bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of marrow cytogenetics and morphology on in vitro hematopoiesis in the myelodysplastic syndromes: comparison between recombinant human granulocyte colony-stimulating factor (CSF) and granulocyte-monocyte CSF
Authors: Nagler A , Binet C , Mackichan ML , Negrin R , Bangs C , Donlon T , Greenberg P
Published: October 2019

Marrow cells from 36 patients with myelodysplastic syndromes (MDS) (13 refractory anemia [RA], 14 refractory anemia with excess of blasts [RAEB], 9 RAEB in transformation [RAEB-T]) were evaluated for their in vitro proliferative and differentiative responsiveness to recombinant human granulocyte colony-stimulating factor (G-CSF) or granulocyte- monocyte CSF (GM-CSF). GM-CSF exerted a stronger proliferative stimulus than G-CSF for marrow myeloid clonal growth (CFU-GM) in these patients (44 v 12 colonies per 10(5) nonadherent buoyant bone marrow cells [NAB], respectively, P less than .025). GM-CSF stimulated increased CFU- GM growth in the 16 patients with abnormal marrow cytogenetics in comparison with the 20 patients who had normal cytogenetics (52 and 30 colonies per 10(5) NAB, respectively, P less than .05), whereas no such difference could be demonstrated with G-CSF (11 and 16 colonies per 10(5) NAB, respectively). In contrast, granulocytic differentiation of marrow cells was induced in liquid culture by G-CSF in 15 of 32 (47% patients), while GM-CSF did so in only 4 of 18 (22%) patients (P less than .025) including, for RAEB/RAEB-T patients: 9 of 18 versus 0 of 9, respectively (P less than .025). For MDS patients with normal cytogenetics, G-CSF- and GM-CSF-induced marrow cell granulocytic differentiation in 12 of 18 (67%) versus 3 of 11 (27%), respectively (P less than .025), contrasted with granulocytic induction in only 3 of 14 (21%) and 1 of 7 (14%) patients with abnormal cytogenetics, respectively. We conclude that G-CSF has greater granulocytic differentiative and less proliferative activity for MDS marrow cells than GM-CSF in vitro, particularly for RAEB/RAEB-T patients and those with normal cytogenetics.
Selective regulation of the activity of different hematopoietic regulatory proteins by transforming growth factor beta 1 in normal and leukemic myeloid cells
Authors: Lotem J , Sachs L
Published: October 2019

The viability of normal bone marrow myeloid precursor cells induced by interleukin-6 (IL-6) or IL-1 alpha and the ability of IL-6 and IL-1 alpha to induce the formation of colonies of granulocytes, macrophages, or megakaryocytes in densely seeded bone marrow cultures was suppressed by transforming growth factor-beta 1 (TGF-beta 1). Induction of normal bone marrow colony formation by IL-3 was much less sensitive to TGF- beta 1, and there was little or no effect of TGF-beta 1 on colony formation induced by macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage CSF (GM-CSF). In different clones of myeloid leukemic cells, TGF-beta 1 suppressed differentiation induced with IL- 6, IL-1 alpha, or lipopolysaccharide (LPS), but did not suppress differentiation induced with IL-3 or GM-CSF. The effect of TGF-beta 1 on differentiation of the leukemic cells can be dissociated from its effect on cell growth. TGF-beta 1 suppressed the production of IL-6 in normal bone marrow cells cultured with IL-1 alpha and the production of IL-6 and GM-CSF in leukemic cells cultured with IL-1 alpha or LPS. The suppression of IL-6 production can explain the suppression by TGF-beta 1 of the effects of IL-1 alpha and LPS that are mediated by IL-6. TGF- beta 1 also suppressed differentiation in clones of myeloid leukemic cells induced with differentiation factor/leukemia inhibitory factor and tumor necrosis factor. In different leukemic clones TGF-beta 1 suppressed or enhanced induction of differentiation with dexamethasone. The results show that TGF-beta 1 can selectively control the activity of different molecular regulators of normal and leukemic hematopoiesis.
Uremic platelets have a functional defect affecting the interaction of von Willebrand factor with glycoprotein IIb-IIIa
Authors: Escolar G , Cases A , Bastida E , Garrido M , Lopez J , Revert L , Castillo R , Ordinas A
Published: October 2019

