bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophilic-chronic myeloid leukemia: a distinct disease with a specific molecular marker (BCR/ABL with C3/A2 junction) [see comments]
Authors: Pane F , Frigeri F , Sindona M , Luciano L , Ferrara F , Cimino R , Meloni G , Saglio G , Salvatore F , Rotoli B
Published: October 2019

Neutrophilic-chronic myeloid leukemia (CML-N) is a rare myeloproliferative disorder that runs a much more benign course than chronic myeloid leukemia, and for which no specific underlying molecular lesion has been described so far. We have analyzed the genomic DNA by Southern blotting and the BCR/ABL hybrid gene transcripts by reverse transcriptase-polymerase chain reaction in three patients with clinical findings of CML-N, who did have a t(9;22) chromosomal translocation. In all patients we have found a rare type of BCR/ABL rearrangement, with a breakpoint between exons c3 and c4 of the BCR gene (corresponding to BCR exons 19 and 20). This was confirmed by hybridization with an oligonucleotide probe spanning the c3/a2 region. This type of junction causes almost the entire BCR gene to fuse with ABL. The junction is in frame and it gives rise to a fusion protein of predicted 230 kD. Our data now provide a molecular diagnostic marker for CML-N, and they are consistent with the notion that the inclusion or exclusion of BCR exons in the fusion protein affects dramatically its capacity to derange myeloid proliferation and differentiation, leading to the appearance of different disease phenotypes.
A variant CD30 protein lacking extracellular and transmembrane domains is induced in HL-60 by tetradecanoylphorbol acetate and is expressed in alveolar macrophages
Authors: Horie R , Ito K , Tatewaki M , Nagai M , Aizawa S , Higashihara M , Ishida T , Inoue J , Takizawa H , Watanabe T
Published: October 2019

We identified and cloned cDNAs for two novel CD30 mRNAs of 2.3 kb that are induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in the human myeloid leukemia cell line HL-60. These transcripts were transcribed from the intronic region just upstream of the exon coding for the transmembrane domain of the CD30 protein. The shorter cDNA had a deletion of 54 nucleotides corresponding to the 3′ region of the transmembrane domain of the CD30 and which was probably caused by alternative splicing. Translation of these transcripts appeared to start from the internal methionine codon at nucleotide position 289 that corresponds to that of 1612 in the CD30 cDNA, and encode a protein of 132 amino acid residues which corresponds exactly to the C-terminal cytoplasmic domain of CD30 protein. The calculated molecular mass of this variant CD30 (CD30v) protein was 14,087. Thus, the predicted CD30v protein retains most of the cytoplasmic region, but lacks the extracellular and transmembrane domains. Northern blots detected the expression of CD30v transcripts only in the lung and the TPA-stimulated HL-60 cell line. Translation of this mRNA in vitro produced a protein of 25 kD. Immunoblotting analysis with HCD30C1, a rabbit polyclonal antibody raised against the cytoplasmic domain of CD30 protein, detected proteins with an apparent Mr 25 kD expressed in TPA-stimulated HL-60 and COS-7 cells that were transfected with both types of CD30v cDNAs. Constitutive phosphorylation of the CD30v protein was demonstrated by in vitro labeling with [32P]. Immunohistochemical studies demonstrated CD30v protein was in alveolar macrophages. Cotransfection experiments using a kappa B-site-dependent reporter construct showed that CD30v can transactivate gene expression through activation of NF kappa B, as was noted on the authentic CD30 protein. Overexpression of the CD30v induced differentiation of HL-60 cells as evidenced by an increased NBT reduction activity. These observations provided new insights into the molecular heterogeneity and biological function of CD30 in myeloid cells.
Targeted T-cell therapy for human leukemia: cytotoxic T lymphocytes specific for a peptide derived from proteinase 3 preferentially lyse human myeloid leukemia cells
Authors: Molldrem J , Dermime S , Parker K , Jiang YZ , Mavroudis D , Hensel N , Fukushima P , Barrett AJ
Published: October 2019

Proteinase 3 is present in high concentration in the primary granules of acute and chronic myeloid leukemia blasts, and may represent a potential T-cell target antigen. We screened proteinase 3 against the binding motif of HLA-A2.1. Based on its high predicted binding, a 9-mer peptide, “PR-1,” was synthesized and tested for binding to HLA-A2.1 using the T2 cell line. PR-1 at 100 micrograms/mL significantly increased expression of HLA-A2.1, with median channel of fluorescence increasing from 22 to 294. Binding half-life was determined to be 1,460 minutes by I125-labeled beta 2-microglobulin incorporation. HLA-A2.1+ peripheral blood mononuclear cells from a normal donor were used to generate a T-cell line specific for PR-1. The line demonstrated 85% PR-1-specific lysis at an E:T ratio of 50:1, compared with 20% lysis without PR-1, using T2 cells as targets. It also showed 79% specific lysis to fresh chronic myelogenous leukemia blasts, 54% to fresh acute myelogenous leukemia blasts, and only background lysis (< 20%) to HLA-A2.1+ normal allogeneic marrow cells. The amount of lysis of HLA-A2.1+ myeloid cells was proportional to cytoplasmic proteinase 3 expression. Thus, HLA-A2.1-restricted cytotoxic T cells, raised against a peptide contained in proteinase 3, preferentially lysed fresh human leukemic cells.
Estimation of the number of hematopoietic precursor cells during fetal mouse development by covariance analysis
Authors: Berger CN , Sturm KS
Published: October 2019

