bluuebunny/crossref_metadata_2025 huggingface dataset:
Response assessment in chronic lymphocytic leukemia after fludarabine plus prednisone: clinical, pathologic, immunophenotypic, and molecular analysis [see comments]
Authors: Robertson LE , Huh YO , Butler JJ , Pugh WC , Hirsch-Ginsberg C , Stass S , Kantarjian H , Keating MJ
Published: October 2019

The goals of this study were to evaluate the response to treatment in chronic lymphocytic leukemia (CLL) according to clinical, pathologic, immunophenotypic, and molecular features, as well as to address the clinical significance of each finding. One hundred fifty-nine CLL patients with either advanced Rai stage III or IV (81 patients) or progressive Rai stage 0 to II (78 patients) were treated with fludarabine (30 mg/m2/d intravenously every day for 5 days) plus prednisone (30 mg/m2/d orally daily for 5 days). Thirty-six patients were previously untreated. The response rates were 12% complete response (CR), 30% nodular complete response (nCR), and 18% partial response (PR). In all patients who achieved a complete response (both CR and nCR) less than 30% of nucleated cells were lymphocytes on marrow aspirate differential analysis; however, nCR patients had residual nodular and/or interstitial lymphocyte involvement on marrow biopsy examination. There was no evidence of leukemic infiltration on marrow biopsy examination in CR patients. With a median follow-up of 35 months, comparison of time to progression in the CR and nCR groups at 2 years showed a projected 87% versus 55% progression-free survival (P less than .03). Residual disease assessment by flow cytometry using simultaneous dual-color staining on blood and marrow lymphocytes was also performed on each patient. Residual disease was determined by the expression of CD5 on B lymphocytes and the monoclonality of surface light-chain expression. After six courses of fludarabine plus prednisone, no residual disease was detected by flow cytometry in 89% of the CRs, 51% of the nCRs, and 19% of the PRs. Clinical residual disease in PR patients with no residual disease detectable by flow cytometry was limited to lymph-adenopathy. Time to progression at 2 years was longer in CR and nCR patients having no residual disease detected by flow cytometry (84% v 39% 2-year progression-free survival, P less than .001). Posttreatment lg gene rearrangement analysis using JH, J kappa, and C lambda probes demonstrated no rearranged bands and a return to the germline configuration in five of seven CRs and two of eight nCRs studied. The molecular studies were concordant with the dual- parameter immunophenotype results and none of the patients who reverted to a germline DNA pattern after treatment have experienced relapse. The absence of detectable minimal residual disease by bone marrow biopsy, dual-color flow cytometry, and lg gene rearrangement analysis is achieveable in CLL with fludarabine and is predictive of the response duration.
Erythropoietin rapidly induces tyrosine phosphorylation in the human erythropoietin-dependent cell line, UT-7
Authors: Komatsu N , Adamson JW , Yamamoto K , Altschuler D , Torti M , Marzocchini R , Lapetina EG
Published: October 2019

UT-7 is a human megakaryoblastic cell line capable of growing in interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (Epo) (Cancer Res 51:341, 1991). We used this cell line and a selected Epo-dependent subcell line (UT-7/Epo) to study the early signal transduction events induced by Epo. When UT-7 cells were exposed to Epo, tyrosine phosphorylation of several proteins (with molecular weight equivalent to that of p85, p110, and p145) was observed. Protein phosphorylation occurred in both a dose- and time-dependent manner. p85 showed a marked increase in phosphotyrosine content within 30 seconds; maximal phosphorylation was observed at 1 minute. Subsequently, tyrosine phosphorylation of p110 and p145 was observed, beginning at 1 minute and reaching plateau at 5 minutes. The degree of phosphorylation of these three proteins gradually decreased thereafter. In addition, in UT-7/Epo cells, Epo induced tyrosine phosphorylation of other proteins that were not observed in Epo-induced UT-7 cells. The concentration of Epo required to induce tyrosine phosphorylation was in the same range of concentration required to stimulate cell growth. Epo was also able to activate p21ras as measured by exchange of guanosine diphosphate for guanosine triphosphate. These data show that tyrosine phosphorylation and P21ras activation are early signals in the Epo-induced mitogenic pathway.
Blasts from patients with acute myelogenous leukemia express functional receptors for stem cell factor
Authors: Broudy VC , Smith FO , Lin N , Zsebo KM , Egrie J , Bernstein ID
Published: October 2019

Stem cell factor (SCF) acts in concert with lineage-specific growth factors to stimulate the growth of hematopoietic colonies. To determine if neoplastic human hematopoietic cells would also respond to SCF, we cultured marrow mononuclear cells from 20 patients with newly diagnosed acute myelogenous leukemia (AML) and two normal donors with SCF, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or combinations of growth factors in semisolid medium, and assessed colony growth. SCF receptors (c-kit receptors) were quantitated by equilibrium binding studies with 125I-SCF, and binding parameters were estimated using the ligand program. The cellular distribution of c-kit receptors was determined by autoradiography. Our results show that SCF alone or in combination with IL-3 or GM-CSF increased both the number and size of colonies in 10 of the patients. Receptors for SCF were identified on the blasts from all 20 AML patients. The number of receptors ranged from 600 to 29,000 per cell. In the majority of patients, both high- and low-affinity binding sites were identified. Neither the number of receptors per cell nor the finding of one or two classes of receptors correlated with growth response to SCF. Autoradiographic analysis of 125I-SCF binding to normal marrow mononuclear cells revealed grains associated with blasts and megakaryocytes. Grain counts on blasts from 10 AML patients and on normal marrow blasts suggested that high-affinity c-kit receptor expression on AML blasts is lower than or similar to that of normal blasts. These results identify c-kit receptors on human AML blasts, and indicate that SCF acts synergistically with IL-3 or GM-CSF to stimulate colony growth from the marrow cells of a portion of patients with AML.
Separation of hematopoietic stem cells into two populations and their characterization
Authors: Ogata H , Taniguchi S , Inaba M , Sugawara M , Ohta Y , Inaba K , Mori KJ , Ikehara S
Published: October 2019

