bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Implications of Spliceosome Mutations: Epidemiology, Clonal Hematopoiesis, and Potential Therapeutic Strategies
Authors: Walter Matthew J. , Graubert Timothy A.
Published: October 2019

Somatic mutations in genes that are involved in pre-mRNA splicing (i.e., spliceosome genes) occur in ~50% of patients with myelodysplastic syndromes (MDS). Commonly mutated spliceosome genes (e.g., SF3B1, SRSF2, and U2AF1)have variable prognostic significance in patients with myeloid malignancies. For example, SRSF2 and U2AF1 mutations are associated with worse and SF3B1 mutations with better clinical outcomes compared to patients without a spliceosome gene mutation. While multiple groups have shown that RNA splicing is altered in patients with spliceosome gene mutations, the contribution of these alterations to disease pathogenesis is largely unknown. Several groups have recently reported that RNA splicing and hematopoiesis are altered in mutant expressing mouse models, providing direct evidence that spliceosome gene mutations contribute to some MDS phenotypes. Spliceosome gene mutations have also been detected in the blood cells of healthy elderly individuals with clonal hematopoiesis who are at an increased risk of developing hematopoietic cancer, further implicating these mutations as early genetic drivers in MDS. Consistent with this possibility, spliceosome gene mutations tend to occur in the founding clone (i.e., present in every tumor cell in a patient) of myeloid malignancies. Collectively, these observations indicate that targeting mutant cells could have a large clinical impact. Several observations indicate that spliceosome gene mutations may create a genetic vulnerability in cells that could be exploited therapeutically. Spliceosome gene mutations are typically heterozygous and mutually exclusive of each other in patients, implying either a redundancy in pathogenic function of mutant genes or that a cell cannot tolerate two spliceosome perturbations at once. Accumulating evidence now suggests that heterozygous mutant cells require appropriate expression levels of the non-mutant residual allele or other splicing factors to maintain cell viability. Therefore, we and others hypothesize that cells harboring spliceosome mutations will have increased sensitivity to pharmacological perturbation of the spliceosome, providing a new treatment approach for patients with these mutations. To examine this, we used sudemycin compounds that bind the SF3B1 protein to modulate pre-mRNA splicing. We show that treatment of U2AF1 mutant mice with sudemycin results in attenuation of mutant-associated hematopoietic progenitor cell expansion compared to control treated mice. Collectively, data from our group and others suggest that cells expressing mutant spliceosome genes have an increased sensitivity to pharmacologic perturbation of splicing. Clinical trials are being designed to treat spliceosome mutant hematological cancers with splicing modulator compounds.

Disclosures

No relevant conflicts of interest to declare.
Ferritinophagy and Cell Cycle Control
Authors: Carlomagno Francesca
Published: October 2019

The autophagic degradation of the iron-storage macromolecule ferritin, called ferritinophagy, is critical to restore the appropriate cellular iron levels and influences systemic iron homeostasis. Under low iron conditions, nuclear receptor coactivator 4 (NCOA4) protein accumulates and promotes, as cargo receptor, ferritinophagy. We have recently demonstrated that mice carrying genetic ablation of NCOA4 were unable to mobilize iron from deposits, featuring tissue iron overload as well as mild anemia. Because of impaired ferritinophagy, NCOA4 null mice displayed a severe microcytic hypochromic anemia and ineffective erythropoiesis when fed with an iron low diet. Conversely, they poorly tolerated an iron rich diet, dying prematurely from iron toxicity. Since in previous studies we discovered that nuclear NCOA4 is a chromatin binding protein that acts as a negative regulator of DNA replication origin activation, inhibiting the MCM2-7 DNA helicase, we also investigated whether NCOA4 could regulate DNA replication as a function of iron bioavailability. Treatment with iron chelators promoted a G1 phase cell cycle arrest, blocking DNA replication origins activation. In cell fractionation experiments, we observed that iron depletion induced not only cytosolic but also nuclear NCOA4 stabilization, and by chromatin immunoprecipitation (CHIP) and co-immunoprecipitation assays, we demonstrated that NCOA4 enriches at canonical DNA replication origins increasing the binding to MCM2-7 complex. Silencing of NCOA4 induced an unscheduled activation of DNA replication under iron-depleted conditions that promotes replication stress and impairs cell viability. In conclusion, our data indicate NCOA4 as a novel key iron responsive protein able to couple DNA replication origin activation to cellular iron levels.

Disclosures

No relevant conflicts of interest to declare.
Modeling Diamond Blackfan Anemia and p53 Activation in the Zebrafish
Authors: Zon Leonard I.
Published: October 2019

Marrow dysplasias are pre-leukemic conditions that include ribosomopathies, a group of rare genetic diseases, or myelodysplasia preceded by clonal expansion in older adults with somatic mutations. To discover novel therapies for ribosomopathies, we performed two chemical suppressor screens using a ribosomal protein mutant zebrafish and human induced pluripotent stem cells (iPSCs) derived from ribosomopathy patients. In the zebrafish screen, we found calmodulin inhibitors rescued the anemia in zebrafish and also rescued the erythroid defect of the mouse (in vivo) and human (in vitro) models of marrow failure. The screen in RPS19 mutant human iPSCs identified a compound, SMER28, which enhanced erythropoiesis in anemia models through an autophagy-dependent mechanism. The compounds identified in our chemicals screens have provided new insight into the etiology of marrow failure, and calmodulin inhibitors will soon be studied in a clinical trial with adult Diamond-Blackfan anemia patients. To study the abnormal clonal hematopoiesis associated with myelodysplasia, we have generated a new system using the zebrafish in which single stem cells and their progeny express unique combinations of fluorescent proteins, allowing them to be tracked over time by their specific color. Mosaic mutagenesis induced by clustered regularly interspaced short palindromic repeats /cas9 and/or gene overexpression in this system led to expansion of specifically colored blood, modeling the clonal hematopoiesis of human myelodysplasia. Our studies should provide new insight into the ribosomopathies and myelodysplasia.