Differentiation of hematopoietic precursor cells results in the formation of clonally related descendent cells. Using the mosaic expression of beta-galactosidase in female mouse fetuses heterozygous for an X-linked lacZ transgene, we analyzed the clonal relationship of the hematopoietic progeny. The proportion of beta-galactosidase positive cells for different T- and B-lymphoid and myeloid cell populations was determined at different stages of fetal development. We found excellent correlations of the proportion of beta-galactosidase expressing cells for all hematopoietic lineages confirming that they share a common ancestry. Therefore, it was possible to estimate the number of common precursor cells (PC) based on binomial distribution and covariance analysis of pairs of different hematopoietic cell populations. Our results obtained from hematopoietic cells at 15.5 to 18.5 days of gestation indicated the presence of 15 to 18 lymphoid and 18 to 22 myeloid/lymphoid specific precursor cells. Statistical analysis of the precursor cell numbers showed a trend of increasing numbers that was highly significant. The precursor cell number was inversely related to maturity of the cell populations analyzed; ie, the lowest number of lymphoid and lymphoid/myeloid precursors was calculated when the most mature CD3+ T-cell population was used for comparison. Determination of PC numbers can therefore be used to assess the relative maturity and developmental potential of individual cell populations.
Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis
Authors: Cohen-Solal K , Villeval JL , Titeux M , Lok S , Vainchenker W , Wendling F
Published: October 2019

Mpl ligand (thrombopoietin [TPO]) is the physiological regulator of platelet production. In mice, mRNA encoding the Mpl ligand (Mpl-L) is predominantly found by Northern blot analysis in the liver and kidney. To investigate the mode of regulation of the Mpl-L gene, we have developed several experimental models of severe thrombocytopenia differing in their kinetics and an opposite model of chronic thrombocytosis. Northern analysis performed at various times after induction of a thrombocytopenic state demonstrates that, whatever the number of circulating platelets, no change in Mpl-L mRNA level occurs in liver and kidney. By ribonuclease protection assays, we analyzed the ratios between mRNAs coding for the wild-type Mpl-L form and various splice variants encoding inactive or nonsecreted Mpl-L proteins. No modification in levels of these various isoforms was detected confirming the data of a previous report. Because the highest level of Mpl-L bioactivity in sera was observed only in mice with drastically reduced numbers of both platelets and megakaryocytes, these results further suggest that not only platelets, but also megakaryocytes, must be involved in the regulation of the level of circulating Mpl-L. In addition, we show that no downregulation of wild-type Mpl-L mRNA and no change in the ratio of Mpl-L mRNA isoforms were detected in mice in which a chronic thrombocytosis was induced. Together, these different models extend and further confirm that the regulation of Mpl-L does not occur at a transcriptional level or by a modulation in the ratios of Mpl-L mRNA isoforms.
Affinity modulation of the platelet integrin alpha IIb beta 3 by alpha- chymotrypsin: a possible role for Na+/Ca2+ exchanger
Authors: Shiraga M , Tomiyama Y , Honda S , Kashiwagi H , Kosugi S , Handa M , Ikeda Y , Kanakura Y , Kurata Y , Matsuzawa Y
Published: October 2019

In the present study, we have investigated the mechanism of affinity modulation of alpha IIb beta 3 by chymotrypsin. We first confirmed that alpha-chymotrypsin could activate alpha IIb beta 3 (approximately 7,000 molecules per platelet) without major intracellular signaling. However, we unexpectedly found that high concentrations of amiloride dose-dependently inhibited 125I-fibrinogen binding to the chymotrypsin-treated platelets, as well as the platelet aggregation (IC50 [50% inhibitory concentration] for fibrinogen binding, 530 mumol/L). In contrast, amiloride did not inhibit alpha IIb beta 3 activation induced by anti-alpha IIb beta 3 monoclonal antibody PT25–2 or AP5. To identify the pathway involved, the effects of alteration of Na+ gradient in platelets were examined. Lowering Na+ gradient by replacing extracellular Na+ with tetramethylammonium (TMA) increased the number of activated alpha IIb beta 3 by twofold, as assessed by fibrinogen-binding assay. The incubation of platelets with ouabain, a Na+/K(+)- adenosine triphosphatase (ATPase) inhibitor, further augmented alpha IIb beta 3 activation. These data suggested that a likely candidate for the pathway was Na+/Ca2+ exchanger. At 140 mmol/L [Na+]o, 45Ca2+ influx to the chymotrypsin-treated platelets was twofold greater than that to non-treated platelets. Replacement of Na+ with TMA further increased the Ca2+ influx, and the increase was inhibited by amiloride dose-dependently. 3′,4′-Dichlorobenzamil (DCB) and bepridil, relatively specific inhibitors of Na+/Ca2+ exchanger, also inhibited the chymotrypsin-induced alpha IIb beta 3 activation, and the IC50 values of these inhibitors for fibrinogen binding were 25 mumol/L and 52 mumol/L, respectively. Moreover, platelet aggregation induced by various physiologic agonists was inhibited by DCB or bepridil, while platelet agglutination by ristocetin was not. Our data newly suggest that Na+/Ca2+ exchanger operating in reverse mode may be directly involved in inside-out signaling that activates alpha IIb beta 3.
Identification of human juvenile chronic myelogenous leukemia stem cells capable of initiating the disease in primary and secondary SCID mice
Authors: Lapidot T , Grunberger T , Vormoor J , Estrov Z , Kollet O , Bunin N , Zaizov R , Williams DE , Freedman MH
Published: October 2019