Two populations of hematopoietic stem cells (HSC) in mouse bone marrow (BM) are defined on the basis of the presence or absence of interleukin- 3 (IL-3) receptor-associated antigen (IL-3RAA). HSC were purified by depletion of mature lymphoid-lineage cells followed by collection of the low-density fraction and sorting of wheat germ agglutinin-binding (WGA+) cells using a fluorescein-activated cell sorter. WGA+ cells were further separated into two populations (IL-3RAA+/WGA+ and IL-3RAA- /WGA+) by a monoclonal antibody (MoAb) against IL-3RAA. IL-3RAA+/WGA+ cells formed CFU-S on day 8; this population consisted mainly of cells in the cycling phase. IL-3RAA-/WGA+ cells form CFU-S on day 12; this population consisted mainly of dormant cells (cells in the G0 phase). When two populations obtained from C3H/HeN mice were injected into lethally irradiated (C57BL/6 x C3H/HeN)F1 mice, donor-derived cells in the peripheral blood (PB) appeared significantly earlier in mice injected with IL-3RAA+/WGA+ cells than in those injected with IL-3RAA- /WGA+ cells, whereas the reconstruction efficiency of IL-3RAA-/WGA+ cells had overtaken that of IL-3RAA+/WGA+ cells 6 weeks after injection. Long-term observation showed no significant difference between these two populations, however. The radioprotective ability (RPA) (30-day survival) of these two populations was therefore compared. The RPA of IL-3RAA-/WGA+ cells was significantly higher than that of IL-3RAA+/WGA+ cells. These findings therefore suggest that the former population is more primitive.
Differential effects of sequential, simultaneous, and single agent interleukin-3 and granulocyte-macrophage colony-stimulating factor on megakaryocyte maturation and platelet response in primates
Authors: Stahl CP , Winton EF , Monroe MC , Haff E , Holman RC , Myers L , Liehl E , Evatt BL
Published: October 2019

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) following interleukin-3 (IL-3) priming has been shown to increase thrombopoiesis. To elucidate the comparative abilities of IL-3 and GM- CSF in influencing megakaryocyte development in vivo, serial bone marrow analyses were performed on rhesus monkeys treated with 5 micrograms/kg/d of IL-3 and 5 micrograms/kg/d of GM-CSF sequentially for 4 days each, simultaneously for 8 days, and as single agents for 8 days. Platelet counts maximally increased to a mean of 7.5 x 10(5)/microL (n = 3) on days 11 through 12 in monkeys treated with sequential IL-3/GM-CSF. In contrast, neither IL-3 alone nor simultaneously administered IL-3/GM-CSF elicited increases in thrombopoiesis between days 3 and 15. GM-CSF elicited a variable platelet response. Megakaryocyte ploidy distributions were significantly (P < .001) shifted between days 7 and 10 in monkeys treated sequentially and between days 3 and 15 in monkeys treated with combined IL-3/GM-CSF and with GM-CSF alone but not in monkeys treated with IL-3 alone. The changes in mean DNA content and megakaryocyte size, as determined by digital image analysis, were larger in monkeys treated with sequential IL-3/GM-CSF and with GM-CSF alone than in simultaneously treated monkeys. In addition, sequentially but not simultaneously treated monkeys showed increased numbers of megakaryocytes on bone marrow biopsy. We conclude that administration of IL-3 followed by GM-CSF treatment increases thrombopoiesis by sequentially increasing megakaryocyte numbers and maturation and that these effects are diminished by simultaneous administration of the two cytokines.
Retinoic acid counteracts both the downregulation of thrombomodulin and the induction of tissue factor in cultured human endothelial cells exposed to tumor necrosis factor
Authors: Ishii H , Horie S , Kizaki K , Kazama M
Published: October 2019

Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) shift the hemostatic balance of endothelial cell surfaces in favor of prothrombotic properties by downregulating thrombomodulin (TM) and inducing tissue factor (TF) expression. We investigated the effects of retinoic acid (RA) on the prothrombotic properties of cultured umbilical vein endothelial cells exposed to TNF-alpha. The approximate 50% downregulation of TM antigen and cofactor activity induced by TNF- alpha (10 U/mL for 24 hours) was completely prevented when the cells were coincubated with both TNF-alpha and 10 mumol/L RA. In accordance with changes in cell surface TM antigen levels, the 70% decrease in TM messenger RNA (mRNA) induced by TNF-alpha was also prevented by 10 mumol/L RA. TNF-alpha induced TF activity of lysed cells (100-fold greater than untreated controls), an effect prevented when the cells were coincubated with both the TNF-alpha and 10 mumol/L RA. The 34-fold increase in TF mRNA levels induced by TNF-alpha (10 U/mL for 3 hours) was only two-fold in the presence of both TNF-alpha and RA. The effects of RA on the regulation of TM and TF expression in the cells exposed to TNF-alpha was dose-dependent from 0.01 to 10 mumol/L RA. The present results suggest that RA may affect on the mRNA level to alter TM and TF expression, effectively counteracting expression of prothrombotic properties of endothelial cells induced by inflammatory cytokines such as TNF-alpha.
Cell lines produce factors that induce fetal hemoglobin in human BFUe- derived colonies
Authors: Constantoulakis P , Walmsley M , Patient R , Papayannopoulou T , Enver T , Stamatoyannopoulos G
Published: October 2019

