 in patients with hairy cell leukemia
Authors: Ho AD , Grossman M , Trumper L , Pezzutto A , Pralle H , Boedewadt-Radzun S , Papendick U , God B , Zwingers T , Hunstein W
Published: October 2019

Plasma levels of soluble T-suppressor/cytotoxic antigen (sCD8) were measured at diagnosis or before systemic treatment in 69 patients with hairy cell leukemia (HCL). The 49 nonsplenectomized patients were characterized by high concentrations of sCD8 antigen as compared with 17 controls (P less than .0001). The median sCD8 level in non- splenectomized patients was 1,050 U/mL (range: 160 to 2,400 U/mL) and was significantly higher (P less than .0001) than the median of 275 U/mL (range: 20 to 1,080 U/mL) in splenectomized patients. The relationship of sCD8 to clinical response to subsequent interferon alpha (IFN alpha) treatment was analyzed. Patients who showed subsequent hematologic response with normalization of all blood counts had significantly lower levels of sCD8 concentrations at diagnosis than those who did not (P = .0056). Furthermore, normalization of sCD8 during IFN alpha treatment paralleled the achievement of normal counts in peripheral blood, whereas soluble interleukin-2 receptor (sIL-2R) levels remained high in most patients after 12 to 15 months of treatment. We speculate that activation of suppressor/cytotoxic T cells might play a role in myelosuppression, and its modulation during treatment with IFN alpha correlates with normalization in peripheral blood counts.
Enrichment of natural suppressor activity in a wheat germ agglutinin positive hematopoietic progenitor-enriched fraction of monkey bone marrow
Authors: Sugiura K , Ikehara S , Gengozian N , Inaba M , Sardina EE , Ogata H , Seong SM , Good RA
Published: October 2019

Natural suppressor (NS) activity, the capacity of unprimed cells to suppress immunologic responses, is present in mouse, rabbit, and human bone marrow (BM). In this study we characterize NS activity in bone marrow cells of the rhesus monkey. Greatest NS activity was found in low-density cells (1.0600 to 1.0655 g/mL) obtained by density centrifugation on a discontinuous Percoll gradient. NS activity was further enriched when cells were separated by affinity for wheat germ agglutinin (WGA). Cells with high affinity to WGA demonstrated potent NS activity, whereas cells with low affinity to WGA had no NS activity. A significant relationship between NS activity and hematopoietic activity was demonstrated using in vitro assays of colony formation (CFU-GM and CFU-MIX). NS activity was not affected by treatment with monoclonal antibodies (MoAbs) to human Fc gamma receptors (Leu, 11a,b,c) or treatment with MoAbs to monkey natural killer cells. These findings extend our prior observations by showing that cells with NS activity, which apparently have WGA receptors, are present not only in murine BM but also in monkey bone marrow, and suggest that such cells may be involved in immunoregulation by primitive cells of BM.
An improved clonal excess assay using flow cytometry and B-cell gating
Authors: Letwin BW , Wallace PK , Muirhead KA , Hensler GL , Kashatus WH , Horan PK
Published: October 2019

In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti- kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non- B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B- cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.
Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts
Authors: Ebi Y , Kasugai T , Seino Y , Onoue H , Kanemoto T , Kitamura Y
Published: October 2019

Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.
Analysis of rodent platelet glycoprotein IIb: evidence for evolutionarily conserved domains and alternative proteolytic processing
Authors: Poncz M , Newman PJ
Published: October 2019

