bluuebunny/crossref_metadata_2025 huggingface dataset:
Coexpression of gamma and beta globin mRNA in cells containing a single human beta globin locus: results from studies using single-cell reverse transcription polymerase chain reaction [published erratum appears in Blood 1994 Aug 15;84(4):1357]
Authors: Furukawa T , Zitnik G , Leppig K , Papayannopoulou T , Stamatoyannopoulos G
Published: October 2019

We developed a method detecting globin gene expression in single cells using reverse transcription polymerase chain reaction. epsilon and gamma globin cDNAs are coamplified by an epsilon gamma primer set whereas gamma and beta globin cDNAs are coamplified by a gamma beta primer set and the individual globin cDNAs are distinguished by restriction enzyme digestion. Analysis of RNA preparations from human fetal liver, neonatal red blood cells (RBCs), or adult RBCs showed the expected mRNA species for each stage of human development. Analysis of single cells from a human erythroleukemia line coexpressing gamma and beta globin chains showed heterogeneity in gamma and beta mRNA contents. The method was subsequently used to test whether only one or more than one globin genes are expressed in cells that contain a single human beta globin locus. We found that about 50% of single cells from MEL x fetal erythroid cell hybrids containing a single human beta globin locus coexpressed gamma and beta globin mRNA. This finding is best explained by assuming that both gamma and beta genes are simultaneously transcribed from the same beta globin locus implying that the LCR can simultaneously interact with more than one globin gene promoter.
Expression and biochemical characterization of human glucose-6- phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes
Authors: Tang TK , Yeh CH , Huang CS , Huang MJ
Published: October 2019

We have developed a system to characterize normal and mutated glucose-6- phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->wG; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.
Mode of inheritance of the higher degree of megakaryocyte polyploidization in C3H mice. I. Evidence for a role of genomic imprinting in megakaryocyte polyploidy determination
Authors: McDonald TP , Jackson CW
Published: October 2019

C3H mice have higher average ploidy megakaryocytes than all other mouse strains tested, but the mode of inheritance of this anomaly is unknown. Therefore, to clarify the genetics of high ploidy megakaryocytes in C3H mice, we measured megakaryocyte DNA content from both male and female offspring from F1, as well as backcross matings. In all, offspring from seven different matings of mice were studied: (1) C57BL X C57BL (the first strain listed is the male parent in each case), (2) B6C3F1 (offspring from C57BL X C3H mating) X C57BL, (3) C57BL X B6C3F1, (4) C57BL X C3H, (5) C3H X B6C3F1, (6) B6C3F1 X C3H, and (7) C3H X C3H. The polyploid megakaryocyte DNA content distributions of the offspring from these matings show that C3H mice have higher percentages of high ploidy megakaryocytes than did all other mice. Also, male mice had significantly higher percentages of high ploidy (32N and 64N) megakaryocytes than did female mice for all matings, except backcross mating no. 6. The megakaryocyte DNA content for individual offspring of a given backcross appeared to form a single, continuous distribution, rather than segregate into two distinct groups, suggesting that the higher megakaryocyte DNA content of C3H mice is caused by involvement of multiple allelles. This conclusion is further supported by our finding that the frequency of high ploidy megakaryocytes among offspring of the various matings was related to the proportion of C3H genotype contributed by the parents, ie, average megakaryocyte DNA content increased linearly (r2 = .88 for male mice and .84 for female mice. P < .0001) with increasing C3H gene dosage; the correlations for both male and female mice were essentially parallel (slope = 0.08 and 0.09, respectively). In addition, we found an effect of genomic imprinting on megakaryocyte DNA content in backcross offspring. The genetic imprinting was characterized by the female parent having a greater influence on the offspring's megakaryocyte DNA content than the male parent, ie, although the overall genetic makeup was the same, female offspring from backcross no. 6 (in which the female was C3H) had higher average megakaryocyte ploidy values than those from backcross no. 5 (in which the female was B6C3F1
Characterization of Leu777Pro and Ile865Thr type IIA von Willebrand disease mutations
Authors: Lyons SE , Cooney KA , Bockenstedt P , Ginsburg D
Published: October 2019

Type IIA von Willebrand disease (vWD) is an autosomal dominant bleeding disorder characterized by a qualitative defect in von Willebrand factor (vWF). A number of missense mutations responsible for type IIA vWD have recently been identified. This report examines the type IIA vWD mutations Leu777-->Pro and Ile865-->Thr by expression of recombinant vWF containing mutant and wild-type (WT) sequences. Recombinant vWF containing the L777P mutation (vWFL777P) showed markedly impaired secretion compared with that for wild-type vWF (vWFWT) after DNA transfection into mammalian cells. Multimer analysis of secreted vWFL777P showed predominantly low molecular weight forms. In contrast, recombinant vWF containing the I865T mutation (vWFI865T) was processed in a pattern similar to vWFWT, with secretion of the full spectrum of vWF multimers. Thus, L777P and I865T are subclassified as type IIA group I and group II mutations, respectively. Analysis of platelet vWF from a patient heterozygous for the L777P mutation shows reduced large vWF multimers in a pattern similar to plasma, consistent with the intracellular transport defect predicted for a group I mutation. An increase in the proportion of high molecular weight multimers observed in type IIA vWD patient plasma, after renal transplantation from a normal donor, suggests that the kidney endothelium may be a major source of plasma vWF.
Stable multilineage hematopoietic chimerism in alpha-thalassemic mice induced by a bone marrow subpopulation that excludes the majority of day-12 spleen colony-forming units
Authors: van der Loo JC , van den Bos C , Baert MR , Wagemaker G , Ploemacher RE
Published: October 2019