Most juvenile chronic myelogenous leukemia (JCML) cells have limited long-term proliferative capacity, and only a minority of immature cells give rise to colonies in semisolid cultures. Clonogenic JCML progenitors cannot be maintained in culture because they differentiate, and within a few weeks the leukemic clone is lost. This makes it difficult to identify the cell that initiates and maintains the disease in patients. To determine the proliferative capacity of JCML cells in vivo, bone marrow (BM), peripheral blood, or spleen cells from eight patients with JCML either at diagnosis or during treatment were transplanted into sublethally irradiated severe combined immune deficient (SCID) mice. JCML cells from all patients homed to the murine BM and proliferated extensively in response to exogenous stimulation with granulocyte-macrophage colony-stimulating factor. Within a few weeks, highly engrafted mice became ill and cachectic due to infiltration of leukemic cells and secretion of tumor necrosis factor-alpha. Murine BM, spleen, and liver were infiltrated with leukemic blasts, and typical JCML colony-forming progenitors could be recovered. Kinetic experiments demonstrated that only a small minority of transplanted cells homed to the murine BM, and that these cells initiated and maintained the disease in vivo by extensive proliferation and differentiation. To characterize the cell-surface phenotype of the JCML initiating cell (JCML-IC), JCML blood or spleen cells were fractionated on the basis of CD34/CD38 marker expression and transplanted into SCID mice. Only immature CD34+ cells could initiate the disease, while mature CD34- cells did not engraft. Within the CD34+ compartment, there was enrichment for JCML-ICs by immature cells with a CD34+/CD38- stem-cell-like phenotype. Mice transplanted with more mature CD34+/CD38+ populations that also contained clonogenic JCML progenitors were poorly engrafted. These results indicate that the JCML-IC is an earlier stage of development than clonogenic JCML progenitors. Additional evidence that the JCML-IC has stem-cell properties comes from secondary transplant experiments that test the self-renewal capacity. The JCML-IC from all three patients tested could successfully reinitiate the disease in secondary murine recipients. Thus, we have developed a functional in vivo model that replicates many aspects of human JCML, and have used this model to identify and characterize JCML-ICs and their stem-cell properties.
Alterations in expression, binding to ligand and DNA, and transcriptional activity of rearranged and wild-type retinoid receptors in retinoid-resistant acute promyelocytic leukemia cell lines
Authors: Rosenauer A , Raelson JV , Nervi C , Eydoux P , DeBlasio A , Miller WH Jr
Published: October 2019

All-trans retinoic acid (tRA), a naturally occurring ligand of the nuclear retinoic acid receptors (RARs), induces differentiation of leukemic cells and clinical complete remission in patients with acute promyelocytic leukemia (APL). This differentiation effect can also be seen in vitro in both fresh leukemic cells and in the unique permanent APL cell line, NB4. However, APL cells become resistant to RA-induced differentiation both in vitro and in patients. Although pharmacodynamic mechanisms of resistance have been reported, there is growing evidence that resistance both in patients, as well as in vitro, can be mediated by changes in the sensitivity of leukemic cells to retinoids. To investigate possible mechanisms of retinoid resistance, we established subclones of NB4 that are stably resistant to both tRA and 9-cisRA. Unlike the previously reported NB4.306 retinoid-resistant cells, these subclones expressed PML/RAR-alpha RNA and protein, but demonstrated altered ligand binding patterns of PML/RAR-alpha and differed in retinoid-induced gene expression. They were significantly less able to stimulate transcription of an RARE driven CAT-reporter gene on induction by tRA and showed altered DNA binding activity on a RARE. These data suggest that NB4 cells selected for resistance to retinoids demonstrate abnormal ligand binding to PML/RAR-alpha that lead to altered transcriptional activation by retinoids.
Genomic structure, chromosomal localization, start of transcription, and tissue expression of the human p40-phox, a new component of the nicotinamide adenine dinucleotide phosphate-oxidase complex
Authors: Zhan S , Vazquez N , Zhan S , Wientjes FB , Budarf ML , Schrock E , Ried T , Green ED , Chanock SJ
Published: October 2019

p40-phox is a newly isolated cytosolic component of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase that copurifies with p67-phox. Although its function is not well defined, preliminary evidence indicates that it is a component of the cytosolic complex. We report the characterization of the human p40-phox gene, which is single copy and spans approximately 18 kb with 10 exons. Based on fluorescent in situ hybridization (FISH) studies and analysis of somatic hybrid cell lines, the chromosomal location of p40-phox is human chromosome 22q13.1. The start of transcription has been mapped to bp -156. The expression of p40-phox message is restricted to hematopoietic cells. In addition to identifying the mRNA transcript on Northern blot analysis in cells known to express components of the NADPH-oxidase, polymorphonuclear leukocytes, monocytes, B lymphoblastoid cell lines, and eosinophils, p40-phox is also expressed in two other cell types of white cell lineage, mast cells, and basophils. In addition, the mRNA for p40-phox is expressed in megakaryocytic cells, but not in erythroid cells.
Molecular genetic analysis of variant phenotypes of the ABO blood group system
Authors: Ogasawara K , Yabe R , Uchikawa M , Saitou N , Bannai M , Nakata K , Takenaka M , Fujisawa K , Ishikawa Y , Juji T , Tokunaga K
Published: October 2019