Uremic patients have an impaired platelet function that has been related to membrane glycoprotein (GP) abnormalities. Using a perfusion system, we have studied the interaction of normal and uremic platelets with vessel subendothelium (SE) under flow conditions. Reconstituted blood containing washed platelets, purified von Willebrand factor (vWF) (1 U/mL), and normal washed red blood cells was exposed to de- endothelialized rabbit segments for 10 minutes at two different shear rates (800 and 1,600 seconds-1). In some experiments a monoclonal antibody to the GPIIb-IIIa complex (EDU3) was added to the perfusates. With normal platelets, the percentage of the vessel covered by platelets (%CS) was 23.1% +/- 3.7% at 800 seconds-1 and 30% +/- 4.3% at 1,600 seconds-1. Platelets were observed in contact or forming monolayers on vessel SE. EDU3 inhibited the spreading of normal platelets. The %CS (11.1% +/- 3.3%) was statistically decreased (P less than .01) and most of the platelets were observed in contact with the vessel surface. These data indicate that, under flow conditions, the interaction of vWF with GPIIb-IIIa can support the spreading of normal platelets in the absence of exogenous fibrinogen. Under the same experimental conditions, the interaction of uremic platelets with SE was markedly impaired at both shear rates studied (P less than .01 v normal platelets). The presence of EDU3 did not modify the interaction of uremic platelets. These results confirm the impairment of the platelet adhesion observed in uremic patients. Furthermore, they indicate the presence of a functional defect in the interaction of vWF with GPIIb-IIIa. The fact that perfusions with normal and uremic platelets in the presence of an antibody to the GPIIb-IIIa complex did not show any differences gives indirect evidence on a functionally normal interaction vWF/GPIb in uremic patients.
Multiple myeloma: increased circulating lymphocytes carrying plasma cell-associated antigens as an indicator of poor survival
Authors: Omede P , Boccadoro M , Gallone G , Frieri R , Battaglio S , Redoglia V , Pileri A
Published: October 2019

In multiple myeloma (MM) an increase in circulating lymphocytes expressing plasma cell-associated antigens (PCAA) has been described. Its prognostic significance was evaluated in this study. The immunologic phenotype of peripheral blood lymphocytes was analyzed with a panel of monoclonal antibodies specific for B, T, natural killer lymphocytes, and PCAA (CD38, PCA1) in 52 MM patients at diagnosis, remission, and during relapse, 18 monoclonal gammopathy of undetermined significance (MGUS), and 25 normal controls. No significant phenotypic alteration was observed in MGUS. In MM, the number of B lymphocytes was in the normal range at diagnosis and during the subsequent phases. A CD4/CD8 ratio decrease, during relapse, was due to both a CD4+ reduction and to an expansion of a subset of CD8+ activated suppressor lymphocytes. CD38+ and PCA1+ lymphocytes at diagnosis were significantly higher than in MGUS, and a further increase was observed during relapse, suggesting a correlation between PCAA expression and disease activity. The prognostic significance of increased PCAA was confirmed by a survival analysis of 32 patients evaluated at diagnosis using a CD38 cutoff of 0.45 x 10(9)/L positive lymphocytes. Median survival for patients with high values was only 14 months, whereas it was not reached at 32 months by those with low values (P less than .0007).
Interleukin-4 (IL-4) inhibits secretion of IL-1 beta, tumor necrosis factor alpha, and IL-6 by human monocytes
Authors: te Velde AA , Huijbens RJ , Heije K , de Vries JE , Figdor CG
Published: October 2019

Monocytes activated by lipopolysaccharide (LPS) and interferon gamma (IFN gamma) rapidly secrete a number of monokines with different functional properties. Interleukin–4 (IL–4), a T-cell derived cytokine, has been shown to reduce the production of monokines with cytostatic activity for tumor cells, chemotactic activity for monocytes, and factors that stimulate thymocyte proliferation. This latter activity is mediated by a number of monokines like IL–1, tumor necrosis factor alpha (TNF alpha), and IL–6. To elucidate which cytokines produced by monocytes are controlled by IL–4, we tested the effect of IL–4 on the secretion of IL–1 alpha, IL–1 beta, TNF alpha, and IL–6 induced by LPS or IFN gamma. IL–4 was found to inhibit the secretion of IL–1 beta and TNF alpha by activated monocytes almost 100%. The secretion of IL–6 was found to be reduced 70% to 85% in the presence of IL–4, whereas there was no effect on the secretion of IL–1 alpha (IL–1 alpha is mainly cell- associated). Time-course experiments demonstrate that IL–4 reduces the secretion of monokines for a prolonged period of time (greater than 40 hours). The reduced secretion of IL–1 beta and TNF alpha was specifically induced by IL–4 because anti-IL–4 antiserum completely restored normal monokine production. These data suggest that IL–4 plays a role in the regulation of immune responses by reducing the production of functionally important monokines.
Transfusion and alloimmunization in sickle cell disease. The Cooperative Study of Sickle Cell Disease
Authors: Rosse WF , Gallagher D , Kinney TR , Castro O , Dosik H , Moohr J , Wang W , Levy PS
Published: October 2019

In 1,814 patients with sickle cell disease who had been transfused, the overall rate of alloimmunization to erythrocyte antigens was 18.6%. The rate of alloimmunization in this group appears to be an explicit function of the number of transfusions received because it increases exponentially with increasing numbers of transfusions. Alloimmunization usually occurred with less than 15 transfusions, although the rate of alloimmunization continued to increase when more transfusions were given. The rate of alloimmunization was less in patients with hemoglobin SC disease and sickle-beta+ thalassemia because these patients had received fewer transfusions. Children less than 10 years old had a slightly lower rate of alloimmunization than patients in other age groups even after correction for the number of transfusions given. Women were more frequently alloimmunized than men; this was largely due to the fact that women received more transfusions than men, but in the age group 16 to 20 years the increase may have been due in part to alloimmunization owing to pregnancy. Forty-five percent of those alloimmunized made antibodies of only one specificity; 17% made four or more antibodies reacting with different antigens. Antibodies to the C and E antigens of the Rh group, the Kell antigen, and the Lewis antigens were most commonly made. These findings may be important in formulating a rational transfusion policy in sickle cell disease.
Biology and clinical significance of cytogenetic abnormalities in childhood acute lymphoblastic leukemia
Authors: Pui CH , Crist WM , Look AT
Published: October 2019