Recently, the full-length primary amino acid sequence for human glycoproteins (GP) IIb and IIIa have been derived from their respective cDNAs. Potential functional domains within these proteins have been proposed based primarily on homology with similar domains in other proteins having known biologic function. To further understand the relationship between structure and function of the platelet fibrinogen receptor, we have begun comparative studies of the human GPIIb/IIIa receptor with the corresponding rodent receptor. The rodent rGPIIb/IIIa receptor differs from the human receptor, having low affinity for R.G.D- containing oligopeptides and not binding at all to the C-terminus of the gamma chain of human fibrinogen. We describe the structure of rodent platelet GPIIb derived from a combination of peptide sequencing, and cDNA and partial genomic DNA sequence analysis. The initial transcript is 1037 amino acid residues, having 78% amino acid identity with its 1039 residue human analog. Both heavy chains have the N- terminal sequence L.N.L.D, agreeing with the consensus derived from other integrin family alpha heavy chains. All 18 cysteine residues occur at positions conserved in human GPIIb and the vitronectin receptor alpha subunit VNR alpha. The putative calcium-binding domains of the GPIIbs have a high level of amino acid identity (92%), supporting the supposition that these regions have a critical biologic role. The final 48 C-terminal amino acid residues of the heavy chain of rodent GPIIb share only 56% identity with its human counterpart, and the proposed cleavage site of human GPIIb into its heavy and light chains is not present in the rodent sequence. Although we demonstrate that rodent GPIIb is split into two subunits during its maturation, this process either involves a different recognition sequence in the rodent or occurs at a different site. Finally, partial genomic DNA sequence analysis indicates that there are at least two rodent GPIIb genes: a normal gene, containing introns in positions similar to those in the human gene, and a processed pseudogene. The human haploid genome contains only a single GPIIb gene.
Cytogenetic analysis of chimerism and leukemia relapse in chronic myelogenous leukemia patients after T cell-depleted bone marrow transplantation
Authors: Offit K , Burns JP , Cunningham I , Jhanwar SC , Black P , Kernan NA , O'Reilly RJ , Chaganti RS
Published: October 2019

Serial cytogenetic studies were performed on 64 patients with chronic myelogenous leukemia (CML) after T cell-depleted allogeneic bone marrow transplantation (BMT). Forty patients with CML in chronic phase (CP) received cytoreduction followed by BMT with HLA-matched T cell-depleted allogeneic marrow. The remaining 24 patients were transplanted in second chronic, accelerated, or blastic phase, or received T cell- depleted grafts with a dose of T cells added back. The Y chromosome and autosomal heteromorphisms were used to distinguish between donor and host cells. Mixed hematopoietic chimerism (presence of donor and host cells) was identified in 90% of patients in first CP. The Philadelphia (Ph) chromosome reappeared in 16 of the 40 first CP CML patients. As expected, patients who had detectable Ph chromosome positive cells at any time during the posttransplant period had a high likelihood of subsequent clinical relapse. Transient disappearance of the Ph positive clone was rarely observed, and was followed by reappearance of the Ph chromosome or clinical relapse. A subset of engrafted patients with greater than 25% host cells within 3 months post-BMT had a significantly shorter survival time free of cytogenetic or clinical relapse compared with other patients. In patients who had received donor T cells added to the T cell-depleted graft, there was a higher proportion of complete chimerism. Clonal progression of Ph positive as well as negative cells was observed and may be the result of radiation induced breakage. Serial cytogenetic studies of patients post-BMT can provide useful information regarding the biologic and clinical behavior of CML.
Mechanisms accounting for the defective natural killer activity in patients with hairy cell leukemia
Authors: Trentin L , Zambello R , Agostini C , Ambrosetti A , Chisesi T , Raimondi R , Bulian P , Pizzolo G , Semenzato G
Published: October 2019