Disclosures

Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
Selective Occurrence of Glutathione Instability in Red Blood Corpuscles of the Various Jewish Tribes
Authors: SZEINBERG A. , SHEBA CH. , ADAM A.
Published: October 2019

1. Experience with GSH stability test performed on blood samples to which glucose has been added is summarized and the procedure is recommended for routine use for the detection of sensitive subjects.

2. Glutathione stability of erythrocytes has been studied in various population groups of Israel. No case with instable GSH has been found among Jewish subjects originating from Eastern, Central or Western Europe. Instability of GSH was however found in about 20 per cent of subjects originating from Iraq and about 5 per cent of subjects originating from Yemen or North Africa. Isolated cases of this abnormality were also discovered among a small number of persons from other Oriental or Mediterranean countries as well as among the Arab inhabitants of Israel.

3. The genetic analysis points to a transmission of glutathione instability by a sex-linked, incompletely dominant gene with variable expressivity.

4. Variable expressivity of glucose-6-phosphate dehydrogenase activity has also been detected in the defective erythrocytes.
Platelet Adsorptive Properties and Platelet Extracts in Thromboplastin Generation
Authors: PERRY SEYMOUR , CRADDOCK CHARLES G.
Published: October 2019

(1) Simple incubation of normal platelets in saline for 10 minutes at 37 C. markedly diminishes their activity in the thromboplastin generation test. This loss of activity is due to the removel of a factor(s) from the platelet and is present in the saline extract.

(2) These "attenuated" platelets retain their effect on the recalcification time, prothrombin consumption, and clotting time of whole blood.

(3) Attenuated platelets, as well as platelets thought to be qualitatively defective, regain normal activity in the TGT after incubation in normal plasma or in plasmas from patients with qualitative platelet defects.

(4) Attenuated platelets, in contrast to qualitatively defective platelets, are restored to normal, as measured by the TGT, after treatment with saline extracts of normal platelets. Attenuated platelets, however, do not function normally after incubation with saline extracts of qualitatively defective platelets.

(5) The possible mechanisms involved are discussed, and it is concluded that the phenomenon involves the adsorption of plasma factor(s) by the platelet. The nature of the plasma factor(s) is not known.
The Effect of Acute Protein Deprivation upon Erythropoiesis in Rats
Authors: BETHARD W. F. , WISSLER R. W. , THOMPSON J. S. , SCHROEDER M. A. , ROBSON M. J.
Published: October 2019

1. Hemoglobin concentration, blood volume, and erythrocyte radioiron uptake were measured in rats subjected to acute protein deficiency.

2. Removal of protein from the diet was followed promptly by hemoconcentration, diminution in blood volume, and drastic reduction in erythropoiesis. These changes were reversible, after 35 days, upon addition of protein to the diet.

3. Protein intake is more essential for maintenance of normal erythropoiesis than is total caloric intake.

4. The data suggest that hemoglobin concentration within the vascular system is more important than red cell volume in regulating erythropoietic rate.
Erythrocyte Osmotic Fragility of Rats Receiving the Thermostable Plasma Erythropoietic Factor
Authors: LINMAN JAMES W. , LONG MARTHA J.
Published: October 2019

1. The thermostable erythropoietic factor in "anemic" rabbit plasma stimulates cellular division of marrow erythrocytic precursors in the normal rat producing erythrocytosis due to microcytes without evidence of augmented hemoglobin synthesis.

2. Study of erythrocyte osmotic behavior in these animals with a quantitative photocolorimetric method failed to demonstrate significant variation from the controls.

3. Osmotic fragility determinations by a direct cell enumeration technic revealed decreased resistance of the microcytes to lysis in hypotonic sodium chloride solutions.

4. It is concluded from these data that the erythrocytes in the normal rat injected with the heat stable plasma erythropoietic factor comprise two distinct cell types. They are microcytes with shortened survival times and normal erythrocytes.

5. The decreased viability of these microcytes is the apparent explanation for the rapid re-establishment of normal erythrocytic equilibrium after the "anemic" plasma extract injections are stopped.
Absorption of Cobalt60-Labeled Vitamin B12 After Subtotal Gastrectomy
Authors: LOEWENSTEIN FRITZ
Published: October 2019

Vitamin B12 absorption has been studied in patients with subtotal gastrectomy by means of the Co60 B12 urinary excretion test. Five patients tested before and after a conventional subtotal gastrectomy showed no significant change in absorption.

Of 22 patients who were tested at various intervals after a conventional (distal) subtotal gastrectomy, 19 had normal vitiamin B12 absorption and three had low absorption that was corrected to normal by giving gastric juice with the test dose. Three patients who had had a proximal gastrectomy showed normal vitamin B12 absorption, indicating that intrinsic factor was being produced by the remaining distal portion of the stomach.
The Efficiency of Oxidative Phosphorylation by Normal and Leukemic Human Leukocytes
Authors: DAVIS VIRGINIA E. , WILSON WILLIAM L. , SPURR CHARLES L.
Published: October 2019

1. A new modification of existing methods has been described for the separation of leukocytes from whole blood which provides a procedure for the rapid isolation of uninjured cells suitable for the study of oxidative phosphorylations.