Virtually all cases of childhood ALL have chromosomal abnormalities and half contain translocations, which are nearly equally divided between random and nonrandom rearrangements. Nonrandom chromosomal abnormalities have been correlated with leukemic cell lineage, the degree of cell differentiation, and the specific gene involved at the molecular level. Many cytogenetic findings have prognostic significance; however, the adverse influence of certain changes, including most chromosomal translocations, may in fact be offset by the greater cytoreductive effects of intensified therapy. Table 4 summarizes the relation of selected karyotypic findings to treatment outcome in patients treated on contemporary protocols. Among all of the chromosomal abnormalities identified in childhood ALL, hyperdiploidy greater than 50 has been associated with the most favorable prognosis. At the opposite end of the spectrum, the treatment outcome for patients with classical Ph+ or hypodiploid ALL is very poor even in programs of intensive chemotherapy; alternative treatment such as bone marrow transplantation should be considered for such patients. Cases with the t(4;11)(q21;q23) also have a very poor clinical outcome, but the adverse prognosis may be limited to the infant or adolescent age groups. The prognostic significance of other nonrandom translocations, such as t(1;19)(q23;p13) and several other abnormalities, needs to be further assessed in larger numbers of patients. Finally, as more is learned about the molecular pathology underlying these rearrangements, it may be possible to develop new therapeutic agents that are specifically targeted to interfere with the aberrant gene products expressed by human leukemic cells.
Induction of monocyte proliferation and HIV expression by IL-3 does not interfere with anti-viral activity of zidovudine
Authors: Schuitemaker H , Kootstra NA , van Oers MH , van Lambalgen R , Tersmette M , Miedema F
Published: October 2019

Myelosuppression is a major symptom in the acquired immunodeficiency syndrome (AIDS). Moreover zidovudine, an anti-retroviral drug used to treat AIDS patients has myelosuppressive side effects. Therefore treatment with IL-3, a multi-lineage hemopoietic growth factor may be beneficial for zidovudine-treated individuals. In this study we examined the effect of IL-3 on human immunodeficiency virus (HIV) expression. The proliferative response to rIL-3 and the effects on the replication of the monocytotropic HIV variant, HTLV-III Ba-L, in the absence or presence of the anti-retroviral drug zidovudine was studied in purified human peripheral blood monocytes. Zidovudine concentrations sufficient for complete inhibition of HIV replication did not affect rIL-3 induced monocyte proliferation. Although rIL-3, like rGM-CSF, was able to augment HIV expression in monocytes, it did not interfere with the anti-retroviral activity of zidovudine. These data indicate that rIL-3 is a potential candidate for use in myelosupportive therapy in AIDS patients treated with anti-retroviral drugs.
Factor IXHollywood: substitution of Pro55 by Ala in the first epidermal growth factor-like domain
Authors: Spitzer SG , Kuppuswamy MN , Saini R , Kasper CK , Birktoft JJ , Bajaj SP
Published: October 2019

Factor IX is a multidomain protein essential for hemostasis. We describe a mutation in a patient affecting the first epidermal growth factor (EGF)-like domain of the protein. All exons and the promoter region of the gene were amplified by the polymerase chain reaction method, and sequenced. Only a single mutation (C----G), that predicts the substitution of Pro55 by Ala in the first EGF domain was found in the patient's gene. This mutation leads to new restriction sites for four enzymes. One new site (Nsi) was tested in the amplified exon IV fragment and was shown to provide a rapid and reliable marker for carrier detection and prenatal diagnosis in the affected family. The factor IX protein, termed factor IXHollywood (IXHW), was isolated to homogeneity from the patient's plasma. As compared with normal factor IX (IXN), IXHW contained the same amount of gamma-carboxy glutamic acid but twice the amount of beta-OH aspartic acid. Both IXHW and IXN contained no detectable free -SH groups. Further, IXHW could be readily cleaved to yield a factor IXa-like molecule by factor Xla/Ca2+. However, IXaHW (compared with IXaN) activated factor X approximately twofold slower in the presence of Ca2+ and phospholipid (PL), and 8- to 12-fold slower in the presence of Ca2+, PL, and factor VIIIa. Additionally, IXaHW had only approximately 10% of the activity of IXaN in an aPTT assay. In agreement with the nuclear magnetic resonance- derived structure of EGF, the Chou-Fasman algorithm strongly predicted a beta turn involving residues Asn-Pro55-Cys-Leu in IXN. Replacement of Pro55 by Ala gave a fourfold decrease in the beta turn probability for this peptide, suggesting a change(s) in the secondary structure in the EGF domain of IXHW. Since this domain of IXN is thought to have one high-affinity Ca2+ binding site and may be involved in PL and/or factor VIIIa binding, the localized secondary structural changes in IXHW could lead to distortion of the binding site(s) for the cofactor(s) and, thus, a dysfunctional molecule.
Differential response of malignant human B-cells to anti-IgM immunoglobulin (anti-mu) and B-cell growth factor: unique direct cytotoxicity of anti-mu on prolymphocytic leukemia cells
Authors: Chan CS , Soehnlen F , Schechter GP
Published: October 2019