Natural killer (NK) cell activity is severely impaired in untreated patients with hairy cell leukemia (HCL). In an attempt to investigate whether this impairment is related to a defect at the target cell binding and/or at the post target cell binding level, we evaluated the peripheral blood mononuclear cells (PBMC) of HCL patients for their ability to: (1) bind and kill K-562 NK-sensitive targets at the single cell binding level; (2) release the NK cytotoxic factor (NKCF) under different in vitro stimuli, including K-562 and phytohemoagglutinin; and (3) kill K-562 targets in a lectin-dependent cellular cytoxicity (LDCC) assay. This study demonstrates that untreated HCL patients' PBMC show a low ability to form conjugates with K-562 targets at the single cell binding level (5.7% +/- 1.0%) with respect to patients studied after treatment (9.3% +/- 1.3%) and controls (15.0% +/- 4.0%); P less than .05 and P less than .001, respectively. A decreased ability to kill the bound target was demonstrated in untreated cases (1.2% +/- 1.1%) versus patients studied after treatment and controls (12.3% +/- 1.6%, 17.0% +/- 3.1% respectively); P less than .001 in both conditions. After activation of effector cells with interleukin-2 (IL- 2) in vitro, an increase in the ability of PBMC to form conjugates with K-562 targets and kill the bound target was demonstrated in each group of patients. Moreover, IL-2 was able to increase the cytotoxicity against NK-sensitive targets in all patients tested. Evaluation of NKCF production showed that untreated patients release low levels of NKCF when PBMC were incubated in the presence of K-562 stimulators (1.8% +/- 0.7%) with respect to patients after interferon-alpha (IFN-alpha) therapy (7.6% +/- 2.1%) and controls (12.9% +/- 2.2%); P less than .02 and P less than .001, respectively. When the recognition mechanisms were bypassed by triggering the cells with lectins in an LDCC assay, we demonstrated an increase of the lytic activity in both groups of patients with respect to the baseline values. However, the cytotoxic capacity observed in untreated patients was significantly lower than that observed in subjects after IFN-alpha therapy and controls (P less than .001). These findings suggest that the impaired NK activity observed in patients with HCL is related to defects both at the target and posttarget cell binding levels.
CD28 ligation in T-cell activation: evidence for two signal transduction pathways
Authors: Ledbetter JA , Imboden JB , Schieven GL , Grosmaire LS , Rabinovitch PS , Lindsten T , Thompson CB , June CH
Published: October 2019

The CD28 homodimer is thought to function as a signal transducing receptor during activation of T cells. Evidence is presented that the degree of aggregation of CD28 on the cell surface regulates two distinct CD28-associated signals. Binding of bivalent CD28 monoclonal antibody (MoAb) 9.3 upregulates lymphokine production by messenger RNA (mRNA) stabilization, without direct initiation of lymphokine mRNA transcription. This signal was not dependent on inositol phospholipid production or activation of a protein tyrosine kinase (PTK). In contrast, further crosslinking of CD28 on the cell surface rapidly induced formation of large amounts of inositol trisphosphate (InsP3) and increased cytoplasmic calcium concentration [( Ca2+]i), but did not stimulate PTK. CD28 crosslinking directly activated a subset of resting T cells, since CD25 (interleukin [IL]-2 receptor alpha chain) mRNA was rapidly induced in purified T cells, and proliferation, even without addition of exogenous IL-2, was sometimes observed. CD25 expression was detected on the cell surface of approximately 20% of CD4+ T cells. The degree of CD28 aggregation required for activation was investigated by preparing soluble 9.3 x 9.3 conjugates ranging in size from approximately 300 Kd to greater than 1,000 Kd, and comparing their function in T-cell proliferation assays with phorbol-12-myristate-13- acetate (PMA), anti-CD3, or IL-2. There was a correlation between conjugate size and proliferation with IL-2, whereas costimulation with PMA or CD3 was optimized at a lower degree of CD28 aggregation. The inositol phospholipid (InsP) generation and increase in [Ca2+]i after CD28 receptor aggregation appeared to proceed through a pathway different from the CD3/T-cell receptor (TCR) pathway since it was enhanced by pretreatment with PMA, while the InsP and [Ca2+]i signal from crosslinking CD3 was suppressed by PMA. Furthermore, the proliferation response to CD28 aggregation was resistant to inhibition by CD3 modulation. Thus, CD28 aggregation appears to trigger a phospholipase C activation pathway that differs from the CD3/TCR-linked pathway.
Prognostic value of lymphocyte homing receptor and S phase fraction in non-Hodgkin's lymphoma
Authors: Jalkanen S , Joensuu H , Klemi P
Published: October 2019