We have investigated the contribution of highly purified day-12 spleen colony-forming units (CFU-S-12) as well as more primitive cells to sustained blood cell production using in vivo and in vitro assays that allow frequency analysis. Normal or day-6 post-5-fluorouracil light- density bone marrow (BM) was sorted on the basis of differences in rhodamine-123 (Rh123) retention or wheat germ agglutinin (WGA) affinity and tested in vivo using a recently developed alpha-thalassemic chimeric mouse model. In addition, short-term and long-term clonal activity was assessed in vitro using a limiting dilution-type long-term BM culture, the cobblestone area forming cell assay. When sublethally irradiated alpha-thalassemic mice were transplanted with as many as 281 purified WGAbright CFU-S-12, derived from a fraction containing 95% of all CFU-S-12 from day-6 post-5-fluorouracil light-density BM of wild- type mice, detectable chimerism was not observed at 6 months posttransplantation. In contrast, only three CFU-S-12 were included in the Rh123dull and WGAdim subpopulations that induced 29% to 58% and 21% to 31% stable multilineage donor-type chimerism of erythrocytes and leukocytes, respectively. The Rh123dull and WGAdim cells were up to 240- fold enriched for long-term repopulating ability (LTRA) as compared with unseparated BM. A comparable level of chimerism was found in the different hematopoietic organs and at the level of BM CFU-S-12. The frequency of the LTRA unit capable of inducing a 10% sustained level of donor-type erythrocytes was calculated to be 1 to 2 per 10(5) BM cells. Several reports have suggested that LTRA and spleen colony formation could be capacities of the same stem cell subset. However, the present results show that the majority of CFU-S-12 have only short-term repopulating ability and are physically separable from more primitive stem cells with long-term multilineage reconstituting capacities.
Dissociation between p93B-myb and p75c-myb expression during the proliferation and differentiation of human myeloid cell lines
Authors: Arsura M , Luchetti MM , Erba E , Golay J , Rambaldi A , Introna M
Published: October 2019

Direct and indirect evidence strongly indicates that the proto-oncogene c-myb plays an important role in the regulation of both the proliferation and differentiation of hematopoietic cells. In addition, recent data suggest that the structurally related B-myb gene is also necessary for the proliferation of these cells. To help understand the relationship between these two related gene products during proliferation and differentiation of myeloid cells, we have studied in parallel the regulated expression of c-myb and B-myb RNAs and proteins in human myeloid cells that were either growth-arrested or induced to differentiate along different pathways. For this purpose, we have produced a polyclonal antibody directed against a fragment of the recombinant B-myb protein. We have thus been able to detect the B-myb protein in human cell lines and have found it to be a 93-kD protein localized in the nucleus. We have chosen two models to study the expression of both c-myb and B-myb mRNAs and proteins during myeloid proliferation and differentiation. One of the models was the HL-60 cell line, which can be induced to differentiate towards the monocytic pathway with either phorbol ester (phorbol myristate acetate) or vitamin D3 and towards the granulocytic pathway with either dimethyl sulfoxide or retinoic acid. In addition, we have studied another recently established human leukemic cell line, called GF-D8, which is strictly dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) for proliferation. The results show that the expression of B- myb RNA and protein closely correlates with proliferation in all experimental setups studied, whereas the c-myb protein levels do not always do so. We observed that the c-myb protein levels decreased well before the decrease of B-myb protein and of proliferation itself during differentiation toward monocytes. Such a difference was not present during granulocytic differentiation, in which c-myb levels decreased, if anything, later than those of B-myb and proliferation. Most striking was the finding that high levels of c-myb RNA and protein, but not of B- myb, were present in the GF-D8 cell line, even after growth arrest by GM-CSF deprivation. These data suggest that B-myb may function solely in the regulation of cellular proliferation, whereas c-myb has additional functions, for example, in the maintenance of an undifferentiated state.
Basic fibroblast growth factor antagonizes transforming growth factor beta-mediated erythroid differentiation in K562 cells
Authors: Burger PE , Dowdle EB , Lukey PT , Wilson EL
Published: October 2019