We examined in vitro tritiated thymidine uptake by B cells from seven prolymphocytic leukemia (PLL), seven chronic lymphocytic leukemia (CLL), and four plasma cell leukemia (PCL) patients in response to culture with anti-human IgM antibody (anti-mu) and B-cell growth factor (BCGF). In contrast to the stimulatory effect observed in normal B-cell cultures, the divalent F(ab')2 anti-mu antibody uniquely inhibited the autonomous proliferation and induced marked cytotoxicity in six of seven PLL cell cultures independent of complement or Fc-receptor- mediated mechanisms. There was neither stimulation or inhibition of the slgM+ CLL or the slgM- PCL cells. The inhibitory effect on the PLL cells was observed at an anti-mu concentration below the stimulatory threshold for normal B cells. Significant impairment of trypan blue exclusion was delayed until 48 hours, with morphological cellular changes suggestive of a programmed cell death mechanism or apoptosis. The cytotoxicity was independent of the slgM-staining intensity or the autonomous and BCGF-augmented DNA synthetic activity of the PLL cells and was similar in patients with de novo PLL or with prolymphocytic transformation of CLL. Cells from a PLL patient were separated by elutriation into two fractions, a BCGF-unresponsive large “transformed” cell fraction with marked autonomous DNA synthesis and a smaller lymphoid cell subset with low 3H-thymidine uptake that could be augmented by BCGF. Both fractions were equally susceptible to the cytotoxic effect of anti-mu. These data suggest that certain slgM- bearing malignant B cells are susceptible to anti-mu-triggered cytotoxicity. They may represent the malignant counterpart of a “tolerogenic” normal B-cell subset, and the unique direct cytotoxicity of anti-mu may provide a therapeutic strategy specifically for PLL.
Recombinant C5a stimulates transcription rather than translation of interleukin-1 (IL-1) and tumor necrosis factor: translational signal provided by lipopolysaccharide or IL-1 itself
Authors: Schindler R , Gelfand JA , Dinarello CA
Published: October 2019

We investigated the effects of recombinant C5a (rC5a) on gene expression and synthesis of interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) in fresh human peripheral blood mononuclear cells (PBMC). Total (cell-associated and secreted) cytokine synthesis was measured. In the strict absence of endotoxin (lipopolysaccharide [LPS]), rC5a resulted in a small but statistically insignificant increase in immunoreactive IL-1 beta and TNF, as well as in IL-1 and IL- 6 bioactivity. On the other hand, rC5a induced marked transcriptional activation of IL-1 beta and TNF in a dose-dependent fashion with an optimal concentration of 50 ng/mL. The rC5a-induced cytokine messenger RNA (mRNA) was not spontaneously translated into protein. At 50 ng/mL, rC5a induced the same levels of mRNA for IL-1 beta and TNF as 1 ng/mL of LPS, whereas LPS induced 12 times more IL-1 beta protein and 70 times more TNF protein than rC5a alone. The C5a-induced mRNA half-life was the same as that induced by LPS. Formyl-Meth-Leu-Phe (fMLP) did not induce cytokine transcription. Pretreatment with rC5a enhanced cytokine synthesis induced by other stimuli. After 2 hours of preincubation with rC5a, PBMC synthesized 3 to 10 times more IL-1 beta and TNF on subsequent stimulation by LPS or IL-1 itself. We conclude that rC5a provides primarily a transcriptional but not translational signal for IL-1 beta and TNF; the half-life of the untranslated mRNA is the same as that of translated message; rC5a-induced transcription upregulates PBMC for enhanced synthesis of these cytokines; and a translational signal can be provided by LPS or IL-1 itself.
All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results [see comments]
Authors: Castaigne S , Chomienne C , Daniel MT , Ballerini P , Berger R , Fenaux P , Degos L
Published: October 2019

Twenty-two patients with acute promyelocytic leukemia were treated with all-trans retinoic acid (RA, 45 mg/m2 per day) for 90 days. Of the 22, four patients were previously untreated, two were resistant after conventional chemotherapy, and 16 were in first (n = 11), second (n = 4), or third (n = 1) relapse. We observed 14 complete response, four transient responses, one failure, and three early deaths. Length of hospitalization and number of transfusions were notably reduced in complete responders. Correction of coagulation disorders and an increase of WBCs were the first signs of all-trans RA efficacy. Morphologic analysis performed at days 0, 15, 30, 45, 60, and 90 showed that complete remissions were obtained without bone marrow (BM) hypoplasia. Presence of Auer rods in the maturing cells confirmed the differentiation effect of the treatment. At remission, the t(15;17) initially present in 20 patients was not found. The in vitro studies showed a differentiation in the presence of all-trans RA in 16 of the 18 tested cases. The single nonresponder to all trans RA in vitro did not respond in vivo. Adverse effects of RA therapy--skin and mucosa dryness, hypertriglyceridemia, and increase of hepatic transaminases-- were frequently noted. We also observed bone pain in 11 patients and hyperleukocytosis in four patients. Whether maintenance treatment consisted of low-dose chemotherapy or all-trans RA, early relapses were observed. Five patients are still in complete remission (CR) at 4 to 13 months. Our study confirms the major efficacy of all-trans RA in M3, even in relapsing patients. Remissions are obtained by a differentiation process.
Autologous bone marrow transplantation in multiple myeloma: identification of prognostic factors
Authors: Jagannath S , Barlogie B , Dicke K , Alexanian R , Zagars G , Cheson B , Lemaistre FC , Smallwood L , Pruitt K , Dixon DO
Published: October 2019