Lymphocyte homing receptors (HRs) mediate lymphocyte binding to high endothelial venules, and control their circulation between the blood and the lymphoid organs. The role of HRs and nuclear DNA content in the spread and prognosis of non-Hodgkin's lymphoma was studied from paraffin-embedded tumor sections of 104 patients followed-up for the minimum of 5 years after the diagnosis. HR expression was analyzed by staining with a monoclonal antibody, Hermes-3, and DNA content by flow cytometry. Ten (10%) lymphomas were HR negative (HR-), 14 (13%) weakly (HR+/-), and 80 (77%) strongly positive (HR+). HR- lymphomas disseminated less often than HR+/- or HR+ lymphomas (P = .03), and their prognosis was more favorable (P = .03), although they often had a large S phase fraction (SPF), indicating a rapid proliferation rate. A large SPF (greater than 12%) was strongly associated with an unfavorable histologic type in Working Formulation (P = .0001) and poor survival (P = .006), whereas DNA aneuploidy was not. The 5-year survival rate corrected for intercurrent deaths was 61% in lymphomas with SPF less than 12% or with HR-, but only 15% if SPF was greater than 12% and HR+ (P less than .0001). In multivariate analysis stage (P less than .001), SPF (P = .002) and HR (P = .003) were the only independent prognostic factors.
Actions of human interleukin-4/B-cell stimulatory factor-1 on proliferation and differentiation of enriched hematopoietic progenitor cells in culture
Authors: Sonoda Y , Okuda T , Yokota S , Maekawa T , Shizumi Y , Nishigaki H , Misawa S , Fujii H , Abe T
Published: October 2019

We studied the effects of recombinant human interleukin-4 (IL-4) on colony formation by enriched hematopoietic progenitors. IL-4 alone did not support colony formation at all. When IL-4 was combined with granulocyte colony-stimulating factor (G-CSF), the number of pure neutrophil colonies increased three times over that supported by G-CSF alone. IL-4 added 5 days after the addition of G-CSF failed to exert this synergistic effect, indicating that IL-4 acts on the early stage of proliferation. The mapping experiments (sequential observation of colony formation) have clearly shown that IL-4 did not initiate progenitor cell proliferation. Based on these data, IL-4 may possess a direct action on progenitor cells; however, it can only act as a costimulant with G-CSF. In contrast, IL-4 had possible inhibitory effects on macrophage colony formation supported by interleukin-3 (IL- 3) and macrophage colony-stimulating factor (M-CSF). In other words, IL- 4 may induce progenitor cells to become sensitive to G-CSF and thereby induce neutrophil differentiation. Delayed addition experiments demonstrated that human IL-4, unlike murine IL-4, could support neither proliferation nor survival of erythroid burst or mixed colony forming cells. Neutrophil colony forming cells only survived and recovered after addition of G-CSF and erythropoietin on day 5 of incubation. On the other hand, IL-3 supported neutrophil, erythroid burst, and mixed colony forming cells as reported previously (Sonoda et al, Proc Natl Acad Sci USA, 85:4360, 1988). These results led us to propose that IL-4 possibly acts with more mature progenitor cells than those of IL-3 or granulocyte-macrophage (GM)-CSF.
Recombinant tumor necrosis factor enhances the proliferative responsiveness of murine peripheral macrophages to macrophage colony- stimulating factor but inhibits their proliferative responsiveness to granulocyte-macrophage colony-stimulating factor
Authors: Chen BD , Mueller M
Published: October 2019

Tumor necrosis factor (TNF) is a protein produced by activated macrophages in response to endotoxin. The effect of recombinant murine TNF (rMuTNF) on the growth of murine tissue-derived macrophage colony- forming units (CFU-M) which are responsive to both macrophage and granulocyte-macrophage colony-stimulating factors (M-CSF and GM-CSF), was studied. TNF alone did not stimulate macrophage proliferation but did prolong their survival in vitro. The proliferative response of CFU- M to M-CSF, however, was greatly enhanced by the presence of TNF. The enhancement effect of TNF is dose-dependent, reaching a maximum at approximately 50 U/mL. In contrast, the proliferative responsiveness of CFU-M to GM-CSF was inhibited by the concurrent addition of rMuTNF. Both effects appear to be caused directly by rMuTNF, rather than by the secondary factor(s) produced by TNF-treated macrophages. TNF treatment also induced a transient downmodulation of M-CSF receptors in cultured macrophages and accelerated their uptake and use of exogenous M-CSF, which may account for, at least in part, the enhanced proliferative activity in response to M-CSF. Short-term treatment (24 hours) was not sufficient to induce either an enhancing or an inhibitory effect upon CFU-M. This study suggests an autoregulatory role for TNF in the production of mature tissue macrophages by selectively enhancing their proliferative response to lineage specific growth factor, M-CSF.
Purification and characterization of factor VIII 372-Cys: a hypofunctional cofactor from a patient with moderately severe hemophilia A
Authors: O'Brien DP , Pattinson JK , Tuddenham EG
Published: October 2019