Basic fibroblast growth factor (bFGF) and transforming growth factor- beta 1 (TGF-beta) have both been shown to act on hematopoietic progenitor cells. bFGF is a hematopoietic cytokine that acts on progenitor cells in concert with other cytokines to promote their proliferation. TGF-beta induces erythroid differentiation in K562 cells. To determine whether bFGF might act on progenitor cells by antagonizing the effects of cytokines that induce differentiation, we determined the effects of bFGF on the TGF-beta-mediated induction of hemoglobin synthesis in K562 cells. bFGF antagonized the TGF-beta- mediated induction of hemoglobin in a dose-dependent manner, with 0.1 ng/mL bFGF inhibiting hemoglobin induction by 40% and 10 ng/mL bFGF completely abrogating hemoglobin production. bFGF was most effective at antagonizing the TGF-beta-mediated induction of hemoglobin if it and TGF-beta were added simultaneously to K562 cells, but delayed addition of bFGF to TGF-beta-treated cultures still resulted in significant inhibition of hemoglobin synthesis. The inhibitory effects of bFGF on hemoglobin production were fully reversible, showing that bFGF did not permanently alter the phenotype of K562 cells. The hemin-mediated induction of hemoglobin synthesis in K562 cells was only partially negated by bFGF. bFGF also diminished the expression of glycophorin A on the surface of K562 cells. These results indicate that bFGF might increase progenitor/stem cell numbers by antagonizing the effects of cytokines that induce differentiation, thereby increasing the pool of proliferating progenitor/stem cells.
Retinoic acid and granulocyte colony-stimulating factor synergistically induce leukocyte alkaline phosphatase in acute promyelocytic leukemia cells
Authors: Gianni M , Terao M , Zanotta S , Barbui T , Rambaldi A , Garattini E
Published: October 2019

In this report we show a strong synergistic interaction between granulocyte colony-stimulating factor (G-CSF) and all-trans retinoic acid (ATRA) on the expression of leukocyte alkaline phosphatase (LAP) in freshly isolated acute promyelocytic leukemia (APL) blasts as well as in NB40 and HL-60 cell lines. The strong synergism observed in these cell types was not evident in two acute leukemia cell lines (K562 and GF-D8), in normal granulocytes, and in monocytes. In freshly isolated leukocytes derived from chronic myelogenous leukemia (CML), in the stable phase of the disease, a weaker interaction between ATRA and G- CSF was documented. The cross-talk between the cytokine and the retinoid was studied in detail in NB4, an immortalized APL leukemia cell line, retaining the 15′17 chromosomal translocation involving the retinoic acid receptor type alpha. The treatment of NB4 cells with G- CSF alone or ATRA alone leads to no increase and to minor induction in LAP activity, respectively. If the cells are treated with the two compounds simultaneously, a dramatic elevation of LAP is observed after 4 days. The synergism between G-CSF and ATRA is evident at concentrations of the retinoid between 10(-7) and 10(-5) mol/L and at concentrations of the cytokine between 1 and 10 ng/mL. The simultaneous presence of the two compounds is necessary to obtain maximal increase of LAP activity and the effect is cell density-dependent. Synergism is specific for G-CSF, and it is not observed with other cytokines and functional inducers of the granulocyte. The augmentation of LAP activity is the consequence of an increased transcriptional rate of the liver/bone/kidney-type (L/B/K-type) alkaline phosphatase gene, as determined by Northern blotting and nuclear run-on analysis using specific cDNA probes. Only one of the two possible alternatively spliced forms of L/B/K-type alkaline phosphatase transcript is detected in NB4 cells after stimulation with G-CSF and ATRA. This mRNA form, which is the one observed in normal polymorphonuclear leukocytes, contains the most upstream leader exon. In NB4 cells, ATRA induces G- CSF, alpha, and beta retinoic acid receptor transcripts, whereas G-CSF has minor effects on the expression of these mRNAs.
Immune status of human immunodeficiency virus seropositive and seronegative hemophiliacs infused for 3.5 years with recombinant factor VIII. The Kogenate Study Group
Authors: Mannucci PM , Brettler DB , Aledort LM , Lusher JM , Abildgaard CF , Schwartz RS , Hurst D
Published: October 2019

Recent studies suggest that treatment of hemophiliacs with highly purified factor VIII concentrates may preserve immune function. To test this hypothesis, we prospectively studied 51 hemophilic patients (21 human immunodeficiency virus [HIV] seropositive and 30 seronegative) who were on home therapy exclusively with recombinant factor VIII (Kogenate, Miles Laboratory, Berkeley, CA) for 3.5 years. Patients, all of whom had been previously treated with plasma-derived factor VIII concentrates, were monitored every 6 months with T-lymphocyte subsets and beta 2-microglobulin levels. Mean rate of change in absolute CD4 cell counts, calculated from regression slopes for individual patients, showed a small but statistically significant decrease over the 3.5-year study period for HIV seropositive hemophiliacs. No decrease in CD4 cell counts was seen in HIV seronegative hemophiliacs when the data for children under age 6 years were excluded from the analysis. beta 2- microglobulin levels and CD8 cell counts remained unchanged. These data show stability of immunologic parameters in HIV seronegative hemophiliacs, and a small decrease in CD4 cell counts in HIV seropositive hemophiliacs treated with recombinant factor VIII.
Effect of T-cell depletion as graft-versus-host disease prophylaxis on engraftment, relapse, and disease-free survival in unrelated marrow transplantation for chronic myelogenous leukemia
Authors: Drobyski WR , Ash RC , Casper JT , McAuliffe T , Horowitz MM , Lawton C , Keever C , Baxter-Lowe LA , Camitta B , Garbrecht F
Published: October 2019