Established cell lines were screened for secretion of activities than can stimulate fetal hemoglobin (HbF) production in adult burst-forming unit-erythroid (BFUe) cultures. Conditioned media from four cell lines, a human teratocarcinoma, an osteosarcoma, a bladder cell carcinoma, and feline leukemia virus (FeLV) A-infected feline fibroblasts (FEF-A cells), consistently increased the relative production of fetal globin in BFUe-derived colonies. In vitro translation of RNA from these cells in Xenopus oocytes yielded products that increased the gamma to gamma+beta ratio in adult erythroid colonies. These results demonstrate that a variety of cell lines produce factors that stimulate the production of HbF in vitro. The genes of such factors could be isolated by expression cloning of cDNA from cell lines using the Xenopus oocyte system.
Circulating cytomegalovirus (CMV) neutralizing activity in bone marrow transplant recipients: comparison of passive immunity in a randomized study of four intravenous IgG products administered to CMV-seronegative patients
Authors: Filipovich AH , Peltier MH , Bechtel MK , Dirksen CL , Strauss SA , Englund JA
Published: October 2019

Forty-two cytomegalovirus (CMV)-seronegative bone marrow transplant (BMT) recipients were randomized in a double-blind fashion to receive one of four commercially available intravenous Ig (IVIgG) products (Gamimmune N, Immune Globulin Intravenous, Gammagard, or Sandoglobulin) at a dose of 500 mg/kg every other week. The four treatment groups were similar in distribution of patient ages, weights, autologous versus allogeneic donor type, and underlying diseases. Every other week administration of IVIgG provided total serum IgG levels within the physiologic range for age. CMV titers by latex agglutination were stable (average geometric mean titer of 18.4 after the second IVIgG dose), with no statistically significant differences among the four product groups. CMV neutralizing activity (CMVNA) and CMV enzyme-linked immunosorbent assay (ELISA) titers were determined on a subset of sera from 27 study patients representing the four product groups. Patient serum samples obtained before IVIgG infusions and 2 weeks after the second IVIgG dose (ie, 3 weeks post-BMT) were assayed for CMVNA and CMV ELISA titers. Geometric mean titers of CMVNA and CMV ELISA varied among the product groups. The highest mean 50% CMVNA was 1:43 for product B, whereas the lowest mean 50% CMVNA was 1:14 for product A; two of the IVIgG product groups showed intermediate 50% mean titers of 1:27 (product C) and 1:26 (product D) for an overall P = .02. CMV ELISA titers (expressed as Paul Ehrlich International units [PEI U]) also showed the highest mean of 2.95 PEI U/mL for product B and the lowest mean of 1.34 PEI U/mL for product A. Intermediate mean values of 2.27 PEI U/mL and 2.03 PEI U/mL were obtained with products C and D, respectively (overall P = .003). The CMV ELISA titers show a minimal correlation (r = .566) to the observed CMVNA titers. We conclude that commercially available IVIgG products provide passive CMVNA, and that the level of circulating CMVNA is affected by the IVIgG product used.
Antilymphocytic antibodies and marrow transplantation. XII. Suppression of graft-versus-host disease by T-cell-modulating and depleting antimouse CD3 antibody is most effective when preinjected in the marrow recipient
Authors: Mysliwietz J , Thierfelder S
Published: October 2019

A hamster antimouse CD3 monoclonal antibody (MoAb) opened the way to experimental studies on the suppression of allograft rejection and cytokine-related morbidity after treatment with antibodies modulating the CD3/T-cell receptor complex (CD3/TCR). Because earlier attempts to suppress graft-versus-host disease (GVHD) in patients by in vitro treatment of donor marrow with anti-CD3 MoAb had remained inconclusive, we used a rat IgG2b antimouse CD3 MoAb (17A2) with fewer side effects to analyze suppression of GVHD in the mouse model. Detailed phenotyping of blood, spleen, and lymphnode T cells after the injection of 400 micrograms 17A2 in C57BL/6 mice showed 60% CD3 downmodulation and 50% T- cell depletion for spleen cells. Injection of these spleen cells, together with bone marrow cells, in fully mismatched preirradiated CBA mice delayed GVHD by only 6 days. Ex vivo treatment of donor cells with 17A2 was not effective. In contrast, conditioning of marrow recipients with a single injection of 17A2 delayed 50% GVHD mortality by 100 days and prevented GVHD altogether after prolonged treatment, with survivors showing complete chimerism and specific transplantation tolerance. This difference in antibody effect contrasts with earlier experiences with nonmodulating but more strongly T-cell-depleting MoAbs of the same isotype, which prevent GVHD no matter whether applied in vitro or injected into donor or recipient mice. Our data indicate that CD3/TCR reexpression in marrow recipients with no circulating 17A2 is the reason why ex vivo donor cell treatment with anti-CD3 MoAb is comparatively ineffective. Our data, which allow separate evaluation of cell-depleting and cell-modulating antibody activity, help to explain previous clinical failure to suppress GVHD and provide evidence in favor of conditioning the marrow recipient with anti-CD3 MoAb as a therapeutic alternative.
Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype [see comments]
Authors: Drach D , Zhao S , Drach J , Mahadevia R , Gattringer C , Huber H , Andreeff M
Published: October 2019