Multiple myeloma remains a universally fatal malignancy with a median survival time not exceeding 3 years. A clinical trial was undertaken to determine feasibility and efficacy of marrow-ablative chemoradiotherapy supported by unpurged autologous bone marrow (ABMT) and to define prognostic variables. Total body irradiation and either melphalan or thiotepa were administered to 55 patients (median age 53 years; range 20 to 66 years). The group of 21 patients with resistance to standard melphalan-prednisone and to continuous infusions of vincristine and Adriamycin with high dose dexamethasone (VAD) included 7 with primary unresponsive disease and 14 with resistant relapse; among the 34 patients achieving remission with the VAD regimen, 14 were in first and 20 in a subsequent remission. Marked cytoreduction by greater than or equal to 75% was observed among all 21 patients with refractory myeloma, whereas further cytoreduction of this magnitude was noted in only 56% of the 34 patients already in remission after VAD. Five of the 6 early deaths among all 55 patients occurred in the 14 patients with resistant relapse, none of whom achieved complete remission and who, as a group, had median durations of relapse-free and overall survival of only 8 and 7 months, respectively. Among the 41 remaining patients, there was only one early death, and 27% achieved complete remission including a 36% incidence among the 14 patients treated in first remission; their projected 4-year survival rate was 82% regardless of their disease status (first or later remission or primary resistance). When information about sensitivity to prior therapy is unavailable, the presence before ABMT of both high beta-2-microglobulin levels (greater than 3 mg/L) and non-IgG isotype helped identify 9 among the 55 patients with a very poor prognosis: all 8 responders relapsed within 9 months, and 8 patients died within 15 months. By contrast, a 4-year projected survival rate of over 70% for the other patients (about 80% of this series) justifies further investigation of this novel treatment approach in comparison with standard dose regimens. Our results indicate that marrow-ablative therapy cannot be recommended for myeloma patients with resistant relapse or those with a combination of risk factors (advanced tumor burden, absence of IgG isotype). The apparent lack of an adverse effect of even marked plasmacytosis in autografts (up to 30%) emphasizes the need for better cytoreduction rather than bone marrow purging.
Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome
Authors: Gerrard JM , Lint D , Sims PJ , Wiedmer T , Fugate RD , McMillan E , Robertson C , Israels SJ
Published: October 2019

Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.
Expression of c-myb and B-myb, but not A-myb, correlates with proliferation in human hematopoietic cells
Authors: Golay J , Capucci A , Arsura M , Castellano M , Rizzo V , Introna M
Published: October 2019

The steady-state expression of three members of the myb family of genes, c-myb, B-myb, and A-myb, was studied in four purified normal human hematopoietic cell types, ie, T and B lymphocytes, monocytes, and granulocytes. The c-myb proto-oncogene is low to undetectable in resting T and B lymphocytes and shows a biphasic induction in both cell types after mitogenic stimulation, with a first peak at 3 hours and a second and stronger induction at 44 to 72 hours. Study of the B-myb gene showed that this gene is low to undetectable in resting T or B cells and is strongly induced after mitogenic stimulation with a peak between 44 and 72 hours in both cell types. Finally, the A-myb gene shows a pattern of expression in lymphocytes different from that of c- myb and B-myb. It is expressed in resting T lymphocytes and its levels gradually decrease after mitogenic stimulation to become undetectable at 48 hours. It is also expressed in a subpopulation of large B lymphocytes but not in in vitro activated B cells. Neither of the three members of the myb family of genes was expressed in monocytes and granulocytes, even after functional activation of these cells. Taken together, these data bring further evidence for the role of c-myb in cellular proliferation and on the basis of the kinetics of its induction relative to thymidine uptake, we hypothesize that it may have a role during G1 progression in addition to that already documented for entry into S phase. Furthermore, our studies indicate that another member of the myb family of genes, B-myb, may also be involved in cellular proliferation, because its expression correlates with the induction of mitogenesis.
Molecular basis of spectrin and ankyrin deficiencies in severe hereditary spherocytosis: evidence implicating a primary defect of ankyrin
Authors: Hanspal M , Yoon SH , Yu H , Hanspal JS , Lambert S , Palek J , Prchal JT
Published: October 2019