Between January 1988 and March 1993, 48 patients received T-cell- depleted marrow grafts from unrelated donors as treatment for chronic myelogenous leukemia (CML). The median age of the population was 31.7 years (range 5.4 to 53) with 17 of 48 patients greater than 40 years of age. Twenty-seven patients were transplanted in chronic phase, 17 in accelerated phase, and 4 in blast crisis. All patients received a standardized preparative regimen of cyclophosphamide, high-dose cytosine arabinoside, methylprednisolone, and total body irradiation. Marrow grafts were depleted of mature T cells with the alpha beta T- cell receptor antibody T10B9 as graft-versus-host disease (GVHD) prophylaxis. All patients also received posttransplant cyclosporine therapy. Twenty-eight of 48 patients were mismatched with their donors for one or more HLA-A, B, DR, or DQ loci by either serology or high- resolution oligonucleotide genotyping. Nine of 28 were mismatched at multiple HLA loci. Durable engraftment was achieved in 94% (45/48) of patients. The actuarial probability of developing grades II to IV and grades III to IV acute GVHD were 39.6% (95% confidence interval (CI) 26.9 to 53.0) and 8.3% (95% CI 6.1 to 10.9) for the entire cohort. There was no difference in the incidence of grades II to IV acute GVHD between patients receiving matched (36.8%) or mismatched (41.4%) marrow grafts (P = .77). The actuarial probability of relapse at 2 years was 8.8% (95% CI 2.1 to 21.6) for the entire cohort and 18% (95% CI 4 to 41) for patients transplanted in either the accelerated or blast crisis phase (advanced disease). One cytogenetic relapse has occurred among patients transplanted in the chronic phase. The probability of disease- free survival at 2 years was 52% (95% CI 24 to 70) for patients transplanted in chronic phase and 46% (95% CI 25 to 73) for patients transplanted with advanced disease. No difference in disease-free survival was observed between patients receiving matched (49%) or mismatched (51%) marrow grafts (P = .90). This study shows that patients receiving unrelated T-cell-depleted marrow grafts for CML can achieve durable engraftment with a low incidence of severe GVHD and apparent preservation of graft-versus-leukemia reactivity. These data also suggest that T-cell depletion may allow patients who might otherwise experience unacceptable toxicity from GVHD-related complications caused by older age or increased HLA disparity to benefit from unrelated marrow grafts.
Hepatitis C virus infection is a risk factor for liver failure from veno-occlusive disease after bone marrow transplantation [see comments]
Authors: Frickhofen N , Wiesneth M , Jainta C , Hertenstein B , Heymer B , Bianchi L , Dienes HP , Koerner K , Bunjes D , Arnold R
Published: October 2019

The contribution of hepatitis C virus (HCV) infection to liver disease after bone marrow transplantation (BMT) was retrospectively evaluated in 61 patients treated with BMT. HCV genome, as well as antibodies to HCV, was analyzed in sera collected before and serially after BMT. Six patients had been infected with HCV before BMT and three patients acquired the infection during or shortly after BMT. All patients infected before BMT died within 10 weeks after transplantation. Five of these six patients (83%) died of veno-occlusive disease (VOD), compared with nine of 52 patients (17%) not infected with HCV (P < .005). Risk factors for VOD other than HCV were not more prevalent in these patients compared with uninfected patients. Parallel to the development of VOD, replication of HCV increased, as demonstrated by rising concentrations of viral RNA in serum. HCV infection acquired during or after BMT caused only mild acute hepatitis C, which progressed to chronic hepatitis C in one patient surviving 10 years after BMT. These data suggest that patients with liver disease caused by HCV infection are at high risk of developing lethal VOD after BMT.
Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia
Authors: Hall GW , Thein S
Published: October 2019

We present in vivo evidence that there is no reduction in beta-mRNA accumulation in patients with nonsense codons in the terminal exon of the beta-globin gene. Using reverse transcriptase/polymerase chain reaction (RT-PCR), beta-globin cDNA was isolated from the reticulocytes of individuals heterozygous for nonsense codon mutations in exons II and III of the beta-globin gene. Clinically asymptomatic individuals heterozygous for mutations causing premature termination of translation in exon II [beta(0)39(C-T) and F/S71/72(+A)] were found to have almost no mutant beta-cDNA, whereas patients with nonsense codon mutations in exon III [beta 121(G-T) and beta 127(C-T)] with the clinical phenotype of thalassemia intermedia had comparable levels of mutant and normal beta-cDNA. Translation of the mutant beta-mRNA from patients with nonsense codon mutations in exon III would give rise to truncated beta- globin chains, which could explain the more severe phenotype seen in these individuals.
Human umbilical vein endothelial cells display high-affinity c-kit receptors and produce a soluble form of the c-kit receptor
Authors: Broudy VC , Kovach NL , Bennett LG , Lin N , Jacobsen FW , Kidd PG
Published: October 2019