The multidrug-resistance gene, MDR1 is expressed in many normal tissues, but little is known about its expression in normal hematopoietic cells. Using the monoclonal antibody C219 and flow cytometric analysis, P-glycoprotein (P-gp) was found to be expressed in all peripheral blood (PB) subpopulations (CD4, CD8, CD14, CD19, CD56) except granulocytes. To specifically determine MDR1 gene expression, these PB subpopulations were isolated by fluorescence-activated cell sorting (FACS) and analyzed for MDR1 mRNA by polymerase chain reaction (PCR). All subsets were positive by PCR, but only minimal MDR1 mRNA was detected in monocytes and granulocytes. Significant efflux of Rhodamine- 123 (Rh-123), a measure of P-gp function, was detected in CD4+, CD8+, CD14+, CD19+, and CD56+ cells but not in granulocytes. Next, PCR- analysis was performed on FACS-sorted bone marrow (BM) cells to assess MDR1 expression in different maturational stages. Precursors (CD34+), early and late myeloid cells (CD33+/CD34+, CD33+/CD34-) as well as lymphocytes of the B-cell lineage (CD19+/CD10+, CD19+/CD10-) expressed the MDR1 gene. BM monocytic cells (CD33++/CD34-) were negative, and a very weak signal was detected in erythroid cells (glycophorin A+). Significant Rh-123 efflux was found in CD34+, CD10+, CD33+, and CD33++ BM cells, but not in glycophorin A+ cells. We conclude that PB and BM lymphocytes, PB monocytes, BM progenitors, and immature myeloid cells, but not late BM monocytes, erythroid cells, and PB granulocytes, express MDR1 mRNA and a functional P-gp. These results have to be taken into account when MDR1 expression is determined in tumor samples containing normal blood cells.
Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome
Authors: van de Kar NC , Monnens LA , Karmali MA , van Hinsbergh VW
Published: October 2019

The epidemic form of the hemolytic uremic syndrome (HUS), beginning with an acute gastroenteritis, has been associated with a verocytotoxin- producing Escherichia coli infection. The endothelial cell is believed to play an important role in the pathogenesis of HUS. Endothelial cell damage by verocytotoxin-1 (VT-1) in vitro is potentiated by the additional exposure of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). Preincubation of human umbilical vein endothelial cells (HUVEC) with TNF-alpha resulted in a 10- to 100-fold increase of specific binding sites for 125I-VT-1. Furthermore, interleukin-1 (IL-1), lymphotoxin (TNF-beta), and lipopolysaccharide (LPS) also markedly increase VT-1 binding. Several hours' exposure to TNF-alpha was enough to enhance the number of VT-1 receptors on the endothelial cells for 2 days. The TNF-alpha-induced increase in VT-1 binding could be inhibited by simultaneous addition of the protein synthesis inhibitor cycloheximide. Glycolipid extracts of TNF-alpha- treated cells tested on thin-layer chromatography demonstrated an increase of globotriaosylceramide (GbOse3cer), a functional receptor for VT-1, which suggests that preincubation of human endothelial cells with TNF-alpha leads to an increase in GbOse3cer synthesis in these cells. We conclude from this study that TNF-alpha and IL-1 induce one (or more) enzyme(s) that is (are) rate-limiting in the synthesis of the glycolipid VT-1 receptor, GbOse3cer. These in vitro studies suggest that, in addition to VT-1, inflammatory mediators play an important role in the pathogenesis of HUS.
Detergent-resistant cytoskeleton of the surface-activated platelet differs from the suspension-activated platelet cytoskeleton
Authors: Smith CM , Burris SM , Rao GH , White JG
Published: October 2019

This study contrasts the protein composition of the detergent-resistant cytoskeleton of platelets fully spread on glass with the cytoskeletal composition of resting platelets and platelets aggregated in suspension with thrombin. Complete Triton X-100-insoluble cytoskeletons were isolated from spread, resting, and suspension-activated platelets in the presence of protease inhibitors, solubilized in sodium dodecyl sulfate/EDTA and analyzed on reduced, one-dimensional polyacrylamide gels. The protein composition of the cytoskeletons differed both qualitatively and quantitatively. Most notable were more extensive incorporation of total protein, talin, and vinculin into the cytoskeleton of spread platelets than the cytoskeleton of suspension- activated platelets. Varying the concentration and time of exposure to thrombin during suspension activation did not mimic the cytoskeletal changes of surface activation. Scanning electron microscopy, measurement of lipid phosphorus content, and varying the duration of Triton extraction did not show incomplete solubilization or nonspecific trapping of constituents in the spread platelet cytoskeleton. Proteolysis of talin was minimal in suspension-activated platelets and in platelets spread for 50 minutes. The differences in the detergent- resistant cytoskeletons of surface- and suspension-activated platelets indicate significant divergence in the physiologies of platelet spreading on surfaces and platelet activation in suspension.
Interleukin-11 promotes accessory cell-dependent B-cell differentiation in humans
Authors: Anderson KC , Morimoto C , Paul SR , Chauhan D , Williams D , Cochran M , Barut BA
Published: October 2019