While varying degrees of spectrin deficiency have been found in the majority of patients with hereditary spherocytosis (HS), a combined severe deficiency of both spectrin and the spectrin-binding protein, ankyrin, has been reported only in two patients with severe HS. To elucidate the molecular basis of these protein deficiencies, we have studied the synthesis, assembly, and the mRNA levels of spectrin and ankyrin in peripheral blood reticulocytes in one of the previously reported probands. Pulse-labeling studies showed that in HS reticulocytes, the synthesis of alpha-spectrin was comparable with control reticulocytes while that of beta-spectrin was increased about fourfold, presumably reflecting increased erythropoietic drive. On the HS reticulocyte membrane, the amount of newly assembled spectrin was reduced to about half of the control values, presumably reflecting a decrease in the synthesis of the spectrin binding protein, ankyrin: the ankyrin synthesis was nearly absent in the cytosol and the amounts of membrane-associated ankyrin were reduced to about half of the normal values. The changes in the amounts of spectrin and ankyrin mRNAs quantitated by slot blot and Northern blot analyses were comparable with changes in the synthesis of these proteins: The alpha spectrin mRNA was within a control range and the beta-spectrin mRNA was slightly increased, while the amounts of ankyrin mRNA were reduced to about 50% of control values. We conclude that the primary defect underlying the combined spectrin and ankyrin deficiency is a deficiency of ankyrin mRNA leading to a reduced synthesis of ankyrin which, in turn, underlies the decreased assembly of spectrin on the membrane.
Toxicity and efficacy of anti-T-cell ricin toxin A chain immunotoxins in a murine model of established graft-versus-host disease induced across the major histocompatibility barrier
Authors: Vallera DA , Carroll SF , Snover DC , Carlson GJ , Blazar BR
Published: October 2019

Graft-versus-host disease (GVHD) was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow and splenocytes into lethally irradiated B10.BR (H-2k) murine recipients. An immunotoxin (IT) composed of a pan T-cell monoclonal antibody called anti-Ly1 (the murine homologue to human anti-CD5) was conjugated to ricin toxin A chain (anti-Ly1-RTA) and used to treat recipient mice. In vitro, IT was as active as free RTA, bound selectively, and inhibited T- cell proliferation even in the absence of potentiators. Mice administered anti-Ly1-RTA in vivo during ongoing GVHD, at a dose of 10 micrograms/d for 5 days, showed lower numbers of splenic Thy1.2+ T cells and significantly improved survival as compared with mice given phosphate-buffered saline (PBS) or irrelevant control RTA IT. Protection was transient because GVHD and weight loss occurred when injections ceased. Survival could not be enhanced by crosslinking RTA30, a low oligosaccharide-containing fraction of purified RTA. Treatment with anti-Ly1-RTA caused a significant elevation in neutrophils, and higher doses were associated with mild hepatotoxicity. In contrast, infusion of identical doses and schedules of another pan T- cell immunotoxin, anti-Thy1.2-RTA, caused a significant decrease in lymphocytes, but not neutrophils; a precipitous increase in weight; a decrease in total plasma protein (TPP); and an increase in pleural and peritoneal effusions reminiscent of vascular leak syndrome (VLS). Although the toxic effects of anti-Thy1.2-RTA were too severe to show a survival advantage in a GVHD model, histopathologic studies showed a definite anti-GVHD effect. The most significant decline in GVHD as compared with the PBS-treated controls was observed in skin, and to a lesser extent, in liver and lung. To investigate the cause of IT toxicity, anti-Thy1.2-RTA was administered intraperitoneally to lethally irradiated B10.BR (H-2k) recipients of syngeneic bone marrow. These recipients showed the same weight gain, hypoproteinuria, and VLS observed in the GVHD model. Death occurred at higher anti-Thy1.2-RTA doses (30 or 50 micrograms/daily injections administered days 8 through 12 posttransplant). Anti-Thy1.2-RTA had a negligible effect on renal function, but histologic studies showed patchy dropout of the renal tubules. Treatment resulted in pulmonary vascular congestion, but there was no pathologic evidence of liver, brain, or colon toxicity. Weight gain was enhanced by irradiation because nonirradiated normal mice did not undergo such a precipitous weight increase.(ABSTRACT TRUNCATED AT 400 WORDS)
Observations on the effect of chimeric anti-CD4 monoclonal antibody in patients with mycosis fungoides
Authors: Knox SJ , Levy R , Hodgkinson S , Bell R , Brown S , Wood GS , Hoppe R , Abel EA , Steinman L , Berger RG
Published: October 2019

Chimeric (murine/human) anti-CD4 monoclonal antibody was infused into seven patients with mycosis fungoides. Successive patients received doses of 10, 20, 40, and 80 mg of antibody twice a week for 3 consecutive weeks. All patients had some clinical improvement, but responses were of relatively short duration. Serum levels of chimeric antibody varied as a function of dose. At the 80-mg dose level, antibody was readily observed in biopsied skin lesions. Although there was coating by antibody of most CD4 positive cells in the blood, there was no significant depletion of CD4 positive cells. Low-level antibody responses against the mouse Ig variable region and human Ig allotypic constant region determinants were observed in several patients, but none were of clinical significance. All but two patients made primary antibody and T-cell proliferative responses to a simultaneously administered foreign protein test antigen. However, there was marked suppression of the mixed lymphocyte reaction. We conclude that at the dose levels studied, a chimeric anti-CD4 monoclonal antibody (1) had some clinical efficacy against mycosis fungoides; (2) was well tolerated; (3) had a low level of immunogenicity; (4) had immediate immunosuppressive effects; and (5) did not induce tolerance to a co- injected antigen.
High risk of severe bleeding in aged patients with chronic idiopathic thrombocytopenic purpura
Authors: Cortelazzo S , Finazzi G , Buelli M , Molteni A , Viero P , Barbui T
Published: October 2019