ABO is clinically the most important blood group system in transfusion medicine and includes many variant phenotypes. To understand the molecular genetic basis of this polymorphic system, we have analyzed genomic DNAs obtained from Japanese individuals possessing variant ABO phenotypes including A2, Ax, Ael, cis-AB, Bx, and Bel. By polymerase chain reaction-single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses, we identified 11 different alleles. These alleles had nucleotide sequences different from those of the previously described 13 different alleles responsible for the common ABO phenotypes. Analysis of the nucleotide sequences of the alleles responsible for those variant phenotypes showed that the amino acid residues at position 266 and 268 may be crucial for transferase specificity, whereas those at positions 214, 216, 223, 291, and 352 may be critical for the activity level. Nine of the 11 alleles, responsible for the A2, Ax, Ael, cis-AB, Bx, and Bel phenotypes, were presumed to be generated from common ABO alleles by single nucleotide mutations such as nonsynonymous substitution, deletion, or insertion. Two other alleles, responsible for the A2 and Ael phenotypes, may have originated by recombination, gene conversionlike events or accumulation of nucleotide substitutions. Our data indicate that different alleles could cause the same ABO variant phenotypes, and that these alleles do not necessarily belong to a single evolutionary lineage.
Quantification of hepatitis C virus-infected peripheral blood mononuclear cells by in situ reverse transcriptase-polymerase chain reaction
Authors: Muratori L , Gibellini D , Lenzi M , Cataleta M , Muratori P , Morelli MC , Bianchi FB
Published: October 2019

Hepatitis C virus (HCV) is known to infect peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, but the proportion of HCV-infected circulating cells is not detectable by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and the pathogenic significance of HCV lymphotropism is still unclear. Therefore, we have devised an in situ RT-PCR technique using fluorescein-labeled HCV-specific primers revealed by flow cytometry. PBMC were isolated from 28 patients with chronic HCV-related liver disease; of these, 6 had previously received an orthotopic liver transplantation (OLT) and were on immuno-suppressive treatment. Fourteen patients (50%) were found positive for HCV genome within PBMC by in situ RT-PCR, the proportion of HCV-infected cells ranging from 0.2% to 8.1%. All 6 OLT patients tested positive. The fluorescent signal, corresponding to the HCV-specific 340-bp amplicon, was confined to part of the cytoplasmic compartment of scattered PBMC. Of these 14 patients, 12 had also negativestrand HCV RNA within PBMC detected by “tagged” RT-PCR. We conclude that HCV may infect a significant proportion of PBMC in chronic hepatitis C patients, especially immunosuppressed OLT cases, and that viral replication within PBMC is a common occurrence. Over time, the persistence of HCV-infected immune system cells might interfere with normal immunologic mechanisms and play a role in the pathogenic processes leading to extrahepatic disorders such as mixed cryoglobulinemia and B-cell malignant lymphoma.
Improved immune reconstitution after allotransplantation of peripheral blood stem cells instead of bone marrow
Authors: Ottinger HD , Beelen DW , Scheulen B , Schaefer UW , Grosse-Wilde H
Published: October 2019

Clinical studies are evaluating possible advantages of allogeneic peripheral blood stem cell transplantation (PBSCT) over bone marrow transplantation (BMT). We compared immune reconstitution after PBSCT (n = 20) and BMT (n = 20) in terms of lymphocyte subset counts and proliferative in vitro responses to mitogens and recall antigens (follow-up: 5 to 11 months posttransplant). Additionally, 10 PBSC harvests and 10 marrow harvests were analyzed for their composition of immunocompetent cells. Compared with BMT patients, PBSCT recipients had PB counts of naive (CD4+CD45RA+) and memory (CD4+CD45RO+) helper T cells and of B cells (CD19+) that were elevated (P < .003, P < .001, and P < .004, respectively) and proliferative responses to phytohemagglutinin (P < .0001), pokeweed mitogen (P < .02), Tetanus toxoid (P < .0005), and Candida (P < .004) that were increased. PBSCT recipients received a mean of 188 (range, 44 to 280) x 10(6) naive helper T cells and 169 (range, 18 to 296) x 10(5) memory helper T cells per kilogram; the corresponding numbers for BMT recipients were 11 (range, 4 to 24) and 10 (range, 1 to 22) x 10(5) cells per kilogram, respectively. The question of whether the documented improved in vitro immune competence after PBSCT is associated with a lower incidence of infectious complications in vivo still needs further study.
Quantitative analysis of von Willebrand factor propeptide release in vivo: effect of experimental endotoxemia and administration of 1- deamino-8-D-arginine vasopressin in humans
Authors: Borchiellini A , Fijnvandraat K , ten Cate JW , Pajkrt D , van Deventer SJ , Pasterkamp G , Meijer-Huizinga F , Zwart-Huinink L , Voorberg J , van Mourik JA
Published: October 2019