Interleukin-11 (IL-11) is a recently described stromal-derived cytokine that supports the growth of an IL-6-dependent murine plasmacytoma line in the presence of antibody to IL-6 and appears to act in a manner similar to IL-6 on hematopoietic stem cells. Because IL-6 is known to promote differentiation of normal human B cells, the role of IL-11 on B- cell differentiation in vitro was characterized. IL-11 does not result in significantly increased DNA synthesis or Ig secretion by purified B cells alone or B cells cultured with Staphylococcus Cowan I, a T-cell- independent B-cell mitogen. In contrast, purified B cells cultured in the presence of pokeweed mitogen (PWM), irradiated T cells, and monocytes show increased DNA synthesis at day 3 and increased IgG and IgM secretion at day 7 of culture; addition of IL-11 further augments Ig secretion without change in DNA synthesis, an effect that can only be partially blocked by monoclonal antibody to IL-6. Similar experiments confirmed that increased IgG secretion was demonstrable when either IL-11 or IL-6 was added to B cells + CD4+/45RA- T cells + monocytes + PWM; in contrast, Ig secretion was low and equivalent when CD4+/45RA+ T cells were cultured with B cells+monocytes+PWM with or without IL-6 or IL-11. Neither IL-6 nor IL-11 could significantly increase phytohemagglutinin (PHA)-induced DNA synthesis by CD4+/45RA- or CD4+/45RA+ T cells. Although PWM or IL-11 induced IL-6 mRNA expression in both CD4+/45RA- T cells and monocytes, in neither cell did IL-11 increase IL-6 mRNA expression over that noted to PWM alone. These observations support the view that IL-11 promotes differentiation of human B lymphocytes only in the presence of accessory T cells and monocytes and that a minor component of this effect may be through stimulation of IL-6 production by CD4+/45RA- T cells and monocytes.
Human macrophage colony-stimulating factor levels are elevated in pregnancy and in immune thrombocytopenia [see comments]
Authors: Yong K , Salooja N , Donahue RE , Hegde U , Linch DC
Published: October 2019

Plasma macrophage colony-stimulating factor (M-CSF) levels were measured by enzyme immunosorbent assay (ELISA) using horse and rabbit polyvalent antibodies raised against human M-CSF purified from urine (hM-CSF). Plasma M-CSF levels in nonpregnant female controls were 364 +/- 69 U/mL (mean +/- SD, n = 20). Pregnancy results in significant elevation of circulating M-CSF levels (541 +/- 164 U/mL, n = 46, P < .0005). M-CSF levels were increased by 28 weeks' gestation and did not increase further in later pregnancy. M-CSF levels were also measured in 20 female controls before and after commencing on the oral contraceptive pill. There was no effect of the contraceptive pill on plasma M-CSF levels (364 +/- 69 U/mL before v 373 +/- 66 U/mL after commencing on the pill). In 28 nonpregnant patients with untreated immune thrombocytopenic purpura, (ITP), plasma M-CSF levels were significantly increased (797 +/- 402 U/mL, n = 28, v 364 +/- 69 U/mL in controls, N = 20, P < .0005). Pregnant ITP patients had higher levels of plasma M-CSF (929 +/- 327 U/mL, n = 25) than nonpregnant patients, but this difference was not significant. Elevated levels of M-CSF in ITP may reflect activation of the reticuloendothelial system (RES), which could result in positive feedback to increase the destruction of platelets. The increase in M-CSF associated with pregnancy could contribute to the exacerbation of latent ITP in pregnancy.
Cytokine dysregulation and acute graft-versus-host disease
Authors: Antin JH , Ferrara JL
Published: October 2019

We suggest that acute GVHD after marrow transplantation reflects (1) host injury due to the conditioning regimen followed by the production of inflammatory cytokines; (2) stimulation of mature donor T cells in the milieu of increased cell surface expression of leukocyte adhesion molecules and HLA molecules, followed by the autocrine production of IL- 2; and, finally, (3) recruitment and activation of additional mononuclear effector cells from donor marrow progenitors, which produce additional inflammatory cytokines, thus sustaining the response. The second step is critical for the amplification of the systemic inflammatory response, and it is absence in autologous, syngeneic, and T-cell-depleted transplants. These T cells may also contribute to the inflammatory cytokine network. Acute GVHD can occur in the absence of primary tissue injury in such settings as transfusion-related GVHD; however, it is likely that a greater HLA disparity between donor and host is required. We propose that inflammatory cytokine production is the final common pathway of acute GVHD. If this model is correct, control of cytokine dysregulation at any of several points should control GVHD. Further studies of GVHD and investigations of cytokine antagonists (eg, IL-4 or IL-10) or combinations of antagonists such as IL-1ra and soluble TNF receptor or pentoxifylline will allow us to determine the validity of this hypothesis.
Effects of therapy with didanosine on hematologic parameters in patients with advanced human immunodeficiency virus disease
Authors: Schacter LP , Rozencweig M , Beltangady M , Allan JD , Canetta R , Cooley TP , Dolin R , Kelley S , Lambert J , Liebman HA
Published: October 2019

Myelosuppression is associated with human immunodeficiency virus (HIV) infection and may also be produced by agents used for the treatment of the disease or the treatment of its complications. Didanosine (ddl; 2′,3′-dideoxyinosine) is a newer purine nucleoside that has recently become available for therapy for HIV infection. The effects of didanosine on peripheral blood counts have been retrospectively evaluated in the first 170 patients treated with this new agent in four phase I trials. Patients treated with didanosine showed statistically significant improvements in hemoglobin levels, white cell counts, and granulocyte and platelet numbers as compared with baseline values. These changes were seen with or without prior therapy with zidovudine, were somewhat more pronounced at higher doses of didanosine, and persisted for up to 1 year. Reported adverse events included peripheral neuropathy, diarrhea, and most notably, pancreatitis. It is concluded that, while some toxic side effects occur, didanosine therapy in HIV infection is associated with an amelioration of HIV-induced myelosuppression.
Long-term outcome of high-dose cytarabine-based consolidation chemotherapy for adults with acute myelogenous leukemia
Authors: Schiller G , Gajewski J , Territo M , Nimer S , Lee M , Belin T , Champlin R
Published: October 2019