The purpose of this study was to estimate the incidence and to establish which factors were associated with an increased risk of hemorrhagic complications in an historic cohort of 117 consecutive and unselected patients with chronic idiopathic thrombocytopenic purpura (ITP). Sixty-eight patients (58%) underwent medical treatment and/or splenectomy and 33 (48% of treated) achieved a complete stable remission. At equivalent platelet count the incidence of major hemorrhagic complications was significantly higher in aged (greater than 60 years) than in younger (less than 40 years) patients (10.4% v 0.4%/pt-y, relative risk = 28.9, P less than .01). A previous hemorrhagic event was identified as another major risk factor for hemorrhage (relative risk = 27.5, P less than .0005), while hypertension and underlying disorders had no influence. We conclude that age more than 60 years and a previous history of bleeding are major risk factors for severe hemorrhages in adults with ITP.
Multiple in vivo effects of interleukin-3 and interleukin-6 on murine megakaryocytopoiesis
Authors: Carrington PA , Hill RJ , Stenberg PE , Levin J , Corash L , Schreurs J , Baker G , Levin FC
Published: October 2019

The in vivo effects of interleukin-3 (IL-3), interleukin-6 (IL-6), and a combination of IL-3 plus IL-6 on murine megakaryocytopoiesis and thrombopoiesis were examined. Human recombinant IL-6 was administered subcutaneously as 14 equal injections of 5,000 units each during a 102- hour period. Murine recombinant IL-3 was given as 8 injections of 80,000 units each during the first 54 hours. Megakaryopoiesis and thrombopoiesis were evaluated 120 hours after initial administration of the cytokines. Platelet levels increased by 20% following IL-3 alone, 35% following IL-6 alone and 61% after administration of both IL-3 and IL-6. Platelet production, as measured by 75Se-selenomethionine incorporation, increased by approximately 120% in animals that had received IL-6 or IL-3 plus IL-6. Megakaryocyte ploidy analysis by two- color flow cytometry showed a shift in the modal ploidy class from 16N to 32N and a significant increase in the frequency of 64N cells only in IL-6 treated animals. Both bone marrow and splenic megakaryocyte colony- forming cells were significantly increased following either IL-3 or IL- 6. Bone marrow megakaryocyte size increased 18%, 43%, and 38%, respectively, after administration of IL-3, IL-6, or the combination of IL-3 plus IL-6. Leukocyte counts and hematocrits were unaffected by either cytokine. Additional groups of mice received the same injection schedule as above and the serial effects on peripheral blood cell levels were assessed for 30 days. Platelet levels, which had been elevated by IL-3 or IL-6, fell to control values within 4 days following the last injection. Animals given IL-6 or IL-3 plus IL-6 were subsequently thrombocytopenic relative to controls on days 7 through 9 following cessation of treatment. Temporary ‘cycling’ of platelet levels was observed for 3 weeks following treatment with IL-6 or the combination of IL-3 plus IL-6. We conclude that IL-6 and to a lesser extent IL-3 stimulate platelet production in vivo and that their combined effects on platelet levels are approximately additive. Following discontinuation of IL-3 or IL-6, the effects are rapidly reversed, presumably by negative feedback mechanisms, resulting in a period of ‘rebound thrombocytopenia’ in mice that had received IL-6.
Stimulation of megakaryocytopoiesis in mice by human recombinant interleukin-6
Authors: Hill RJ , Warren MK , Stenberg P , Levin J , Corash L , Drummond R , Baker G , Levin F , Mok Y
Published: October 2019