Stem cell factor (SCF) is a hematopoietic growth factor produced by fibroblasts and endothelial cells that stimulates the growth of primitive hematopoietic cells. SCF triggers cell growth by binding to the c-kit receptor. Because endothelial cells can respond to certain hematopoietic growth factors, we tested human umbilical vein endothelial cells for display of the c-kit receptor and examined the effect of SCF on endothelial cell proliferation, adhesion molecule expression, and production of tissue factor. Quantitative binding experiments with 125I-SCF showed both high-affinity (Kd = 42 pmol/L) and low-affinity (Kd = 1.7 nmol/L) c-kit receptors. There were approximately 1,100 high-affinity c-kit receptors, and 5,400 low- affinity c-kit receptors per endothelial cell. Enzyme immunoassays showed that endothelial cells released soluble c-kit receptor and SCF. The transmembrane form of SCF was detected by indirect immunofluorescence analysis using monoclonal or polyclonal anti-SCF receptor antibodies. The addition of SCF (100 ng/mL) did not alter endothelial cell proliferation over a 7-day period. Similarly, there was no change in the release of tissue factor or expression of inducible endothelial adhesion molecules (intercellular adhesion molecule-1, endothelial-leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1) measured by enzyme-linked immunosorbant assay at 4 and 24 hours after SCF addition. The neutralizing anti-c-kit receptor monoclonal antibody SR-1 blocked binding of 125I-SCF to the c- kit receptor by 98% but did not alter endothelial cell proliferation or adhesion-molecule expression. c-kit receptors were also detected on adult endothelial cells lining small blood vessels in normal human lymph nodes. These data indicate that normal human endothelial cells produce SCF and show high-affinity c-kit receptors that have the capacity to dimerize. The lack of response to exogenous SCF may be because of intracellular activation of the c-kit receptor via autocrine production of SCF. Alternatively, SCF and c-kit may play a role other than stimulation of proliferation, adhesion-molecule display, or tissue factor production by endothelial cells. The production of soluble c-kit receptors by normal human endothelial cells may serve to regulate the bioactivity of SCF within the bone marrow microenvironment.
The human myeloid cell nuclear differentiation antigen gene is one of at least two related interferon-inducible genes located on chromosome 1q that are expressed specifically in hematopoietic cells
Authors: Briggs RC , Briggs JA , Ozer J , Sealy L , Dworkin LL , Kingsmore SF , Seldin MF , Kaur GP , Athwal RS , Dessypris EN
Published: October 2019

We have previously shown that the human myeloid cell nuclear differentiation antigen (MNDA) is expressed at both the antigen and mRNA levels specifically in human monocytes and granulocytes and earlier stage cells in the myeloid lineage. A 200 amino acid region of the MNDA is strikingly similar to a region in the proteins encoded by a family of interferon-inducible mouse genes, designated Ifi-201, Ifi- 202, Ifi-203, etc, that are not regulated in a cell- or tissue-specific fashion. However, a new member of the Ifi-200 gene family, D3, is induced in mouse mononuclear phagocytes but not in fibroblasts by interferon. The same 200 amino acid region, duplicated in the mouse Ifi- 200 gene family, is also repeated in the recently characterized human IFI 16 gene that is constitutively expressed specifically in lymphoid cells and is induced in myeloid cells by interferon gamma. The 1.8-kb MNDA mRNA, which contains an interferon-stimulated response element in the 5′ untranslated region, was significantly upregulated in human monocytes exposed to interferon alpha. Characterization of the MNDA gene showed that it is a single-copy gene and localized to human chromosome 1q 21–22 within the large linkage group conserved between mouse and human that contains the Ifi-200 gene family. The IFI 16 gene is also located on human chromosome 1q. Our observations are consistent with the proposal that the MNDA is a member of a cluster of related human interferon-regulated genes, similar to the mouse Ifi-200 gene family. In addition, one mouse gene in the Ifi-200 gene family and the human MNDA and IFI 16 genes show expression and/or regulation restricted to cells of the hematopoietic system, suggesting that these genes participate in blood cell-specific responses to interferons.
Shear stress enhances the proteolysis of von Willebrand factor in normal plasma
Authors: Tsai HM , Sussman II , Nagel RL
Published: October 2019

While von Willebrand factor (vWF) is secreted from endothelial cells as a very large polymer, it circulates as a series of multimers that are reducible to a 225-kD polypeptide and three proteolytic fragments of 189, 176, and 140 kD. Cleavage at the Tyr-842/Met-843 bond of the vWF polypeptide creates the 140- and 176-kD fragments. In the process of understanding vWF multimer formation, the role of shear stress in vWF proteolysis was investigated in this study. A shear-rate-dependent loss of the largest multimers was observed when normal plasma was perfused through long capillary tubings achieving shear rates normally encountered in the circulation. The shear-dependent vWF change was not observed when purified vWF or normal plasma containing calcium chelator EGTA or EDTA was perfused. As the large multimers decreased, an increase in the smaller multimers, including 200- and 350-kD bands, was detected. Elution and immunoblotting studies with peptide-specific antibodies LJ-7745 and VP-1 showed that the 200-kD band was a dimer of the 140-kD fragment, whereas the 350-kD band was a dimer of the 176-kD fragment. When analyzed after disulfide bonds were reduced, sheared plasma showed an increase in the 176- and 140-kD fragments, but not the 189-kD fragment. Finally, shearing of purified vWF enhanced its proteolytic cleavage when it was subsequently incubated with the cryosupernatant fraction of normal plasma or with cathepsin G, a leukocyte granule serine protease. These results show that shear stress is capable of enhancing the susceptibility of vWF to proteolytic cleavage. It promotes vWF proteolysis in normal plasma at a site that generates the 140-kD/176-kD fragments, leading to a decrease in multimer size. Shear stress might be involved in modulating the size of vWF in the circulation.
Involvement of the sphingomyelin pathway in autocrine tumor necrosis factor signaling for human immunodeficiency virus production in chronically infected HL-60 cells
Authors: Rivas CI , Golde DW , Vera JC , Kolesnick RN
Published: October 2019