2. This method has been employed in a study of the relative efficiency and yield of oxidative-linked phosphorylations mediated by normal and leukemic or immature leukocytes. The maximum aerobic phosphorylating capacity was exhibited by chronic lymphocytic leukemic leukocytes, followed in decreasing order of activity by acute monocytic leukemic leukocytes and chronic myelocytic leukemic leukocytes. Oxidative phosphorylation was not demonstrated with normal leukocytes.

3. Results of this study suggest that expression of leukocyte metabolic data on a unit nitrogen basis more accurately reflect the morphologically obvious size differences among the various leukocytes than presentation of data on a unit cell basis.

4. The aerobic phosphorylations mediated by leukemic leukocytes were found to be dependent upon substrate addition and were depressed by low levels of dinitrophenol. Under the experimental conditions employed in this study, glucose-6-phosphate was formed in stoicheiometric amounts. These results indicate that leukemic leukocytes are capable of the aerobic esterification of inorganic phosphate accompanying the oxidation of selected Kreb's cycle intermediates.
On the Occurrence of a Plasminogen-like Substance in Human Tissues
Authors: KOWALSKI E. , KOPEC M. , LATALLO Z. , ROSZKOWSKI S. , SENDYS N.
Published: October 2019

Evidence is presented for the occurrence of a plasminogen-like proenzyme in human tissues.

When homogenates of human connective tissue are incubated with SK they dissolve fibrin clots far more easily than control homogenates without SK. This dissolution of fibrin clots by tissue extracts is a proteolytic reaction similar to the dissolution of fibrin clots by plasma enzyme.

The chemical features of tissue "plasminogen" are the same as of blood plasminogen. It can be extracted at very high and very low PH, precipitated at pH 5.3 (isoelectric point), and inhibited by ions of heavy metals in exactly the same manner as blood plasminogen.

A similar behavior was found in the case of tissue plasminogen in accordance with recent views on the complex nature of the blood fibrinolytic system. The occurrence of tissue inhibitors of plasmin was proven, and it was shown, on comparing the activity of human and bovine tissues, that there is the possibility that in tissues there also exist a "proactivator" and "plasminogen" in accordance with Müllertz and Astrup.

Tissue "plasminogen" occurs chiefly in organs rich in connective tissue, like the aorta wall, fascia, etc. In other organs the "plasminogen" activity is negligible.

It is suggested that tissue "plasminogen" may be of significance in the pathogenesis of certain diseases of connective tissue, e.g. rheumatoid arthritis, in which proteolysis is postulated to be an important pathogenic factor.
Blood Coagulation in Acute Iron Intoxication
Authors: WILSON SLOAN J. , HEATH HELEN E. , NELSON PAUL L. , ENS G. GEORGE
Published: October 2019

Acute intestinal iron intoxication was produced in rabbits and the levels of serum were correlated with changes in blood coagulation.

Acute intestinal iron intoxication resulted in a prolongation of the coagulation time or a complete absence of coagulation, thrombocytopenia, hypoprothrombinemia, and qualitative changes in the fibrinogen. Clot retraction was decreased to absent.

The most marked defect occurred in fibrinogen. In the postiron period the fibrin clot was rust-colored, friable, and fragmented easily. The iron content of the fibrin was tremendous. The physiologic activity of the fibrinogen was decreased and coagulation prolonged.

Fibrinolytic studies revealed no increase in the lysis of the fibrin.

The decrease in the physiologic activity of the fibrinogen frequently produced a hemorrhagic level of prothrombin as measured by the one-stage method. The prothrombin as measured by the two-stage method, although decreased, was not in the hemorrhagic zone. Modification of the one-stage method, correlated with the prothrombin values as determined by the two-stage technic, revealed a defect in both prothrombin and fibrinogen.
Hemoglobin H-Thalassemia Disease
Authors: WOLFF JAMES A. , MICHAELS RICHARD H. , VON HOFE FREDERICK H.
Published: October 2019

A child of Italian parentage is presented as an example of Hgb H-thalassemia disease. A family study is included. Previous documented reports have included individuals of Chinese, Thai, Greek and Filipino parentage. Genetic aspects of the association of Hgb H with thalassemia are discussed. It is suggested that the study of other patients who present a clinical picture resembling mild Cooley’s anemia may reveal the presence of hemoglobin variants.
Therapy of Polycythemia Vera with Myleran
Authors: WALD NIEL , HOSHINO TAKASHI , SEARS MARY E.
Published: October 2019

Myleran was used in the therapy of nine relapses of polycythemia vera in five patients. Clinical examinations, blood studies, and, in three instances, radioisotope tracer tests before and after treatment demonstrated the effectiveness, safety and simplicity of the treatment. Further trial of Myleran therapy in polycythemia vera seems warranted.
The Efficiency of Oxidative Phosphorylation by Normal and Leukemic Human Leukocytes
Authors: Davis Virginia E. , Wilson William L. , Spurr Charles L.
Published: October 2019

Davis, Virginia E., Wilson, William L. and Spurr, Charles L.: The efficiency of oxidative phosphorylation by normal and leukemic human leukocytes. Blood 13:367-375 (April), 1958.