Modern induction chemotherapy produces 60% to 80% complete remissions in adults with newly diagnosed acute myelogenous leukemia. A major challenge is to eradicate subclinical disease in remission and prevent leukemic relapse. We analyzed the long-term results of high-dose cytarabine-anthracycline consolidation chemotherapy without maintenance treatment and examined the effect of major prognostic factors, including age, sex, history of preleukemia, and cytogenetics. Two hundred twenty-seven patients with newly diagnosed acute leukemia were enrolled on two sequential studies conducted from 1982 to 1991. One hundred fifty-one patients (67%) achieved a complete remission. One hundred twenty-three patients were eligible for high-dose cytarabine- based consolidation administered in two to three courses. After a median follow-up of 4.8 years, 40 patients remain alive, with 28 in continued remission. Median remission duration for all eligible patients is 12.8 months, and actuarial leukemia-free survival (LFS) at 5 years is 26% +/- 8%. Advanced age and male sex were negative prognostic indicators for LFS. For patients < or = 45 years of age, 5- year LFS was 35% +/- 13%, as compared with 18% +/- 11% for patients greater than age 45 (P = .03). Toxicity of consolidation chemotherapy included treatment-related death in nine patients and serious neurotoxicity in five. Our results show an improved LFS for younger patients treated with high-dose cytarabine-based consolidation. There was no apparent benefit for older patients compared with reported data with less intensive regimens.
Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells
Authors: DelaFlor-Weiss E , Richardson C , Ward M , Himelstein A , Smith L , Podda S , Gottesman M , Pastan I , Bank A
Published: October 2019

Gene therapy in humans requires the transplantation of genetically modified cells, and it is important to select only those cells capable of expressing high levels of protein from the transferred gene. Expression of the human multiple drug resistance (MDR) gene confers resistance to a variety of compounds in vitro and in vivo. To determine the feasibility of conferring recipient erythroid cells with the MDR phenotype, we have transduced mouse erythroleukemia cells (MELC) with the MDR gene in a retroviral vector. We show here that MELC clones resistant to exposure to colchicine (an MDR-responsive agent) can be isolated, and demonstrate high levels of MDR RNA and protein expression. Increasing doses of colchicine increase the level of MDR RNA and protein expression significantly. These results indicate that it is possible to transfer and express the human MDR phenotype in mouse erythroid cells by retrovirally mediated gene transfer, and that drug selection can be used to enrich or purify populations of cells containing and expressing this gene.
Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa
Authors: Lamphear BJ , Fay PJ
Published: October 2019

Factor IXa was shown to inactivate both factor VIII and factor VIIIa in a phospholipid-dependent reaction that could be blocked by an antifactor IX antibody. Factor IXa-catalyzed inactivation correlated with proteolytic cleavages within the A1 subunit of factor VIIIa and within the heavy chain (contiguous A1-A2-B domains) of factor VIII. Furthermore, a relatively slow conversion of factor VIII light chain to a 68-Kd fragment was observed after prolonged incubation. Sites of cleavage were identified within the A1 domain at Arg336-Met337 and within the factor VIII light chain at Arg1719-Asn1720. Factor IXa failed to cleave isolated factor VIII heavy chains, yet cleaved isolated factor VIII light chain. In addition, the purified A1/A3-C1-C2 dimer derived from factor VIIIa was a substrate for factor IXa; however, cleavage of the A1 subunit occurred at less than 30% the rate of cleavage of A1 in trimeric factor VIIIa. These data suggest that factor VIII light chain contributes to the binding site for factor IXa and also support a role for a heavy chain determinant located within the A2 subunit in the association of factor VIIIa with factor IXa. Furthermore, the capacity of factor IXa to proteolytically inactivate its cofactor, factor VIIIa, suggests a mode of regulation within the intrinsic tenase complex.
Serum platelet-reactive IgG of autoimmune thrombocytopenic purpura patients is not F(ab')2 mediated and a function of storage
Authors: Karpatkin S , Xia J , Patel J , Thorbecke GJ
Published: October 2019

Serum platelet-reactive and glycoprotein (GP) IIb-GPIIIa-reactive IgG and F(ab')2 was examined in 39 patients with classic autoimmune thrombocytopenic purpura (ATP), two patients with anti-PLA1 antibody and 25 control subjects in an enzyme-linked immunosorbent assay. IgG was purified by diethyl aminoethyl chromatography and centrifuged at 100,000g before testing of the supernatant. Significant IgG binding (threefold to fourfold control IgG binding) was noted with 8 of 17 ATP patients' IgG, 2 anti-PLA1 IgGs, and 2 ATP patients with multiple platelet transfusions. However, F(ab')2 fragments of nine of nine positive ATP IgGs were nonreactive; F(ab')2 from the two anti-PLA1 and two multiply transfused ATP IgGs were as reactive as their intact IgG. Antiplatelet or anti-GPIIb-GPIIIa reactivity of ATP IgG could be adsorbed to fixed platelets or solid-phase GPIIb-GPIIIa and eluted with 0.1 mol/L glycine, pH 2.5. However, binding of IgG to GPIIb-GPIIIa could not be inhibited with F(ab')2 of ATP IgG or Fc fragments of control subjects. When platelet- or GPIIb-GPIIIa-reactive ATP IgG was applied to a Sephacryl 300 gel filtration column, no reactivity was noted in the 7S region, whereas anti-PLA1 localized to this region. Antiplatelet or anti-GPIIb-GPIIIa reactivity was noted in the void volume and accompanied by a high molecular weight protein region. An immunoblot of the void volume fraction with goat antihuman IgG (gamma chain) antibody showed high molecular weight bands greater than 250 Kd, which after reduction converted to a 55-Kd heavy-chain band. Fresh samples of ATP and control IgG processed within 1 to 2 days of blood withdrawal had no reactivity for GPIIb-GPIIIa. After storage at -20 degrees C for greater than 3 months, 5 of 19 ATP IgG became reactive, whereas 16 of 16 controls were nonreactive. Thus, platelet-reactive IgG of ATP sera appears to be caused by the development of IgG aggregates held together by disulfide bonds that develop on storage, and is not F(ab')2 mediated.
Responsiveness of chronic lymphocytic leukemia B cells activated via surface Igs or CD40 to B-cell tropic factors
Authors: Fluckiger AC , Rossi JF , Bussel A , Bryon P , Banchereau J , Defrance T
Published: October 2019