Tumor necrosis factor (TNF) is a potent inducer of human immunodeficiency virus (HIV) proviral transcription and subsequent mature virus production. Recent investigations have shown that TNF can use a signal transduction pathway in HL-60 cells involving sphingomyelin hydrolysis to ceramide with subsequent stimulation of a ceramide-linked kinase. When sphingomyelinase was added exogenously to activate this cascade in HIV-1-infected HL-60 cells, it mimicked TNF- induced HIV production. Phospholipases A2, C, or D, which do not generate ceramide, had no effect; however, a synthetic ceramide analog added exogenously potently induced HIV production. In addition, anti- TNF antibodies blocked much of the effect of both sphingomyelinase and the synthetic ceramide analog on virus expression, suggesting that, although signaling is initiated through the sphingomyelin pathway, it is sustained by autocrine TNF synthesis. Thus, direct activation of the sphingomyelin pathway recapitulated the effect of TNF on both HIV and TNF production. These studies indicate that the sphingomyelin pathway is involved in TNF signaling for HIV production in chronically infected myeloid cells.
Paracrine downregulation of Fc gamma RIII in human monocyte-derived macrophages induced by phagocytosis of nonopsonized particles
Authors: Liao G , Simone J , Simon SR
Published: October 2019

Monocytes and macrophages can take up nonopsonized particles through a direct phagocytic process and IgG-coated particles through combined mechanisms of nonspecific phagocytosis and internalization mediated by specific Fc receptors for IgG (Fc gamma R) on the plasma membrane. In this study, we report the effect of phagocytosis of nonopsonized latex beads on the levels of expression of Fc gamma receptors in human monocyte-derived macrophages (MDM) as measured by flow cytometry (fluorescence-activated cell sorter [FACS]). Macrophages were exposed to green fluorescent 1-micron polystyrene beads before labeling for Fc gamma RI, Fc gamma R-II, and Fc gamma R-III, respectively, with 32.2, 2E1, and 3G8 monoclonal antibodies (MoAbs) and a phycoerythrin (PE)- conjugated secondary Ab to permit dual-channel analysis of fluorescence intensity by FACS. Macrophages that had phagocytosed at least one bead showed reduced levels of surface Fc gamma R-III but not Fc gamma R-I or Fc gamma R-II when compared with cells that had never been exposed to beads. Moreover, cells that were not in direct contact with beads, but that shared medium with cells that had phagocytosed beads also had reduced levels of Fc gamma R-III but not Fc gamma R-I or Fc gamma R-II, suggesting a cytokine-mediated mechanism of Fc gamma R-III downregulation. Phagocytosis of 1-micron beads alone stimulated macrophages to release tumor necrosis factor-alpha (TNF-alpha). The medium from macrophages phagocytosing beads could stimulate other macrophages not in direct contact with beads to release TNF-alpha as well, but such paracrine-triggered release could be reduced by more than 50% if the medium from the phagocytosing cells was first treated with a neutralizing anti-TNF-alpha antibody. Moreover, the paracrine downregulation of Fc gamma R-III described above could also be blocked if the neutralizing anti-TNF-alpha antibody was added to the medium of phagocytosing cells. Treatment of macrophages with recombinant human TNF-alpha in the absence of beads induced decreased levels of Fc gamma R-III but not of Fc gamma R-II. These results show that paracrine downregulation of Fc gamma R-III is mediated by a TNF-alpha-dependent mechanism.
Expression gradient in sheep alpha alpha and alpha alpha alpha globin gene haplotypes: mRNA levels
Authors: Vestri R , Pieragostini E , Ristaldi MS
Published: October 2019

We previously found that in sheep alpha alpha-, alpha alpha alpha-, and alpha alpha alpha alpha-globin gene haplotypes, in which individual genes encode distinct allelic variants, the gene expression determined at protein level progressively decreases from the 5′ to the 3′ end. In the present study, through direct DNA analysis, we definitively established the gene relative position in the cluster and we verified the occurrence of the gradient also at the mRNA level. We measured the relative abundance of the alpha 113His-mRNA in alpha alpha and alpha alpha alpha haplotypes, in which the gene coding for the alpha 113His chain occupies the second and the third position, respectively. The alpha 113His-mRNA levels were about 20% and 8% to 9%, respectively, which closely paralleled alpha-chain levels. We conclude that the expression gradient occurs also at the mRNA level and is most likely of transcriptional origin.
Expression and characterization of recombinant anti-Rh(D) antibodies on filamentous phage: a model system for isolating human red blood cell antibodies by repertoire cloning
Authors: Siegel DL , Silberstein LE
Published: October 2019