P. 369, line 11: for "reagent bank," read "reagent blank."

P. 370, line 5: for "3 µM DNP," read "3 µM DPN."

P. 370, table 1, column 1: for "DNP," read "DPN."
Chromatin Clumping in Mature Leukocytes: A Hitherto Unrecognized Abnormality
Authors: GUSTKE SUSAN S. , BECKER GARY A. , GARANCIS JOHN C. , GEIMER NICHOLAS F. , PISCIOTTA ANTHONY V.
Published: October 2019

A 62-year-old man and a 73-year-old woman were found to have a nuclear abnormality characterized by exaggerated clumping of the chromatin in the mature granulocytic and erythroid cells. The mature granulocytes showed loss of normal segmentation. The abnormality was not noted in other family members and appeared to be related to an atypical leukemic process. The nuclear abnormality was shown to be associated with a defect in cellular production within the marrow. Granulocyte precursors and normoblasts incorporated 3H-TdR but did not divide. Electron microscopy suggested the distribution of more than normal quantities of heterochromatin to mature leukocytes. Ferrokinetic studies disclosed ineffective erythropoiesis. The sharing of this abnormality by the myeloid and erythroid cells suggests a common stem cell origin.

A functional defect of phagocytosis and cellular aggregation was demonstrated in the neutrophils, but bacterial killing was not affected. The nuclear anomaly described herein may be a pre-leukemic abnormality.

Since this paper was submitted for publication, we have treated two more patients with an identical nuclear defect; an 82-year-old woman and a 79-year-old man. The woman succumbed of acute leukemic transformation, while the man is still alive.
Subcellular Distribution of 51Cr and Characterization of its Binding Sites in Human Platelets
Authors: STEINER MANFRED , BALDINI MARIO G.
Published: October 2019

Subcellular fractionation of human platelets labeled with radioactive sodium chromate was utilized to study distribution and characterization of the 51Cr binding sites in these cells. The cytoplasmic fraction of the platelets contained the major portion of the radioactivity while microsomal and mitochondrial fractions played a minor role in the binding of chromate. The stromal sediment had approximately one third of the total radioactivity. Only about 20 per cent of the 51Cr taken up by the platelets was in a free ionic form and was in the cytoplasmic fraction. The major portion of the 51Cr in the platelets was bound to nucleotides in the platelet cytoplasm. Nucleotides of the α-granules had no radioactive chromate. Hypertonic media and platelet antiserum produced marked release of 51Cr from the platelets, whereas aggregating agents produced none.

In physiologic conditions, elution of 51Cr from labeled platelets is very limited because there is only little 51Cr in free ionic form in the cells. Release of considerable amount of 51Cr from the platelets can occur only in case of cell damage with release of nucleotides from the cytoplasmic pool. This was seen with hypertonic solutions and with platelet antibody. Platelet degranulation, as it occurs with aggregating agents, is not associated with release of 51Cr since the granular pool of nucleotides is not labeled by this compound.
A monoclonal antibody-defined membrane antigen complex is required for neutrophil-neutrophil aggregation
Authors: Schwartz BR , Ochs HD , Beatty PG , Harlan JM
Published: October 2019

We examined the aggregation responses of normal neutrophils treated with the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produced dose-dependent inhibition of neutrophil aggregation in response to phorbol myristate acetate, zymosan-activated plasma, and N-formyl-methionylleucylphenylalanine. We conclude that the membrane glycoprotein complex recognized by MoAb 60.3--designated CDw18- -is required for neutrophil-neutrophil aggregation in vitro.
Lack of CD45 antigen on blast cells in childhood acute lymphoblastic leukemia is associated with chromosomal hyperdiploidy and other favorable prognostic features [see comments]
Authors: Behm FG , Raimondi SC , Schell MJ , Look AT , Rivera GK , Pui CH
Published: October 2019

The leukocyte common antigen (CD45) was detected on the surface of leukemic cells in 217 (87%) of 249 cases of newly diagnosed childhood acute lymphoblastic leukemia (ALL). All 55 cases of T-lineage ALL, compared with 159 of 191 B-lineage cases, expressed the CD45 antigen (P = .0005). The frequency of CD45 expression did not differ between cases of early pre-B (CD19+, cytoplasmic mu-) and pre-B (CD19+, cytoplasmic mu+) ALL. Cases of ALL lacking CD45 had significantly lower leukocyte counts (P = .002) and serum lactic dehydrogenase (LDH) levels (P = .007) and were more likely to have leukemic cell hyperdiploidy greater than 50 (P less than .0001) or a DNA index greater than 1.15 (P less than .0001), as compared with cases positive for the antigen. Of the 130 patients whose follow-up duration was sufficient for analysis of event-free survival, the 53 with the highest levels of CD45 expression (greater than or equal to 90%) were the most likely to have an adverse event on intensive multiagent chemotherapy. Patients without detectable CD45 had a negligible risk of failure. This study suggests a relationship between the expression of the CD45 antigen on leukemic lymphoblasts and other biologic factors that influence prognosis in ALL.
Transplantation of bone marrow cells from transgenic mice expressing the human MDR1 gene results in long-term protection against the myelosuppressive effect of chemotherapy in mice
Authors: Mickisch GH , Aksentijevich I , Schoenlein PV , Goldstein LJ , Galski H , Stahle C , Sachs DH , Pastan I , Gottesman MM
Published: October 2019