We have purified factor VIII from a patient with moderately severe hemophilia A (FVIII, 4 U/dL; FVIII:Ag, 110 U/dL) and subjected the protein to Western blot analysis after time course activation with thrombin. The cross reacting material-positive (CRM+) FVIII has the normal distribution of heavy and light chains before thrombin activation, and, after incubation with the enzyme, appropriate cleavages are made at positions 740 and 1689. However, the normal thrombin cleavage at position 372 in the heavy chain of this molecule does not occur. This result is consistent with the demonstration in the patient's leukocyte DNA of a C to T transition in codon 372, leading to the substitution of a cysteine for an arginine residue at the heavy chain internal cleavage site. The severely impaired functional activity of this molecule confirms that the heavy chain of FVIII must be proteolysed in order to effect full cofactor activation in vivo. However, a threefold activation was detected when this protein was incubated with thrombin. No evidence of thrombin-mediated cleavage at position 336 in the heavy chain was detected, in contrast to the variant recombinant B domainless-molecule, FVIII 372-Ile, described by Pittman and Kaufman (Proc Natl Acad Sci USA 85:2429, 1988). Using gel permeation studies of the FVIII/von Willebrand factor (vWF) complex before and after thrombin activation, we have demonstrated that the 40 Kd A2 domain of wild type FVIII dissociates from vWF after cleavage by the enzyme. In contrast, incomplete dissociation was detected in the case of FVIII 372-Cys. We conclude that the functional defect in FVIII 372-Cys is a consequence of the resistance to proteolysis of the internal scissile bond in the heavy chain.
Reticulocytes II: Reexamination of the in vivo survival of stress reticulocytes
Authors: Noble NA , Xu QP , Hoge LL
Published: October 2019

Very young reticulocytes are released into the circulation in response to the stress of anemia. These stress reticulocytes have shortened in vivo survival when transfused into normal recipients, and are generally considered to be abnormal because they have skipped a terminal cell division. We reevaluated one aspect of their abnormality: that of in vivo survival. Using methodology that accounted for all cells transfused, in vivo survival of both normal and stress reticulocytes was investigated in both normal and anemic recipients. The experiments demonstrate that: (1) survival of reticulocytes is normal only when normal reticulocytes are injected into nonanemic animals; (2) intrinsic properties of stress reticulocytes lead to their immediate removal from the circulation by normal recipients to a significantly greater extent than by anemic recipients; and (3) both stress and normal reticulocytes are removed at an accelerated rate over time by anemic recipients. Taken together, the data indicate that in the course of becoming anemic, an adaptation occurs that allows cells produced during anemia to circulate considerably longer in anemic animals than they could in normal nonanemic animals. Other studies disclosed that increased reticulocyte survival in anemic animals could not be attributed to reticuloendothelial overload, but is induced by adaptation of the spleen, decreasing its removal of stress reticulocytes.
Poor prognosis of children with pre-B acute lymphoblastic leukemia is associated with the t(1;19)(q23;p13): a Pediatric Oncology Group study
Authors: Crist WM , Carroll AJ , Shuster JJ , Behm FG , Whitehead M , Vietti TJ , Look AT , Mahoney D , Ragab A , Pullen DJ
Published: October 2019

The prognostic significance of chromosomal translocations, particularly t(1;19) (q23;p13), was evaluated in children with pre-B and early pre-B acute lymphoblastic leukemia (ALL). Patients were treated on a risk- based protocol of the Pediatric Oncology Group (POG) between February 1986 and May 1989. An abnormal clone was detected in 46% (130 of 285) of pre-B cases and 56% (380 of 679) of early pre-B cases. Translocation of any type was associated with a worse treatment outcome than other karyotypic abnormalities: 15 of 66 versus 3 of 64 failed therapy in the pre-B group (P = .001), and 37 of 141 versus 23 of 239 failed in the early pre-B group (P less than .001). The t(1;19) (q23;p13) occurred significantly more often in cases of pre-B ALL with a clonal abnormality than in early pre-B ALL cases (29 of 130 v 5 of 380, P less than .001). Among the 285 pre-B cases in which bone marrow was studied cytogenetically, those with t(1;19) had a significantly worse treatment outcome than all others (11 of 29 v 27 of 256 have failed therapy, P less than .001). This difference is significant (P less than .001) after adjustment for leukocyte count, age, and other relevant features. Cases with the t(1;19) also had a worse prognosis than pre-B patients with other translocations (4 of 37 have failed, P less than .01) or with any other karyotypic abnormality (7 of 101 have failed, P less than .001). We conclude that chromosomal translocations confer a worse prognosis for non-T, non-B-cell childhood ALL, and that the t(1;19) is largely responsible for the poor prognosis of the pre-B subgroup.
Direct sequence analysis of the 14q+ and 18q- chromosome junctions in follicular lymphoma
Authors: Cotter F , Price C , Zucca E , Young BD
Published: October 2019