The production of human anti-red blood cell (RBC) Igs in vitro from immunized individuals would greatly facilitate the genetic analysis of the human immune response to RBC antigens and also provide useful serologic reagents. Technical difficulties inherent in human B-cell immortalization have led to the development of molecular approaches that bypass the need for cell transformation. By cloning human Ig gene segments into bacterial expression vectors, libraries are created of filamentous phage particles displaying Fab fragments on their surfaces. Libraries have been screened with purified, soluble antigen and selected clones genetically manipulated in Escherichia coli to produce soluble Fab fragments. Our goal has been to adapt this technique to the study of RBC autoantibodies and alloantibodies that have specificities against unpurifiable membrane-bound antigens. To test the feasibility of this approach, two sets of phage were created, one set expressing a human anti-Rh(D) Ig and the other expressing a human antitetanus toxoid Ig. After verifying the presence of functional phage-displayed Fabs through biochemical, flow cytometric, and electron microscopic analyses, a model library was constructed comprising one anti-Rh(D)- expressing phage per 10(4) antitetanus toxoid-expressing phage. A method was developed for screening the library with intact Rh(D)- positive RBCs. After four rounds of panning, anti-Rh(D) specificity was enriched more than 10,000-fold to a final frequency of approximately 100%. Plasmid DNA derived from anti-Rh(D) phage was used to produce milligram quantities of soluble recombinant anti-Rh(D) Fabs purified by nitrogen cavitation and nickel-chelation affinity chromatography. The authenticity of the Fabs was confirmed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting, which showed bands with molecular weights of approximately 50 kD and 26 kD under nonreducing and reducing conditions, respectively. Binding of recombinant anti-Rh(D) Fabs to Rh(D)-positive RBCs was demonstrated by flow cytometry and by an agglutination assay. Our results suggest that repertoire cloning by cell-surface enrichment may have broad application to the study of the human immune response to erythroid antigens in addition to membrane-bound antigens present on other hematopoietic cells.
Requirement for nuclear factor (NF)-kappa B p65 and NF-interleukin-6 binding elements in the tumor necrosis factor response region of the granulocyte colony-stimulating factor promoter
Authors: Dunn SM , Coles LS , Lang RK , Gerondakis S , Vadas MA , Shannon MF
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor produced by mesenchymal and myeloid cells following activation by inflammatory stimuli. It has previously been shown that a region of the G-CSF promoter, (-200 to -165) containing the decanucleotide CK-1 element and two repeated sequences that resemble nuclear factor (NF)- interleukin-6 (IL-6) binding sites, is required for activation of the G- CSF gene by tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta. We now show that the NF-kappa B p65 protein can bind to and activate this TNF response region. There are several unusual features of this p65 interaction with the TNF response region. First, NF-kappa B p65 but not the related NF-kappa B p50 binds to the CK-1 element and a p50/65 hybrid protein that relies on the p50 rel homology domain for DNA binding does not transactivate the TNF response region. Second, p65 transactivation of this region is cell specific and requires not only its own binding site but also the NF-IL6 consensus sites. NF-IL6 also binds to the TNF response region of the G-CSF promoter. Electrophoretic mobility shift studies show that p65 and NF-IL6 can bind cooperatively to the TNF response region. The ability of this region to respond to TNF-alpha or p65 is correlated with the ability to form the p65/NF-IL6 ternary complex.
Role of cyclic nucleotides in rapid platelet adhesion to collagen
Authors: Polanowska-Grabowska R , Gear AR
Published: October 2019

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3′5′-cyclic monophosphate (cAMP) and guanosine 3′5′-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4- fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2- fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.
Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable upregulation
Authors: Sellak H , Franzini E , Hakim J , Pasquier C
Published: October 2019

We compared the effects of phorbol 12-myristate 13-acetate (PMA) and thrombin with those of nonlytic concentrations of reactive oxygen species (ROS) generated by hypoxanthine (HX)-xanthine oxidase (XO) on the adhesion properties of human umbilical cord vein endothelial cells (HUVEC) to resting polymorphonuclear neutrophils (PMN). PMN adherence to HX-XO-treated HUVEC was increased approximately twofold to 2.5-fold relative to untreated HUVEC, both immediately and after 2 hours. It was not additive to that induced by PMA or thrombin stimulation of HUVEC. ROS-induced adherence was not due to platelet-activating factor (PAF) or P-selectin expression, as it was neither antagonized by BN52021 (PAF receptor antagonist) nor inhibited by anti-P-selectin monoclonal antibody (MoAb), contrary to the increased adhesion of PMA- and thrombin-stimulated HUVEC. PMN preincubated with mannose-6-P or N- acetylneuraminic acid (sialic acid), but not mannose or galactose-6-P, showed reduced adherence to ROS-treated HUVEC, suggesting that carbohydrate molecules were expressed on the latter and served as the ligand for the PMN L-selectin. Intercellular adhesion molecule (ICAM- 1), constitutively present on the surface of resting HUVEC, was involved in the PMN adherence to ROS-treated HUVEC, since this adherence was inhibited by anti-ICAM-1, anti-CD11a, anti-CD11b, and anti-CD18 MoAbs. A non-CD18, non-ICAM-1-dependent mechanism is also involved in this adherence, since effects of these MoAbs were not additive; moreover, combinations of anti-CD18 and anti-ICAM-1 MoAbs with mannose-6-P and sialic acid completely inhibited PMN adherence. The increased binding of PMN to HX-XO-exposed HUVEC observed here involved IC-AM-1, but was independent of its upregulation, and another non-ICAM-1-dependent mechanism, in which carbohydrates expressed on HUVEC recognize L-selectin on PMN.
Prothrombin synthesis and degradation in rat hepatoma (H-35) cells: effects of warfarin
Authors: Zhang P , Suttie JW
Published: October 2019