Many human cancers that are initially responsive to chemotherapy eventually fail to respond to treatment. For some drugs, dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow (BM) limit cytotoxic therapy. Approaches to prevent or circumvent BM toxicity are therefore a high priority of research on dose escalation protocols. In this study, we have transplanted BM cells from transgenic mice that constitutively express physiologic amounts of a functional human multidrug resistance (MDR1) cDNA to lethally irradiated C57BL/6 x SJL F1 mice (n = 36). From 6 weeks to 10 months after the transplant, all animals contained MDR1 DNA in spleen and BM specimens as indicated by Southern blot analysis, and expressed MDR1 messenger RNA in BM samples as detected by slot blot analysis. In addition, these animals were resistant to the myelosuppressive effect of doxorubicin, daunomycin, taxol, vinblastine, vincristine, etoposide, and actinomycin D, whereas control animals that were reconstituted with normal BM were drug sensitive. Finally, the chemoprotection afforded by the MDR1 gene could readily be reversed by adding chemosensitizers such as cyclosporin A and R-verapamil to chemotherapy. Hence, it appears that BM cells expressing the human MDR1 gene maintain this function after transplantation to host animals for a minimum of 10 months, and confer multidrug resistance to these BM recipients. This selective advantage conferred by expression of the MDR1 cDNA suggests a strategy for the use of MDR1 gene therapy in cancer chemotherapy and for the introduction of otherwise nonselectable genes into BM.
Polyclonal hematopoiesis in interferon-induced cytogenetic remissions of chronic myelogenous leukemia
Authors: Claxton D , Deisseroth A , Talpaz M , Reading C , Kantarjian H , Trujillo J , Stass S , Gooch G , Spitzer G
Published: October 2019

Interferon (IFN) therapy of early chronic myelogenous leukemia (CML) frequently produces partial or complete cytogenetic remission of the disease. Patients with complete cytogenetic remission often continue on therapy for several years with bone marrow showing only diploid (normal) metaphases. We studied hematopoiesis in five female patients with major cytogenetic remissions from CML during IFN therapy. Clonality analysis using the BstXI PGK gene polymorphism showed that granulocytes were nonclonal in all patients during cytogenetic remission. BCR region studies showed rearrangement only in the one patient whose remission was incomplete at the time of sampling. Granulopoiesis is nonclonal in IFN-induced remissions of CML and may be derived from normal hematopoietic stem cells.
UV irradiation can induce in vitro clonal anergy in alloreactive cytotoxic T lymphocytes
Authors: Kobata T , Ikeda H , Ohnishi Y , Urushibara N , Takahashi TA , Sekiguchi S
Published: October 2019

The alloreactive cytotoxic T lymphocytes (CTL) were generated by coculturing peripheral blood mononuclear cells (PBMC) with allogeneic Sa cells (an Epstein-Barr virus [EBV]-transformed B-cell line). The CTL did not proliferate in response to UV-B-irradiated Sa cells unless exogenous interleukin-2 (IL-2) was present, although they could kill the UV-B-irradiated Sa cells. The results indicate that UV-B-irradiated Sa cells do not provide sufficient signals for the proliferation of the CTL while they can be recognized by CTL and induce high-affinity IL-2 receptor (IL-2R) expression on them. The alloreactive CTL could be rendered anergic by previous exposure to UV-B-irradiated Sa cells. The alloreactive CTL previously stimulated with UV-B-irradiated Sa cells failed to proliferate in response to nontreated Sa cells. Proliferation of the anergic CTL could not be restored by Sa cells and exogenous IL-2 but by the combination of phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (A23187). The anergic CTL showed a considerably low cytotoxic activity against Sa target cells. The expression of TCR on the anergic CTL was downregulated while expression of high-affinity IL- 2R was upregulated. Their CD28 and CD8 expression were unchanged. In addition, the proliferative response and cytotoxicity of the anergic CTL to Sa cells could be restored after the cells had been rested for 7 days to allow reexpression of TCR. These results suggest that downregulation of T-cell receptor (TCR) and impairment in the post-IL- 2/IL-2R signaling pathway are relevant to the clonal anergy induced in the alloreactive CTL by stimulation of UV-B-irradiated Sa cells.
C-MYC translocations in de novo B-cell lineage acute leukemias with t(14;18)(cell lines Karpas 231 and 353)
Authors: Nacheva E , Dyer MJ , Fischer P , Stranks G , Heward JM , Marcus RE , Grace C , Karpas A
Published: October 2019