Recent studies performed in the laboratory have established that interleukin-4 (IL-4) used in combination with anti-CD40 monoclonal antibody (MoAb) 89 presented on Ltk- mouse fibroblasts stably expressing human Fc gamma RII/CDw32 (referred to as the CD40 system) sustains long-term proliferation of normal human B cells. In the present study, B-cell chronic lymphocytic leukemias (B-CLLs) activated through slgs or CD40 were examined for their capacity to proliferate and differentiate in response to various cytokines. Our results indicate that the outcome of IL-4 stimulation on the in vitro growth of B-CLL depends on the signalling pathway used for their activation. Whereas IL-4 did not display any growth-stimulatory effect on B-CLL activated by Ig cross-linking agents, it could stimulate DNA synthesis and enhance the viable cell recovery when leukemic B cells were cultured in the CD40 system. Most B-CLL samples were induced for IgM synthesis upon Staphylococcus aureus strain Cowan I stimulation. This Ig response was potentiated by IL-2 and antagonized by IL-4. Anti-CD40 MoAb used alone or in combination with cytokines (IL-1 alpha to IL-6, interferon gamma, tumor necrosis factor gamma, and transforming growth factor beta) failed to induce Ig secretion from B-CLL cells. No evidence for Ig isotype switching was obtained with the cytokines listed above, regardless of the mode of activation. Taken together, our results suggest that B-CLL cells can be partially released from their apparent maturation block by IL-2 and Ig cross-linking agents. In contrast, combinations of IL-4 and cross-linked anti-CD40 antibodies induced entry of B-CLL cell into cycle, but poorly stimulated their differentiation into Ig secreting cells.
Rhom-2 expression does not always correlate with abnormalities on chromosome 11 at band p13 in T-cell acute lymphoblastic leukemia
Authors: Fitzgerald TJ , Neale GA , Raimondi SC , Goorha RM
Published: October 2019

A frequent site for nonrandom recombination in T-cell acute lymphoblastic leukemia (T-ALL) is chromosome 11 at p13. The molecular characterization of a (7;11)(q35;p13) translocation showed that the translocation breakpoint was 2 kb 5′ to the T-ALLbcr locus resulting in the juxtaposition of the T-cell receptor (TCR) beta gene to the rhom-2 gene locus. Northern blot analysis did not detect expression of the rhom-2 gene in the leukemic blasts of the (7;11) translocation. However, using a sensitive polymerase chain reaction (PCR)-based assay, the (7;11) translocation showed a trace expression of rhom-2 at a level of 0.01% of TCR-beta message. Because rhom-2 is considered a proto- oncogene, the significance of the trace expression of rhom-2 in the (7;11) translocation was investigated by comparing the level of rhom-2 expression in 7 additional T-ALLs, normal thymocytes, and CEM (pre-T) and HPB (mature-T) cell lines using the PCR assay. The CEM cells, normal thymocytes, and one patient, whose blasts had no cytogenetic abnormality of chromosome 11, did not express rhom-2 indicating that rhom-2 is not normally expressed in T cells. The other six T-ALLs fell into three categories: (1) two T-ALLs overexpressed rhom-2 in the presence of a translocation; (2) two T-ALLs had trace expression in the presence of a translocation; and (3) two T-ALLs had trace expression with no observable abnormalities on chromosome 11 at p13. Therefore, the data indicate that not all translocations at the T-ALLbcr locus result in overexpression of rhom-2. To account for the sharp contrast in rhom-2 expression seen in these T-ALLs, a model is proposed with a negative regulatory element in the T-ALLbcr locus that is disrupted in some of the cases leading to overexpression of rhom-2.
Morphology in patients with severe aplastic anemia treated with antilymphocyte globulin
Authors: Tichelli A , Gratwohl A , Nissen C , Signer E , Stebler Gysi C , Speck B
Published: October 2019