The results of studies with cultured endothelial cells have shown that most von Willebrand factor (vWF) synthesized is directly secreted (constitutive pathway) and consists of both mature vWF, its precursor molecule pro-vWF, and the cleaved vWF prosequence. Only fully processed, functionally mature vWF is stored within the cell, together with the propeptide, and leaves the cell only on stimulation (regulated secretion). Both in resting and stimulated cultured endothelial cells, the stoichiometry of the released propeptide to the released mature vWF is essentially equimolar. In the present study, we have measured the molar ratio of propeptide to mature vWF in vivo, both under resting conditions and conditions that reflect activation of the endothelium. To this end, we devised a method that allows the measurement of the propeptide (vW antigen II) on a quantitative, is, molar basis, using purified recombinant propeptide as a standard. Our results show that the molar concentration of the propeptide in normal plasma is about one tenth of the concentration of mature vWF (expressed as half-dimer concentration). This ratio is approximately 1:1 in the medium of cultured endothelial cells. On administration in healthy subjects of either 1-deamino-8-D-arginine vasopressin or endotoxin, both agents being known to elicit an intravascular increase of vWF, the molar ratio of propeptide to mature vWF increased fourfold to fivefold. The propeptide concentration returned to baseline values after about 6 to 7 hours of injection of each stimulus, whereas the increase of mature vWF was much more sustained. Because the respective half-lives of mature vWF and its propeptide clearly differ, measurement of the concentration of these proteins could provide a means to assess the extent of activation of the endothelium under physiological and pathophysiological conditions.
Recipient humoral immunity against leukoreduced allogeneic platelets is suppressed by aminoguanidine, a selective inhibitor of inducible nitric oxide synthase
Authors: Bang A , Speck ER , Blanchette VS , Freedman J , Semple JW
Published: October 2019

Leukoreduced allogeneic platelet transfusions have been previously shown to initially stimulate an in vitro cellular cytotoxicity and subsequently Induce the formation of immunoglobulin G (IgG) antidonor alloantibodies. To further characterize these responses and determine if they are related, recipient BALB/c H-2d mice were treated with aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase (iNOS), and transfused weekly with 2 x 10(8) C57BL/6 H2b platelets. In control, non-AMG-treated mice, transfusion significantly (P < .01) increased serum levels of interferon-gamma (IFN-gamma) by day 1 posttransfusion (PT). IFN-gamma returned to pretransfusion levels by day 3 PT, and its production was not affected by AMG treatment. Serum interleukin-4 (IL-4), on the other hand, was undetectable before and during the transfusion protocol. By day 3 PT, recipient spleen cells could mediate in vitro anti-P815 (auto), anti-EL4 (allo), and anti-R1.1 (third-party MHC) cytotoxicity, and these responses were maximal by day 7 PT. Concurrently, a significant reduction in the vitro ability of recipient splenocytes to respond to Concanavalin A (ConA) was observed; this was not seen with lipopolysaccharide (LPS) stimulation. Elevated levels of NO2- were found in the ConA culture supernatants from transfused mice at day 3 PT. Serum antidonor alloantibodies were detected by the fifth platelet transfusion. AMG treatment of recipient mice significantly inhibited the transfusion. Induced cytotoxicity and ConA-stimulated NO2- production, and restored ConA-induced proliferation to normal levels. AMG appeared to selectively inhibit platelet-induced alloantibody production in that it did not affect antibody production induced by transfusions with 10(5) allogeneic leukocytes or by immunization with a foreign protein antigen, human gamma globulin, in adjuvant therapy. These results indicate that an in vivo AMG-sensitive mechanism is essential for recipients to initiate a humoral IgG immune response against allogeneic platelets.
Demonstration of antibodies to human T-cell lymphotropic virus-I tax in patients with the cutaneous T-cell lymphoma, mycosis fungoides, who are seronegative for antibodies to the structural proteins of the virus
Authors: Pancake BA , Wassef EH , Zucker-Franklin D
Published: October 2019

Although most patients with the cutaneous T-cell lymphoma, mycosis fungoides (MF), are seronegative for human T-cell lymphotropic virus-I or -II (HTLV-I/II) when tested by assays that measure only antibodies to the viral structural proteins, the majority of such patients harbor HTLV-I-related pol and tax proviral sequences that encode proteins not included in routinely used serologic tests. Tax mRNA has also been detected in their peripheral blood mononuclear cells (PBMC). Therefore, it seemed possible that these patients have antibodies to the tax protein. To investigate this, enzyme-linked immunosorbent assays (ELI-SAs) and Western blot assays were set up, using as antigens the full-length HTLV-I tax cloned from the prototypic HTLV-I-infected cell line, C91PL, and from PBMC of a MF patient, as well as a synthetic peptide made to the carboxy-terminal 20 amino acids of tax-I. Of 60 MF patients whose PBMC were shown to be positive for tax proviral DNA and mRNA, 50 (83%) were shown to have tax antibodies. The antigen derived from the MF patient was most useful in detecting such antibodies. These results demonstrate the need for including other HTLV-related antigens in addition to gag and env in serologic tests used to identify HTLV-infected individuals. The findings underscore the fact that individuals considered seronegative on the basis of currently used tests can be infected with HTLV.
Expression of pertussis toxin adenosine diphosphate-ribosyltransferase in a T-cell hybridoma reduces metastatic capacity
Authors: Driessens MH , van Rijthoven EA , La Riviere G , Roos E
Published: October 2019