Rare de novo acute leukemias of mature B cells may exhibit the chromosomal translocation t(14;18)(q32.3; q21.3). We report the preliminary characterization of two cases that exhibited not only t(14;18)(q32.3;q21.3) but also the novel translocation t(8;9)(q24.1;p13.3), involving the C-MYC locus with unknown sequences at 9p13.3. From these cases, two Epstein-Barr virus negative cell lines (Karpas 231 and 353) with features identical to those seen in fresh cells from the patient have been derived. Both cell lines have complex karyotypes: in addition to both t(14;18)(q32.3;q21.3) and t(8;9)(q24.1;p13.3), cell line Karpas 231 exhibited three-way translocation t(1;3;11) (q42.3;q27.1;q23.1), whereas Karpas 353 exhibited t(1;3;7)(p32.1;q21.1;q22.1). Both cases retained immunophenotypes characteristic of mature B cells with no evidence for commitment to other hematopoietic lineages. Both cases expressed abundant, normal-sized C-MYC transcript, but no rearrangement of C-MYC DNA sequences could be detected using probes that span 80 kb around the C-MYC coding sequences. Breakpoints within the BCL-2 gene were divergent. In Karpas 353 the BCL-2 breakpoint occurred within the 3′ untranslated major breakpoint region (mbr) of the gene, whereas, in Karpas 231, breaks in both the 3′ mbr and in the region 5′ of the gene were detected. The cytogenetic combination of t(14;18)(q32.3;q21.3) and t(8;9)(q24.1;p13.3) has been previously reported in diffuse B cell lymphomas. This combination may be a new recurrent abnormality, of central pathogenic importance in the transformation of B cells to high grade malignancies through simultaneous deregulation of BCL-2 and C-MYC genes, constitutive expression of C-MYC being driven by currently unknown DNA sequences on chromosome 9p13.3. The presence of other complex translocations including those affecting 11q23.1 may further accelerate the process of acute transformation.
Characterization of posttransplant lymphomas that express T-cell- associated markers: immunophenotypes, molecular genetics, cytogenetics, and heterotransplantation in severe combined immunodeficient mice
Authors: Waller EK , Ziemianska M , Bangs CD , Cleary M , Weissman I , Kamel OW
Published: October 2019

Immunosuppressed individuals are at high risk for the development of hematologic malignancies. The typical lymphomas arising in organ transplant recipients are B-cell non-Hodgkin's lymphomas that contain Epstein-Barr virus (EBV) DNA sequences. We investigated the characteristics of posttransplant lymphomas that lacked expression of the usual markers associated with EBV transformation. We describe four large-cell lymphomas seen recently at our institution. Two of these four cases were CD4+, one was CD8+, and in one staining for CD4 and CD8 expression was not performed. One CD4+ lymphoma was a CD30+, EBV- large- cell lymphoma from a 65-year-old kidney transplant recipient, the second was an EBV+ large-cell lymphoma from a 25-year-old heart transplant patient. Two T-cell lymphomas were EBV+ and had clonal T- cell receptor beta gene rearrangements. The other two lymphomas expressed T-cell markers CD4 and CD43, and lacked expression of B-cell markers CD19, CD20, CD21, CD22, CD23, and surface Ig. Both CD4+ lymphomas were tumorigenic after their heterotransplantation into severe combined immunodeficient (SCID) mice. Cytogenetics, immunophenotyping, and genotyping of the secondary tumors from SCID mice showed their clonality and identity with the patients' primary tumors. Novel CD4+ lymphoma cell lines, LH521/4 and LK418/4, were established from tumors that had been passaged in SCID mice. An immunodeficient environment may facilitate the growth of these T-cell or biphenotypic lymphomas; the etiology of their genesis can include transformation with EBV and other, as yet unidentified mechanisms.
Adhesion to high endothelial venules: a model for dissemination mechanisms in non-Hodgkin's lymphoma
Authors: Stauder R , Hamader S , Fasching B , Kemmler G , Thaler J , Huber H
Published: October 2019

The interaction of human lymphoma cells with high endothelial venules (HEVs) on sections of lymphatic tissues was studied in 44 cases of non- Hodgkin's lymphoma (NHL) with the in vitro HEV binding assay. The relative adherence ratio (RAR) of lymphoma cells to HEVs as related to that of reactive lymphocytes was 0.29 to 4.64 in 38 cases of B chronic lymphocytic leukemia (CLL), 1.15 and 1.54 in two cases of immunocytic NHL, 1.12 and 0.70 in two cases of centrocytic NHL, 1.98 in one case of a peripheral T-NHL, whereas plasma cell leukemia cells adhered very weakly (RAR 0.1). Among the patients suffering from CLL a pronounced HEV binding ability of tumor cells correlated significantly with the more unfavorable Binet stages B and C (median 1.32) as well as with a widespread lymphatic dissemination, which strongly indicates a hematogenous, HEV-mediated spread (median 1.34). In contrast, weak adherence to HEVs was associated with Binet stage A (median 0.85; P < .05) and with a lacking or only localized clinical involvement of lymph nodes (median 0.84; P < .01). Thus, specific HEV recognition processes even operate in lymphoid neoplasms and via this mechanism seem to influence the dissemination of tumors.
Effect of thrombin, the thrombin receptor activation peptide, and other mitogens on vascular smooth muscle cell urokinase receptor mRNA levels
Authors: Reuning U , Little SP , Dixon EP , Bang NU
Published: October 2019