Vitamin K is a substrate for the enzyme catalyzing the carboxylation of specific glutamyl residues to gamma-carboxyglutamyl residues in hepatic precursors of a limited number of plasma proteins, including prothrombin. The gamma-carboxylation of these proteins can be blocked by the anticoagulant warfarin; and in the bovine and human, warfarin treatment results in the secretion of under-gamma-carboxylated forms of prothrombin into plasma. In the rat, this response is not seen, but plasma prothrombin concentrations are drastically decreased. This response has now been studied in rat hepatoma (H-35) cells in which prothrombin secretion is decreased 90% by incubation in the presence of warfarin. Neither prothrombin mRNA levels nor the apparent rate of prothrombin message translation were decreased when cells were cultured in the presence of warfarin rather than of vitamin K. The pool of intracellular prothrombin precursors is increased threefold by warfarin treatment, and this pool is rapidly secreted when vitamin K is administered. In contrast, continued incubation in the presence of warfarin resulted in the degradation of 60% of this pool in 24 hours. When transport of secretory proteins to the golgi apparatus was blocked with Brefeldin A, this precursor pool was gamma-carboxylated in the presence of vitamin K and no degradation occurred. Lysosomal enzyme inhibitors did not block the degradation, and the data suggest that, in rat hepatocytes, under-gamma-carboxylated prothrombin is specifically targeted to a pathway of protein degradation located in the endoplasmic reticulum.
Antibodies in sulfonamide-induced immune thrombocytopenia recognize calcium-dependent epitopes on the glycoprotein IIb/IIIa complex
Authors: Curtis BR , McFarland JG , Wu GG , Visentin GP , Aster RH
Published: October 2019

Drug-dependent IgG antibodies (DDAb) induced by sulfamethoxazole (SMX) and sulfisoxazole (SIX) were identified by flow cytometry in 15 patients who developed thrombocytopenia while taking one of these medications. Fourteen of the 15 DDAb were specific solely for the glycoprotein (GP)IIb/IIIa complex, and 13 of these reacted wholly or in part with epitopes present only on the intact GPIIb/IIIa heterodimer. None of 12 SMX-induced DDAb cross-reacted with SIX, but one of three SIX-induced antibodies reacted with SMX. Each of 10 SMX-induced DDAb tested reacted with the N1-acetyl metabolite of SMX, but only one reacted fully with the N4-acetyl derivative. Detection of the SMX- and SIX-dependent antibodies was facilitated by using bovine serum albumin (BSA) to achieve suspension of these weakly soluble drugs in an aqueous medium. Our findings indicate that DDAb induced by SMX and SIX, in contrast to those induced by quinidine and quinine, are mainly specific for GPIIb/IIIa and react preferentially with calcium-dependent epitopes present only on the intact GPIIb/IIIa heterodimer.
Frequent p53 gene involvement in splenic B-cell leukemia/lymphomas of possible marginal zone origin
Authors: Baldini L , Fracchiolla NS , Cro LM , Trecca D , Romitti L , Polli E , Maiolo AT , Neri A
Published: October 2019

A phenotypic and molecular evaluation was made of 15 patients with mature B-cell leukemia/lymphoma showing exclusive spleen and bone marrow involvement. According to French-American-British criteria, these cases could not be classified as classical B-cell chronic lymphocytic leukemia, hairy cell leukemia and its variant forms, splenic lymphoma with villous lymphocytes, or leukemic phase non- Hodgkin's lymphoma (NHL; follicular or intermediate type). The immunophenotype pattern (high surface Ig and CD25 expression, and little or no reactivity with CD5, CD23, and CD11c) and cytomorphologic features of these neoplasms suggested an origin in the marginal zone of the spleen. Molecular analysis did not show any involvement of the dominantly acting oncogenes generally associated with lymphoid malignancies (c-myc, bcl-2, bcl-1, Ras), but mutations of the p53 tumor suppressor gene involving exons 5, 6, and 8 were found in 6 cases (6 of 15, 40%). In 4 cases, the p53 alterations consisted of a point mutation leading to amino acid substitution. In the remaining 2 cases, an insertion or deletion resulting in a frame-shift of the protein was observed. In all but 1 of the cases, the wild-type sequence at the mutation site was barely visible, implying the loss of the normal p53 allele in leukemic cells. All of the cases showed a clinical course compatible with that of low-grade NHL, regardless of the p53 loss/mutation. Overall, our data suggest the existence of a form of splenic B-cell leukemia/lymphoma of possible marginal zone origin in which p53 inactivation may play an important pathogenetic role.