T-cell hybridomas are highly metastatic, and their in vitro invasiveness correlates with metastatic capacity. Invasion is blocked by pertussis toxin (PT), which adenosine diphosphate (ADP)-ribosylates G1-proteins, and we have provided evidence that the PT-sensitive signal stimulates leukocyte function-associated antigen-1 (LFA-1)-mediated adhesion required for invasion. PT pretreatment of TAM2D2 T-cell hybridoma cells reduced metastasis, but only to a limited extent. In the present study, we have transfected the cDNA of the PT ADP-ribosyltransferase S1 subunit into TAM2D2 cells to abrogate G1-protein function permanently. We report here a substantial reduction in the metastatic capacity of two transfectants, S05 and S09, in which 88% and 95% of the G1-proteins was ADP-ribosylated. Two-thirds of the mice injected with S09 cells were tumor-free. Metastasis to the liver was almost completely prevented and less metastases were formed in the spleen and kidneys. Metastasis formation by S05 cells in liver and spleen was much reduced, but in lymph nodes and peritoneal tissues, metastases occurred with a frequency similar to that of controls. We conclude that G1-proteins play an important role in T-cell hybridoma metastasis. We propose that the reduction in metastasis is due to diminished entry of tumor cells from the blood into tissues.
FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells
Authors: Turner AM , Lin NL , Issarachai S , Lyman SD , Broudy VC
Published: October 2019

FLT3 ligand is a hematopoietic growth factor that plays a key role in growth of primitive hematopoietic cells. FLT3 receptor mRNA is found in early hematopoietic progenitors and in human myeloid leukemia blasts. Much less is known about the surface expression of FLT3 receptor on human hematopoietic cells. Using human 125I-FLT3 ligand, we have identified and characterized surface FLT3 receptors on normal and malignant human hematopoietic cells and cell lines. Our results showed that surface display of FLT3 receptor was greatest in fresh myeloid leukemia blast cells and myeloid leukemia cell lines. Erythroleukemic and megakaryocytic leukemia cell lines (n = 5) bound little to no 125I-FLT3 ligand. Scatchard analysis of 125I-FLT3 ligand binding data shows that three myeloid leukemia cell lines, ML-1, AML-193, and HL-60, as well as normal human marrow mononuclear cells, exhibit high affinity FLT3 receptors. Crosslinking of 125I-FLT3 ligand to FLT3 receptors on the surface of ML-1 myeloid leukemia cells indicates that the FLT3 ligand. The rates of FLT3 ligand internalization and degradation were determined by binding 125I-FLT3 ligand to ML-1 cells and acid stripping to distinguish surface bound from internalized ligand. Internalized 125I-FLT3 ligand was detected within 5 minutes after binding to ML-1 cells. In addition, we evaluated the effect of FLT3 ligand on megakaryocytic colony growth and nuclear endoreduplication, alone or in the presence of thrombopoietin. FLT3 ligand did not promote colony forming unit megakaryocyte (CFU-Meg) colony growth or megakaryocyte nuclear maturation, nor did FLT3 ligand augment the effects of thrombopoietin on these measures of megakaryopoiesis. These data indicate that the FLT3 receptor shares several characteristics with the c-kit receptor including dimerization and rapid internalization. However, the more restricted cellular distribution of the FLT3 receptor may target the effects of FLT3 ligand to primitive hematopoietic cells and to myeloid and lymphoid progenitor cells, in contrast to the pleiotropic effects of the c-kit receptor ligand, stem cell factor.
Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies
Authors: Bi L , Sarkar R , Naas T , Lawler AM , Pain J , Shumaker SL , Bedian V , Kazazian HH Jr
Published: October 2019

Previously we created two strains of factor VIII-deficient mice by insertion of a neo gene into (1) the 3′ end of exon 16 and (2) exon 17 of the factor VIII gene. Affected mice of both strains have no plasma factor VIII activity, yet are healthy with no spontaneous bleeding. Factor VIII-deficient females bred with affected males survive pregnancy and delivery. We used reverse transcriptase-polymerase chain reaction of liver RNA to characterize factor VIII mRNA processing. Factor VIII mRNA of the exon 16 knockout strain contains neo sequences plus 17 bp of intron 16 due to use of a cryptic donor site in intron 16. All factor VIII mRNA of the exon 17 knockout strain lacks exon 17 and neo sequences. In skipping exon 17, the intron 16 donor site or a cryptic donor site 46 bp 3′ to the intron 16 donor site are used. Thus, factor VIII deficiency in exon 16 knockout mice is due to truncated protein, while in exon 17 knockout mice it is due to either truncated or partially deleted protein. After immunizing exon 16 knockout mice with human recombinant factor VIII, two monoclonal antibodies were obtained that recognize <7 100 pg of mouse factor VIII light chain. Assay of cryoprecipitate from the plasma of affected mice failed to show factor VIII light chain.
High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles
Authors: Miyazaki Y , Nomura S , Miyake T , Kagawa H , Kitada C , Taniguchi H , Komiyama Y , Fujimura Y , Ikeda Y , Fukuhara S
Published: October 2019

Previous studies have demonstrated that a high level of shear stress can produce platelet aggregation without the addition of any agonist. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A coneplate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and the remnant platelets showed the exposure of procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor (vWF) to platelet glycoprotein lb, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C (PKC) promoted shear-dependent microparticle formation. Epinephrine did not influence microparticle formation, although it enhanced platelet aggregation by high shear stress. These findings suggest the possibility that local generation of microparticles in atherosclerotic arteries, the site that pathologically high shear stress could occur, may contribute to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.
Analysis of Epstein-Barr virus gene polymorphisms in normal donors and in virus-associated tumors from different geographic locations
Authors: Khanim F , Yao QY , Niedobitek G , Sihota S , Rickinson AB , Young LS
Published: October 2019