Bovine vascular smooth muscle cells (SMC) express the urokinase-type plasminogen activator receptor (u-PAR) claimed to be important in cell invasion. Receptor numbers and affinity are regulated by thrombin and several other mitogens involved in SMC proliferation. We investigated the effects of these mitogens on u-PAR mRNA levels. On continuous thrombin stimulation the u-PAR message in SMC was 10 +/- 2.3-fold elevated reaching a maximum between 6 and 9 hours and declining to control values within 48 hours. Thrombin present for 30 minutes on the cell surface produced similar effects. Stimulation with the thrombin receptor activation peptide S-F-L-L-R-N representing the NH2-terminus of the tethered ligand also increased u-PAR mRNA levels with an identical time course. D-Phe-Pro-Arg-chloromethyl ketone (PPACK) active site blocked thrombin and the catalytically inactive thrombin mutant S205A did not affect u-PAR mRNA levels. Thrombin stimulation also resulted in a 2 +/- 0.2-fold transient increase in thrombin receptor mRNA preceding the rise in u-PAR message. Transforming growth factor beta 1 (TGF beta 1) and platelet-derived growth factor (PDGF) showed similar time courses for the elevation of u-PAR mRNA levels with a maximal 5.5 +/- 0.9 and 12 +/- 2.5-fold increase, respectively. Basic fibroblast growth factor (bFGF) and phorbol myristate acetate (PMA) showed a more prolonged effect increasing u-PAR mRNA levels 8 +/- 2.0- fold and 12.3 +/- 2.5-fold, respectively, within 6 hours but remaining 5 to 10-fold elevated at 48 hours. In order to decide if the u-PAR mRNA increase was due to message stabilization or a consequence of transcriptional activation we used the RNA polymerase II inhibitor 5,6- dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) during the stimulation experiments. u-PAR mRNA levels on TGF beta 1 stimulation of SMC decayed after the addition of DRB indicating that enhancement of transcriptional activity was involved in the induction. In contrast, the time course of u-PAR mRNA elevation on thrombin, bFGF, and PMA stimulation was not significantly altered in the presence of DRB suggesting that in these latter cases u-PAR mRNA message accumulation was at least in part due to mRNA stabilization. Increased transcriptional activity, mRNA stabilization and expression of u-PAR protein on the SMC surface in response to growth factors may facilitate enhanced cell surface protease activity, cell migration, and development of atheromatous lesions.
Chronic granulomatous disease and glutathione peroxidase deficiency, revisited
Authors: Newburger PE , Malawista SE , Dinauer MC , Gelbart T , Woodman RC , Chada S , Shen Q , van Blaricom G , Quie PG , Curnutte JT
Published: October 2019

We have restudied two kindreds that formed the basis of the original report of autosomal recessive chronic granulomatous disease (CGD) associated with leukocyte glutathione peroxidase deficiency. Case 1 from the original study and the surviving brother of the originally reported case 2 both have severe CGD, with no detectable respiratory burst activity in purified intact neutrophils. However, their leukocytes exhibit normal glutathione peroxidase enzyme activity and gene expression. Examination of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase components known to be defective in CGD reveals no detectable cytochrome b558 nor any membrane activity in a cell-free NADPH oxidase assay system. Molecular analysis of the genes encoding cytochrome b558 subunits shows, in case 1, a C-->T substitution at nucleotide 688 of the gene encoding the gp91-phox subunit of cytochrome b558, resulting in a termination signal in place of Arginine-226. Levels of gp91-phox mRNA are markedly decreased despite normal levels of gene transcription, indicating a post- transcriptional effect of the nonsense mutation on mRNA processing or stability. The X-linked form of CGD developed in this cytogenetically normal female due to the uniform inactivation of the normal X chromosome in her granulocytes, indicated by the expression in her granulocyte mRNA of only one allele of a glucose-6-phosphate dehydrogenase polymorphisms for which she is heterozygous in genomic DNA. Case 2 (of the present study) has distinct mutations in each allele of the p22-phox gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Interleukin-6 and Epstein-Barr virus induction by cyclosporine A: potential role in lymphoproliferative disease
Authors: Tanner JE , Menezes J
Published: October 2019

Posttransplant patients undergoing prolonged cyclosporine A (CsA) immunosuppressive therapy have been reported to have increased incidence of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. We undertook experiments to analyze the possible actions of CsA during EBV-infection of human peripheral blood mononuclear cells (PBMC). EBV-infected B cells cultured with CsA demonstrated increased EBV B-cell outgrowth as compared with those cultured without CsA. PBMC, after infection with EBV and CsA treatment, demonstrated increased interleukin-6 (IL-6) activity in the culture supernatant. The induction of IL-6 appears to differ within the various lymphocyte populations. In monocytes, IL-6 expression appears preferentially induced by EBV and is initiated by the binding of the two major virion glycoproteins, gp350 and gp220. Expression of IL-6 in T cells appears to be due mainly to CsA. B cells also express IL-6 after EBV exposure, but not after CsA treatment. EBV-immortalized B-cell lines cultured with CsA exhibited both an increased number of cells expressing viral lytic-cycle antigens and increased amounts of lytic-cycle proteins. IL-6, which is induced by CsA in PBMC, was also capable of inducing the lytic viral cycle in several EBV-immortalized cells. CsA, in promoting both increased numbers of lytic EBV B cells and an EBV paracrine factor, IL-6, within the microenvironment of EBV B cell:T cell and EBV B cell:monocyte interactions, may result in increased EBV B-cell immortalization and ultimately lead to the promotion of B-cell lymphomas in immunosuppressed patients.
Long-term follow-up of patients with hairy cell leukemia after treatment with 2'-deoxycoformycin
Authors: Kraut EH , Grever MR , Bouroncle BA
Published: October 2019

Twenty-four patients with advanced hairy cell leukemia treated with 2'- deoxycoformycin (dCF) were studied after achieving complete remission to determine the impact of treatment on survival, disease-free survival, long-term complications of treatment, and response to retreatment. At a median follow-up time of 82 months (range, 54 to 104 months), 23 of 24 patients remain alive. One patient has died of recurrent disease refractory to treatment. Of the remaining 23 patients, 11 have relapsed at a median time of 30 months (range, 7 to 80 months) after treatment completion. Of these 11 patients, 7 have been retreated with dCF or 2'-chlorodeoxyadenosine (2-CdA), including one patient that was retreated twice. All seven patients have responded, with five patients achieving second complete remission. Two patients have had normalization of blood cell counts, but repeat bone marrows have not been performed. No serious infections have been seen in dCF-treated patients during follow-up. One case of Hodgkin's disease and three cases of skin malignancies have developed in these 24 patients. From initiation of treatment, survival is 93 months (range, 63 to 116 months). We concluded that dCF significantly prolongs the survival of patients with advanced hairy cell leukemia without resultant long-term complications. It is too early to predict if this therapy will be curative for the patients still in remission.
Retinoids (all-trans and 9-cis retinoic acid) stimulate production of macrophage colony-stimulating factor and granulocyte-macrophage colony- stimulating factor by human bone marrow stromal cells
Authors: Nakajima H , Kizaki M , Sonoda A , Mori S , Harigaya K , Ikeda Y
Published: October 2019