One hundred and seventeen patients with severe aplastic anemia (SAA) were treated at our institution between 1976 and 1990 with antilymphocyte globulin (ALG) therapy. Seventy-nine (68%) are alive and probability of survival at 14 years, according to Kaplan and Meier, is 62% +/- 12%. Twenty-six patients developed a late clonal complication: 11 had a myelodysplastic syndrome (MDS) and 17 had paroxysmal nocturnal hemoglobinuria (PNH); two patients had both. The cumulative risk at 10 years is 42%. The development of MDS/PNH after SAA directly affects survival. The probability of being alive at 14 years is 81% +/- 10% for patients with stable disease and 36% +/- 13% for those with clonal evolution (P = .001). To look for predictive signs, we reevaluated peripheral blood and bone marrow cytomorphology at presentation, during regeneration, and in remission. We examined the peripheral blood values for hemoglobin, reticulocytes, granulocytes, thrombocytes, mean corpuscular volume (MCV), and fetal hemoglobin, as well as bone marrow for cellularity, erythropoiesis, myelopoiesis, and megakaryopoiesis. ALG therapy induces slow and incomplete recovery. Although in “remission,” ALG patients have lower hemoglobin values, higher reticulocyte counts, lower granulocyte and platelet values, and a higher MCV and fetal hemoglobin than normal controls. They retain a reduced number of megakaryocytes and a persistence of atypical monocytes in bone marrow morphology as stigmata of their disease. Patients with late clonal complications show distinct morphologic abnormalities: patients with PNH have higher MCVs, higher granulocyte and reticulocyte counts, and more dyserythropoiesis at diagnosis and a lower hemoglobin with an increased proportion of erythroblasts in the bone marrow in “remission.” Patients who later developed MDS are not different from the total patient population at diagnosis. After therapy, these patients are characterized by the presence of ring sideroblasts and atypical monocytes during regeneration and by a persistent increase in MCV, a higher fetal hemoglobin, lower granulocyte values, and megakaryocytic dysplasia during “remission.” Thus, routine morphologic follow-up examination of blood and bone marrow can discover patients at risk for late hematologic complications after ALG therapy.
Stem cell factor, interleukin-3, and interleukin-6 promote retroviral- mediated gene transfer into murine hematopoietic stem cells
Authors: Luskey BD , Rosenblatt M , Zsebo K , Williams DA
Published: October 2019

The efficiency of retroviral-mediated gene transfer into hematopoietic stem cells (HSC) is dependent on the survival and self-renewal of HSC in vitro during retroviral infection. We have examined the effect of prestimulation of bone marrow with various cytokines, including the product of the Steel gene, Steel factor or stem cell factor (SCF) (the ligand for the c-kit receptor) on the efficiency of retroviral transduction of the human adenosine deaminase (hADA) cDNA into murine HSC. Bone marrow cells were prestimulated for 48 hours with hematopoietic growth factors, then cocultivated with the packaging cell line producing the ZipPGK-ADA simplified retrovirus for an additional 48 hours with continued growth factor exposure. Nonadherant cells from these cocultures were injected into lethally irradiated recipients. The content of day 12 colony-forming unit-spleen (CFU-S12) in SCF/interleukin 6 (IL-6)-prestimulated and cocultured bone marrow was more than threefold greater than that of IL-3/IL-6-prestimulated bone marrow cells. All mice receiving bone marrow cells infected with the PGK-ADA virus after prestimulation with IL-3/IL-6 or SCF/IL-6 demonstrated hADA expression in the peripheral blood after full hematopoietic reconstitution. While all recipients of IL-3/IL-6- prestimulated bone marrow expressed hADA at 4 months posttransplant, in three independent experiments examining a total of 33 mice, in most recipients of SCF/IL-6-prestimulated and infected bone marrow cells, the expression of human enzyme was higher than IL-3/IL-6 mice. Southern blot analysis of DNA from hematopoietic tissues from these same mice prepared at least 4 months posttransplantation also demonstrated a higher infection efficiency of HSC as measured by proviral integration patterns and genome copy number analysis. These results suggest that the higher level of hADA expression seen in mice receiving marrow prestimulated with SCF/IL-6 before retroviral infection is due to more efficient infection of reconstituting HSC. Other growth factor combinations were also studied; however, prestimulation with SCF/IL-6 or IL-3/IL-6 appeared optimal. Using retroviral-mediated gene transfer and viral integration patterns, Steel factor (SCF) in combination with IL-6 appears to increase the survival and self-renewal of reconstituting hematopoietic stem cells and proves useful in effecting expression of foreign genes in transplant recipients. Such pretreatment may also be useful in the application of retroviral transfer methods to human cells.
Expression and function of adhesion molecules on human hematopoietic stem cells: CD34+ LFA-1- cells are more primitive than CD34+ LFA-1+ cells
Authors: Gunji Y , Nakamura M , Hagiwara T , Hayakawa K , Matsushita H , Osawa H , Nagayoshi K , Nakauchi H , Yanagisawa M , Miura Y
Published: October 2019

Advances in fluorescence-activated cell sorter technology have brought about multicolor analysis of cell phenotypes. To clarify the phenotypes of human hematopoietic stem cells (HSCs), we initially prepared novel antibodies against CD34 and labeled one of them (4A1) with allophycocyanin (APC). With this, we analyzed the phenotypes of CD34+ HSCs and showed that primitive HSCs or CD34+CD33- cells expressed adhesion molecules such as CD43, CD44, CD11a, CD11c, CD18, and leukocyte adhesion molecule (LAM-1). The more primitive hematopoietic cells or CD34+CD38- cells also expressed CD11a and CD18 with an incidence of 20% to 30%. To clarify the role of adhesion molecules in HSCs, we examined the colony forming capacity after long-term culture with allogeneic irradiated stromal layers. Among CD34+CD33- cells, CD18+ cells gave rise to colony-forming cells (CFCs) on stromal layers, but reached a maximum at week 2, after which the number of generated CFCs decreased. On the other hand, CD18- cells generated less CFCs than CD18+ cells at 2 to 3 weeks, but increased after 4 weeks of culture. When CD18 or CD11a antibody was added to a coculture system of CD34+CD33- cells with stromal layers, the number of generated CFCs decreased significantly compared with the no antibody control. Leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) was expressed on some populations of hematopoietic cells and contributed to the proliferation by interacting with stromal cells. However, more primitive cells capable of reconstituting hematopoiesis did not express LFA-1. These data provide a rationale for the administration of anti- LFA-1 antibody after bone marrow transplantation for reducing the graft failure.