While Epstein-Barr virus (EBV) infection is associated with the development of certain lymphoid and epithelial tumors, the role of the virus in the pathogenesis of these malignancies remains unknown. It has been suggested that EBV strain variation may contribute to tumor development. Two major strains of EBV, type 1 and type 2, have been identified on the basis of genetic polymorphisms and other minor genetic variations give rise to distinct EBV isolates. We analyzed EBV strain variation in healthy individuals and compared them with EBV isolates present in lymphoid and epithelial neoplasms from the same geographic regions. In particular, the incidence of the 30-bp latent membrane protein (LMP1) gene deletion, recently implicated in the development of more aggressive forms of virus-positive lymphomas and Hodgkin's disease [HD], was examined in the normal population. While a preferential association of the LMP1 deletion with the type 2 strain of EBV was observed, there was no increased incidence of virus isolates carrying this deletion in HD, Burkitt's lymphoma, or virus-associated carcinomas compared with the appropriate normal population. A polymorphism in the BamHI F region of the EBV genome, previously identified in Chinese populations, was found at increased incidence in European HD biopsies. Overall, we found that most of the EBV gene polymorphisms detected in EBV isolates from healthy virus carriers occurred with similar frequency in virus-associated tumors from the same geographical region.
Early recurrence or persistence of autoimmune diseases after unmanipulated autologous stem cell transplantation
Authors: Euler HH , Marmont AM , Bacigalupo A , Fastenrath S , Dreger P , Hoffknecht M , Zander AR , Schalke B , Hahn U , Haas R , Schmitz N
Published: October 2019

Autologous stem cell transplantation with or without in vitro lymphocyte depletion has been suggested as a new treatment option for severe autoimmune diseases. We describe five patients with autoimmune diseases (CREST syndrome, myasthenia gravis and Hashimoto's thyroiditis, systemic lupus erythematosus, atopic dermatitis, and rheumatoid arthritis) who underwent autologous bone marrow (n = 1) or peripheral blood progenitor cell (n = 4) transplantation with unmanipulated grafts as treatment for the autoimmune disease in one case or as treatment for a malignant disorder with a concomitant autoimmune disorder in four cases. In all patients serological and clinical signs of the autoimmune disease recurred early or persisted. These observations should be regarded as a cautionary note concerning the efficacy of high-dose therapy followed by transplantation of unmanipulated autologous stem cells for treatment of severe autoimmune diseases.
Posttransplant T-cell lymphoproliferative disorders--an aggressive, late complication of solid-organ transplantation
Authors: Hanson MN , Morrison VA , Peterson BA , Stieglbauer KT , Kubic VL , McCormick SR , McGlennen RC , Manivel JC , Brunning RD , Litz CE
Published: October 2019

T-cell non-Hodgkin's lymphomas are an uncommon occurrence after solid-organ transplantation. We describe a morphologically and immunophenotypically distinct group of T-cell lymphoproliferative disorders that occurred late in the course of six patients with solid-organ transplants. The patients ranged in age from 31 to 56 years (median, 43). Three were male; all were splenectomized. The interval from transplant to the diagnosis of lymphoma ranged from 4 to 26 years (median, 15). Symptoms at presentation were related to sites of involvement. Pulmonary, marrow, and CNS involvement were present in five, four, and one case, respectively. No patient had lymphadenopathy. Five patients had an elevated lactate dehydrogenase level (range, 226 to 4,880 IU/L; median, 1,220 IU/L). Five of six patients had a leukoerythroblastic reaction. All cases had large-cell histology and frequently contained cytoplasmic granules. Those cases tested expressed CD2, CD3, and CD8 and were negative for B-cell antigens. T-cell receptor beta- and gamma-chain genes were clonally rearranged in three of three and one of three cases, respectively. All T-cell posttransplant lymphoproliferative disorders (T-PTLDs) studied were negative for Epstein-Barr virus (EBV), human T-cell leukemia/lymphoma virus type 1 (HTLV-1), human T-cell leukemia/lymphoma virus type 2 (HTLV-2), and human herpes virus type 8 (HHV-8) genomes. Treatment with acyclovir (three patients) or chemotherapy (three patients) resulted in two responses. All patients had an aggressive course, with a median survival duration of 5 weeks. In conclusion, a clinically aggressive T-PTLD may be a late complication of solid-organ transplantation and does not appear to be related to EBV, HTLV-1, HTLV-2, or HHV-8 infection.
Transduction of hematopoietic cells by foamy virus vectors
Authors: Hirata RK , Miller AD , Andrews RG , Russell DW
Published: October 2019

Foamy viruses are retroviruses of the spumavirus family that are often isolated from primary cultures of primate cells. We previously constructed vectors based on human foamy virus (HFV) and found that they were able to transduce a wide variety of vertebrate cells by integration of the vector genome. Here we show that several types of hematopoietic cells are efficiently transduced by an HFV vector that encodes alkaline phosphatase (AP). These cell types include transformed cell lines and primary hematopoietic progenitors from mice, baboons, and humans. The transduction rates of HFV vectors compare favorably with those obtained by murine leukemia virus vectors, which suggests that HFV vectors may be effective in the treatment of hematologic diseases by gene therapy.
Detection and Titration of Human Herpesvirus-8–Specific Antibodies in Sera From Blood Donors, Acquired Immunodeficiency Syndrome Patients, and Kaposi's Sarcoma Patients Using a Whole Virus Enzyme-Linked Immunosorbent Assay
Authors: Chatlynne Louise G. , Lapps William , Handy Michael , Huang Yao Q. , Masood Rizwan , Hamilton Ann S. , Said Jonathan W. , Koeffler H.P. , Kaplan Mark H. , Friedman-Kien Alvin , Gill Parkash S. , Whitman James E. , Ablashi Dharam V.
Published: October 2019