Although the t(14;18) chromosome translocation has been demonstrated to be a highly consistent feature of follicular lymphomas, the underlying mechanism generating this fusion has remained uncertain. To examine this question further, a polymerase chain reaction strategy has been devised to permit the amplification and direct sequencing of the resultant 14q+ and 18q- reciprocal junctions. Direct sequence analysis of amplified 14q+ junctions established that 7 of 11 tumors contained a bcl-2 (mbr) sequence fused to an immunoglobulin JH region (five were J6 and two were J5). One of these junctions had an unusual configuration with the bcl-2 and JH sequences separated by a recognizable DH region. This finding suggests that at least some of the junctional sequences, previously thought of as N insertions, may be fragments of unrecognized DH regions. It was also possible to amplify and sequence 18q- junctions using a primer based on the DH recombination signal sequences. Several 18q- junctions were shown to consist of DH/bcl-2 (either mbr or mcr) fusions. In two tumors the 14q+ and 18q- junctions were fully sequenced, and it was demonstrated that the bcl-2 sequence was conserved during mbr and mcr translocations. This contrasts with previous analyses that demonstrated either loss or duplication of several bases at the breakpoints in the bcl-2 gene.
Association between clonogenic cell growth and clinical risk group in B- cell chronic lymphocytic leukemia
Authors: Dadmarz R , Rabinowe SN , Cannistra SA , Andersen JW , Freedman AS , Nadler LM
Published: October 2019

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a disease with a variable clinical course, despite the fact that the neoplastic cells in this disorder are homogeneous with respect to morphology, immunophenotype, and cell cycle stage. To further investigate the heterogeneity observed in the clinical behavior of B-CLL, we determined the phenotype and growth requirements of clonogenic cells from 28 patients with B-CLL from low-, intermediate-, and high-risk groups as defined by the Rai staging system. Using methyl-cellulose as a semi- solid media with feeder cells and/or growth factors, colonies were observed with one or more of the culture conditions tested in 25 of 28 CLLs. Phenotypic analysis of colonies demonstrated that the clonogenic cells uniformly expressed la, CD19, CD20, CD5, and the identical light chain as the original CLL cell cultured. However, heterogeneity was observed in clonogenic B-CLL cell growth among the three different CLL risk groups. Clonogenic cells from patients with low-risk CLL required either irradiated unstimulated T cells, with or without conditioned media (CM) or irradiated activated T cells alone for colony formation. Both the number of colonies (227 +/- 15) as well as the number of cells per colony (220 +/- 82) were large, with a mean cloning efficiency of 0.39%. In contrast, clonogenic cells from patients with intermediate- and high-risk CLL required the combination of both irradiated activated T cells and CM. As compared with the low-risk CLLs, both the number and size of the colonies formed by the intermediate- (74 +/- 17, 70 +/- 39) and high- (83 +/- 28, 40 +/- 14) risk groups were significantly lower (P less than .0001). Similarly, the mean cloning efficiency was significantly reduced to 0.15% and 0.14%, respectively. None of the recombinant cytokines (interleukin 1 [IL-1] to IL-7, tumor necrosis factor, alpha and gamma-interferon, B-cell growth factor, and granulocyte macrophage colony-stimulating factor) alone or in combination with each other could entirely replace the stimulatory effect of the activated T cells. These data suggest that clinical progression of B-CLL is associated with a loss of clonogenic potential in the circulating pool of neoplastic cells, which require as yet undefined factors provided by activated T cells and CM.
Detection of Epstein-Barr virus genome in benign polyclonal proliferative T cells of a young male patient
Authors: Yoneda N , Tatsumi E , Kawanishi M , Teshigawara K , Masuda S , Yamamura Y , Inui A , Yoshino G , Oimomi M , Baba S
Published: October 2019