Retinoic acids (RAs) exert pleiotropic effects on cellular growth and differentiation. All-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), a stereoisomer of ATRA, induce differentiation of leukemic cell lines and cells from patients with acute myelogenous leukemia (AML) in vitro. Despite information on the effects of RAs on hematopoietic cells, little is known about how RAs act on the hematopoietic microenvironment, especially on bone marrow stromal cells. Based on recent observations that various cytokines produced mainly by bone marrow stromal cells regulate hematopoiesis, we analyzed the effects of RAs on cytokine production by these cells. ATRA or 9-cis RA treatment of human bone marrow stromal cell line KM101, which produces macrophage colony-stimulating factor (M-CSF) and granulocyte- macrophage colony-stimulating factor (GM-CSF) constitutively, enhanced mRNA levels of both cytokines in a dose-dependent manner. Both RAs also stimulated M-CSF production from primary cultures of human bone marrow stromal cells. Both retinoic acid receptor (RAR)-alpha and retinoid X receptor (RXR)-alpha were expressed constitutively in KM101 cells. ATRA did not affect the expression of either receptor, whereas 9-cis RA increased RXR-alpha mRNA expression in a dose-dependent manner, but did not affect levels of RAR-alpha mRNA. These findings may have important biologic implications for both the role of RAs in hematopoiesis and the therapeutic effects of ATRA on the hematopoietic microenvironment in patients with acute promyelocytic leukemia (APL).
Role of the B domain for factor VIII and factor V expression and function
Authors: Pittman DD , Marquette KA , Kaufman RJ
Published: October 2019

Factor V and factor VIII are homologous cofactors in the blood coagulation cascade that have the domain structure A1-A2-B-A3-C1-C2, of which the B domain has extensively diverged. In transfected COS-1 monkey cells, expression of factor VIII is approximately 10-fold less efficient than that of factor V, primarily because of inefficient protein secretion and, to a lesser extent, reduced mRNA expression. To study the functional significance and effect of the B domain on expression and activity, chimeric cDNAs were constructed in which the B domains of factor V and factor VIII were exchanged. Expression of a factor VIII chimera harboring the B-domain of factor V yielded a fully functional factor VIII molecule that was expressed twofold more efficiently than wild-type factor VIII because of increased mRNA expression. Thus, sequences within the factor VIII B domain were not responsible for the inefficient secretion of factor VIII compared with factor V. Expression of a factor V chimera harboring the B domain of factor VIII was slightly reduced compared with wild-type factor V, although the secreted molecule had significantly reduced procoagulant activity correlating with dissociated heavy and light chains and resistance to thrombin activation. Interestingly, the factor V chimera containing the factor VIII B domain was efficiently activated by Russell's viper venum (RVV). A factor V B domain deletion (residues 710- 1545) molecule also exhibited significantly reduced procoagulant activity caused by resistance to thrombin cleavage and activation, although this molecule was activatable by RVV. These results show that, in contrast to factor VIII, thrombin activation of factor V requires sequences within the B domain. In addition, thrombin activation of factor V occurs through a different mechanism than activation by RVV.
YC-1, a novel activator of platelet guanylate cyclase
Authors: Ko FN , Wu CC , Kuo SC , Lee FY , Teng CM
Published: October 2019

YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] inhibited the aggregation of and ATP release from washed rabbit platelets induced by arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), and thrombin in a concentration-dependent manner. YC-1 also disaggregated the clumped platelets caused by these inducers. The thromboxane B2 formation caused by collagen, PAF, and thrombin was inhibited by concentrations of YC-1 that did not affect formation of thromboxane B2 and prostaglandin D2 caused by AA. YC-1 suppressed the increase of intracellular Ca2+ concentration and generation of inositol 1,4,5-trisphosphate caused by these five aggregation inducers. Both the cAMP and cGMP contents of platelets were increased by YC-1 in a concentration- and time-dependent manner. Like sodium nitroprusside, YC- 1 potentiated formation of cAMP caused by prostaglandin E1 but not that by 3-isobutyl-1-methylxanthine. Adenylate cyclase and cAMP phosphodiesterase activities were not altered by YC-1. Activity of cGMP phosphodiesterase was unaffected by YC-1. Activities of guanylate cyclase in platelet homogenate and cytosolic fraction were activated by YC-1, whereas particulate guanylate cyclase activity was unaffected. The antiplatelet effect of sodium nitroprusside but not that of YC-1 was blocked by hemoglobin and potentiated by superoxide dismutase. After intraperitoneal administration for 30 minutes, YC-1 prolonged the tail bleeding time of conscious mice. These data indicate that YC-1 is a direct soluble guanylate cyclase activator in rabbit platelets. It may also possess antithrombotic potential in vivo.