The in vivo effects of purified human recombinant interleukin-6 (IL-6) on murine megakaryocytopoiesis were examined. IL-6 was administered subcutaneously to Swiss Webster mice, followed by evaluation of bone marrow megakaryocyte ploidy, size and frequency, and median platelet volume 24, 48, and 72 hours after the initiation of IL-6 administration. In addition, bone marrow megakaryocyte morphology was examined using electron microscopy at 72 hours. IL-6 (10,000 U per subcutaneous injection) was administered three times during the first 24 hours, three times during the second 24 hours, and twice during the last 24-hour period. IL-6 bioactivity (10 U/ng) was determined using the IL-6-dependent murine hybridoma cell line B9. Megakaryocyte ploidy distribution, measured by two-color flow cytometry, demonstrated a shift in the modal ploidy class from 16N to 32N and a significant increase in the relative frequency of 64N megakaryocytes 48 and 72 hours (but not 24 hours) after initiation of IL-6 administration (cumulative doses of 60,000 and 80,000 U at 48 and 72 hours, respectively). In addition, ploidy levels were increased in animals that received a cumulative IL-6 dose of only 40,000 U (evaluated after 72 hours). The size of recognizable bone marrow megakaryocytes, determined by the cross-sectional areas of plastic embedded bone marrow megakaryocytes, was increased at the 48-hour (60,000 U IL-6) and 72- hour (80,000 U IL-6) time points. Megakaryocyte frequency, measured by flow cytometry, was unaffected at all time points and doses of IL-6. Median platelet volume, measured by electrical impedance, was not consistently altered by administration of IL-6. Electron microscopic examination of bone marrow megakaryocytes showed an increase in the proportion of megakaryocytes with a wide, peripheral, organelle- deficient zone from 20% +/- 9% (SD) in control animals to 50% +/- 7% (SD) (P less than .02) in animals that received IL-6. No changes were observed in the distribution of the demarcation membranes. IL-6 is a potent stimulator of murine megakaryocytopoiesis, in vivo, and appears to act early in megakaryocyte differentiation.
Leukemia inhibitory factor can potentiate murine megakaryocyte production in vitro
Authors: Metcalf D , Hilton D , Nicola NA
Published: October 2019

Receptors for murine leukemia inhibitory factor (LIF) were demonstrated on immature and mature murine megakaryocytes. LIF alone had no effects in culture on the survival or proliferation of normal murine megakaryocytes or their precursors. However, combination of LIF with multipotential-colony-stimulating factor (Multi-CSF) (interleukin-3) enhanced the megakaryocyte colony formation able to be stimulated by Multi-CSF; the enhancement involved all types of megakaryocyte colony and resulted in the formation of increased numbers of megakaryocytes. These observations provide a possible basis for the observation that, when LIF is injected in vivo, elevations are observed in megakaryocyte numbers and platelet levels.
Myeloid cell kinetics in mice treated with recombinant interleukin-3, granulocyte colony-stimulating factor (CSF), or granulocyte-macrophage CSF in vivo
Authors: Lord BI , Molineux G , Pojda Z , Souza LM , Mermod JJ , Dexter TM
Published: October 2019

Myeloid cell kinetics in mice treated with pure hematopoietic growth factors have been investigated using tritiated thymidine labeling and autoradiography. Mice were injected subcutaneously with 125 micrograms/kg granulocyte colony-stimulating factor (G-CSF) (in some cases 5 micrograms/kg), or 10 micrograms/kg of granulocyte-macrophage CSF (GM-CSF), or interleukin-3 (IL-3) every 12 hours for 84 hours. 3HTdR labeling was performed in vivo after 3 days of treatment. G-CSF increased the peripheral neutrophil count 14-fold and increased the proportion and proliferation rate of neutrophilic cells in the marrow, suppressing erythropoiesis at the same time. Newly produced mature cells were released into the circulation within 24 hours of labeling, compared with a normal appearance time of about 96 hours. By contrast, GM-CSF and IL-3 had little effect on either marrow cell kinetics or on the rate of release of mature cells, although GM-CSF did stimulate a 50% increase in peripheral neutrophils. Monocyte production was also increased about eightfold by G-CSF and 1.5-fold by GM-CSF, but their peak release was only slightly accelerated. While the peripheral half- lives of the neutrophilic granulocytes were normal, those of the monocytes were dramatically reduced, perhaps due to sequestration in the tissues for functional purposes. The stimulated monocyte production in the case of G-CSF required an additional five cell cycles, a level that might have repercussions on the progenitor compartments.
Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa
Authors: Austin RC , Rachubinski RA , Ofosu FA , Blajchman MA
Published: October 2019

Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III- Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT- III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III- Hamilton is capable of only the first of these events.
High-dose cyclophosphamide, carmustine, and etoposide followed by autologous peripheral stem cell transplantation for patients with relapsed Hodgkin's disease [published erratum appears in Blood 1991 Dec 15;78(12):3330]
Authors: Kessinger A , Bierman PJ , Vose JM , Armitage JO
Published: October 2019

Between February 1986 and March 1990, 56 patients with relapsed Hodgkin's disease treated with high-dose cyclophosphamide, carmustine, and etoposide (CBV) received an autologous peripheral stem cell transplantation (PSCT) rather than an autologous bone marrow transplantation (ABMT) because each patient had a marrow abnormality, either hypocellularity or tumor involvement. At least 6.5 x 10(8) [corrected] mononuclear cells/kg patient weight were collected from the peripheral blood of each patient, cyropreserved, and returned intravenously following CBV administration. Three patients had an early death 2, 22, and 25 days after PSCT. The actuarial event-free survival for these 56 patients at 3 years was 37% and was at least as good as that reported for relapsed Hodgkin's disease patients treated with CBV and ABMT. The 30 patients who had no marrow metastases at the time of PSC harvesting had an actuarial event-free survival of 47%, while those 26 patients with marrow metastases had a significantly different actuarial event-free survival of 27% (P = .02). CBV and PSCT for patients with relapsed Hodgkin's diseases who have marrow hypocellularity in traditional harvest sites or histopathologic evidence of BM metastases can result in long-term event-free survival.