Epstein-Barr virus (EBV) DNA was detected in polyclonal T cells that proliferated transiently in a 21-year-old male (referred to as H.J.) who underwent an apparently benign lymphocytosis (white blood cells, 31 x 10(6)/microL; lymphocyte, 79%) with fever, tonsillar swelling, lymphadenopathy, and hepatosplenomegaly. The symptoms and signs subsided mostly within a month of hospitalization. The major population of the lymphocytes at admission was positive for CD3, CD8 (4/8 ratio, 0.16), WT31, and DR antigen. Eight percent of the leukocytes were too blastoid to be classified as atypical lymphocytes of infectious mononucleosis (IM). The blastoid lymphocytes and the duration and degree of the lymphocytosis and hypergammaglobulinemia appeared inconsistent with IM, whereas the EBV serology indicated either EBV primary infection or a secondary alteration of normal seropositive EBV immunity. The genomic analysis of T-cell receptor beta chain in the peripheral blood mononuclear cells (PBMC) at admission with a C beta probe did not show a monoclonal rearrangement. EBV genome was detected in these cells, using the BamHI W and K probe, but not in the cells after discharge. Analysis of the EBV terminal repeat junctional sequence, using Xho I fragment of the latent membrane protein (LMP) probe binding with the terminus, did not show monoclonal or oligoclonal populations. EBV-associated nuclear antigen (EBNA) was detected in 36% of the PBMC at admission, but not in the later cells. These EBNA- positive cells were found to form rosette with sheep erythrocytes. The PBMC of six acute IM patients contained neither EBV DNA nor EBNA- positive cells. The observations in this case show a unique type of EBV infection in T cells that has not been previously reported.
Altered expression of neutrophil peripheral benzodiazepine receptor in X-linked chronic granulomatous disease
Authors: Zavala F , Veber F , Descamps-Latscha B
Published: October 2019

This study was aimed at determining whether the peripheral benzodiazepine receptor (PBZDR), which is abundantly expressed on mononuclear phagocytes, is involved in host defense mechanisms depending on phagocyte membrane-associated NADPH-oxidase complex. Analysis by reversible and covalent binding of PBZDR expression on human neutrophils shows that it is modulated during NADPH-oxidase activation with phorbol 12-myristate 13-acetate. Based on a series of 17 patients with chronic granulomatous disease (CGD), results show that PBZDR expression is dramatically impaired in X-linked CGD, an inherited disorder due to a mutation on the gene coding for cytochrome b558 NADPH- oxidase component, whereas it is unaffected in autosomal recessive CGD where cytochrome b558 is normally expressed, suggesting a link between PBZDR and cytochrome b558 expressions. PBZDR can be assigned by covalent binding to an 18-Kd membrane protein. These results suggest that the neutrophil PBZDR, which can accommodate the widely prescribed anxiolytic drug Valium (diazepam), is involved in host defense against pathogens, a function that could be affected by neuroimmune interactions.
Deoxygenation-induced cation fluxes in sickle cells: II. Inhibition by stilbene disulfonates
Authors: Joiner CH
Published: October 2019

Deoxygenation-induced cation movements in sickle cells were inhibited 80% to 85% by the anion transport inhibitor, 4,4′-diisothiocyano- 2,2′disulfostilbene (DIDS). Morphologic sickling was not altered by DIDS treatment, demonstrating that morphologic sickling was not sufficient to produce cation leaks in sickle cells. DIDS inhibition of deoxygenation-induced cation flux was not affected when l- replaced Cl- , indicating that conductive anion movements did not limit cation flux in deoxygenated cells treated with DIDS. Inhibition was irreversible after preincubation with DIDS at 37 degrees C for 20 minutes, and was not affected by the oxygenation state of cells at the time of drug exposure. Sulfate self-exchange was inhibited at lower DIDS concentrations than was deoxygenation-induced flux. Incubation of cells with DIDS at 4 degrees C produced progressive blockade of sulfate exchange, but did not alter deoxygenation-induced cation fluxes. Other stilbene disulfonates, including compounds incapable of covalent reactions, also inhibited deoxygenation-induced cation movements, although several other inhibitors of anion exchange did not. Dissociation of the inhibition of anion exchange and deoxygenation- induced cation flux indicates that the DIDS effect on deoxygenation- induced cation movements does not involve the well-characterized stilbene binding site of the anion exchanger. These data provide evidence for a membrane constituent on the external surface of oxygenated sickle cells capable of interacting with DIDS to prevent the increase in cation permeability associated with sickling.
Value of day 100 screening studies for predicting the development of chronic graft-versus-host disease after allogeneic bone marrow transplantation
Authors: Loughran TP Jr , Sullivan K , Morton T , Beckham C , Schubert M , Witherspoon R , Sale G , Sanders J , Fisher L , Shulman H
Published: October 2019