A human herpesvirus-8 (HHV-8) enzyme-linked immunosorbent assay (ELISA) with a whole virus lysate as antigen was developed and used to measure the seroprevalence rate and levels of IgG antibodies to HHV-8 in sera/plasma of various patient groups and blood donors. The virus antigen was prepared from the KS-1 cell line, which produces lytic virus, and therefore contains a broad array of viral proteins. Seroprevalence studies using this ELISA showed the following: 10 of 91 blood donors (11%) had an average HHV-8 antibody titer of 118; 67 of 72 (93%) classic Kaposi's sarcoma (KS) patients were positive with an average titer of 14,111; and 57 of 62 (92%) KS/human immunodeficiency virus (HIV) patients were positive with an average titer of 4,000. A study on a very limited number of serial serum samples from patients before and after diagnosis with KS showed highly elevated antibody titers to HHV-8 virus after KS lesions developed. Preliminary data show that 50% of the sera from HIV-1+ homosexual patients contain IgG antibodies to HHV-8 suggesting that this population is at high risk for developing KS. Antibody results correlated well with the confirmatory immunofluorescent assays (IFA) using KS-1 cells as the substrate. This HHV-8 IgG antibody detection ELISA is sensitive and specific and does not cross-react with Epstein-Barr virus (EBV) or other human herpesviruses. The results of this HHV-8 antibody survey suggest that this rapid ELISA assay can be used to screen large numbers of sera to find those at risk for developing KS.
Erythropoietin mRNA Expression in Human Fetal and Neonatal Tissue
Authors: Dame Christof , Fahnenstich Hubert , Freitag Patricia , Hofmann Dietmar , Abdul-Nour Thair , Bartmann Peter , Fandrey Joachim
Published: October 2019

Based on animal experiments, a switch of the erythropoietin (EPO) production site from the liver in the fetus to the kidneys in the adult has been postulated. To study the switch in humans, we have quantitated EPO mRNA expression in liver, kidney, spleen, and bone marrow of human fetuses and neonates by means of a competitive polymerase chain reaction (PCR). Tissue samples from 66 routine postmortem examinations were obtained. EPO mRNA was expressed in 97% of the tissue specimen derived from the liver (n = 66) and in 93% of those from the kidneys (17 weeks of gestation until 18 months after birth; n = 59). For the first time the EPO gene was found expressed in vivo in human spleen (96% of 64 samples) and in fetal and neonatal bone marrow (81% of 21 samples). EPO mRNA expression in the kidneys increased significantly beyond 30 weeks of gestation (P < .05). Although there was a slight decrease in EPO mRNA content per g liver tissue towards birth, the liver accounted for about 80% of the total body EPO mRNA. The contribution of the spleen and bone marrow were minor compared with liver and kidneys. Our results indicate that in humans the liver is the primary site of EPO gene expression not only in fetal, but also in neonatal life. A significant increase of renal EPO mRNA expression after 30 weeks of gestation might indicate the beginning switch.

© 1998 by The American Society of Hematology.
Autografting With Philadelphia Chromosome–Negative Mobilized Hematopoietic Progenitor Cells in Chronic Myelogenous Leukemia
Authors: Carella Angelo M. , Lerma Enrica , Corsetti Maria T. , Dejana Anna , Basta Palmina , Vassallo Franca , Abate Monica , Soracco Monica , Benvenuto Federica , Figari Osvaldo , Podestà Marina , Piaggio Giovanna , Ferrara Raimondo , Sessarego Mario , Parodi Caterina , Pizzuti Michele , Rubagotti Alessandra , Occhini Domenico , Frassoni Francesco
Published: October 2019

Intensive chemotherapy given in early chronic phase of chronic myelogenous leukemia (CML) has resulted in high numbers of circulating Philadelphia (Ph) chromosome–negative hematopoietic progenitor cells (HPC). We have autografted 30 consecutive patients with CML in chronic phase with HPC collected in this way to facilitate restoration of Ph-negative hematopoiesis in bone marrow after high-dose therapy. Hematopoietic recovery to greater than 0.5 ×109/L neutrophils and to greater than 25 × 109/L platelets occurred in all patients, a median of 13 (range, 9 to 32) days and 16 (range, 6 to 106) days postautograft, respectively. Regenerating marrow cells were Ph-negative in 16 (53%) patients and greater than 66% Ph-negative in 10 (33%) patients. Twenty-eight patients are alive 6 to 76 months (median, 24 months) after autografting. Three patients have developed blast crisis from which 2 have died. Eight patients are in complete cytogenetic remission at a median of 20 (range, 6 to 44) months with a median ratio BCR-ABL/ABL of 0.002 (range, <0.001 to 0.01). Eight patients are in major cytogenetic remission at a median of 22 (range, 6 to 48) months. No patient died as a consequence of the treatment. All patients had some degree of stomatitis that was severe in 15 (50%) patients. Gastrointestinal and hepatic toxicities were observed in about one fourth of patients. Thus, autografting with Ph-negative mobilized HPC can result in prolonged restoration of Ph-negative hematopoiesis for some patients with CML; moreover, most autograft recipients report normal or near normal activity levels, suggesting that this procedure need not to be associated either with prolonged convalescence or with chronic debility.