We prospectively evaluated 169 patients with a number of screening studies performed between 71 to 121 days after allogeneic marrow transplantation to detect the development of chronic graft-versus-host disease (GVHD). Group 1 patients (n = 78) were asymptomatic and had normal physical examinations at the time of screening and, with a minimum of 8 years follow-up, have not developed chronic GVHD. Group 2 patients (n = 38) had signs and symptoms of chronic GVHD at time of testing. Group 3 patients (n = 53) were similar to those in group 1 in having no clinically evident GVHD at the time of testing, but later developed clinical chronic GVHD. Using time to an event analysis, we compared patients in groups 1 and 3 to determine which of 17 clinical and laboratory factors evaluated at screening accurately predicted the development of subsequent chronic GVHD. Multivariate analyses showed several factors to have independent predictive value. In the first model, results of oral biopsies were excluded since these were done only in one half of the patients. Predictive factors in this analysis included: (1) histologic findings of GVHD on skin biopsy, relative risk 3.23 (95% confidence interval 1.75 to 5.94), P = .0002; and (2) history of grade II through IV acute GVHD, relative risk 3.12 (95% confidence interval 1.72 to 5.64), P = .0002. When oral biopsy results were included in the second model, independent risk factors included: (1) histologic findings of GVHD on skin biopsy, relative risk 5.96 (95% confidence interval 1.95 to 18.19), P = .0017; and (2) low numbers of immunoglobulin A (IgA)-bearing plasma cells detected by direct immunofluorescence in salivary gland areas on oral biopsy, relative risk 11.53 (95% confidence interval 2.51 to 52.03), P = .0017. Our study demonstrates the value of day 100 screening studies for predicting subsequent development of clinical chronic GVHD.
Accumulation of methotrexate and methotrexate polyglutamates in lymphoblasts at diagnosis of childhood acute lymphoblastic leukemia: a pilot prognostic factor analysis
Authors: Whitehead VM , Rosenblatt DS , Vuchich MJ , Shuster JJ , Witte A , Beaulieu D
Published: October 2019

Lymphoblasts in bone marrow samples, obtained from 43 children with acute lymphoblastic leukemia at diagnosis, were incubated with 1.0 mumols/L [3H] methotrexate for 24 hours in vitro. Nonexchangeable methotrexate and methotrexate polyglutamates were separated and quantitated. Event-free survival at 5 years was 38% +/- 9% for all 43 patients (27 failures), and 44% +/- 10% for the 35 with non-T, non-B- cell acute lymphoblastic leukemia (20 failures). Of these 35 children, those whose lymphoblasts accumulated more than 100 pmol methotrexate and 500 pmol methotrexate polyglutamates per billion cells experienced better 5-year event-free survival than those whose lymphoblasts did not (65% +/- 12% v 22% +/- 9%, P = .010). This difference characterized “good-risk” patients who were female (P = .014), less than age 7 at diagnosis (P = .005), or had low initial white blood cell counts (less than 20 X 10(9)/L, P = .018). Findings were similar for the 43 children with acute lymphoblastic leukemia and for the “good-risk” children in this total group. Thus, the ability of lymphoblasts to accumulate methotrexate and form methotrexate polyglutamates may be important to the curative properties of current therapy of acute lymphoblastic leukemia in children, particularly for “good-risk” patients. In such patients, inherent rather than acquired drug resistance may be the initial event leading to treatment failure.