bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the CD95 (APO-1/Fas) system in T cells from human immunodeficiency virus type-1-infected children
Authors: Baumler CB , Bohler T , Herr I , Benner A , Krammer PH , Debatin KM
Published: October 2019

Increased apoptosis of CD4+ T cells is considered to be involved in CD4+ T-cell depletion in human immunodeficiency virus type-1 (HIV-1)- infected individuals progressing toward acquired immunodeficiency syndrome (AIDS). We have recently shown that CD95 (APO-1/Fas) expression is strongly increased in T cells of HIV-1-infected children. In this report we provide further evidence for a deregulated CD95 system in AIDS. CD95 expression in HIV-1+ children is not restricted to previously activated CD45RO+ T cells but is also increased on freshly isolated naive CD45RA+ T cells. In addition, specific CD95-mediated apoptosis is enhanced in both CD4+ and CD8+ T cells. Furthermore, levels of CD95 ligand mRNA are profoundly increased. Specific T-cell receptor/CD3-triggered apoptosis in HIV-1+ children is more enhanced in CD8+ than in CD4+ T cells. Accelerated activation induced cell death of T cells could partially be inhibited by blocking anti-CD95 antibody fragments. These data suggest an involvement of the CD95 receptor/ligand system in T-cell depletion and apoptosis in AIDS and may open new avenues of rational intervention strategies.
Lack of the t(2;5) or other mutations resulting in expression of anaplastic lymphoma kinase catalytic domain in CD30+ primary cutaneous lymphoproliferative disorders and Hodgkin's disease
Authors: Wood GS , Hardman DL , Boni R , Dummer R , Kim YH , Smoller BR , Takeshita M , Kikuchi M , Burg G
Published: October 2019

The t(2;5) (p23;q35) chromosomal translocation has been found in a high proportion of lymph node-based CD30+ large cell lymphomas of T-cell lineage. This translocation is believed to result in the expression of a fusion protein containing the catalytic domain of anaplastic lymphoma kinase (ALK) under the control of the promoter for nucleophosmin, a nucleolar phosphoprotein. Expression of ALK activity, which does not normally occur in lymphocytes, is postulated to be involved in the pathogenesis of lymphomas bearing the t(2;5) translocation. Several primary cutaneous lymphoproliferative disorders and Hodgkin's disease are also known to contain CD30+ large lymphoid cells. To determine the role of the t(2;5) translocation in these diseases, we developed a DNA- based polymerase chain reaction (PCR)/Southern blot assay to detect this translocation at the genomic level in lymphomatoid papulosis (14 cases), primary cutaneous CD30+ large cell lymphoma of T-lineage (10 cases) and Hodgkin's disease (13 cases). Two cases of pityriasis lichenoides were also studied. The t(2;5) translocation was not present in any of these specimens. To determine if some other somatic mutation might have resulted in inappropriate expression of ALK catalytic domain, we devised an RNA-based reverse transcriptase-PCR assay to detect transcripts encoded by this ALK region. None were found in the six additional cases of lymphomatoid papulosis that were studied. In aggregate, these results strongly suggest that inappropriate expression of ALK is not involved in the pathogenesis of these CD30+ lymphoproliferative disorders, and that lymph node-based CD30+ large cell lymphoma is a disease that is biologically distinct from skin- based CD30+ lymphoproliferative disorders and Hodgkin's disease. Using methods developed for this report, we also cloned and sequenced the t(2;5) genomic junctional sequences present in the SUP-M2 and SU-DHL-1 cell lines. These intron sequences will be useful for mapping t(2;5) breakpoint clusters.
High-level reconstitution of respiratory burst activity in a human X- linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox
Authors: Ding C , Kume A , Bjorgvinsdottir H , Hawley RG , Pech N , Dinauer MC
Published: October 2019

The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit- spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high- level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.
Increased erythrocyte phosphatidylserine exposure in sickle cell disease: flow-cytometric measurement and clinical associations
Authors: Wood BL , Gibson DF , Tait JF
Published: October 2019

Increased exposure of phosphatidylserine (PS) in erythrocytes has been postulated to contribute to the pathophysiology of sickle cell disease because of its possible effects on blood coagulation, cell adhesion, and cell clearance. We developed a flow-cytometric assay to measure exposure of PS on the outer face of the erythrocyte membrane based on addition of fluorescein-annexin V to whole-blood specimens. The assay correlated linearly with binding of 125I-annexin V (r2 = .95, n = 125 samples). Normal donors (n = 30) showed virtually no annexin-positive cells (0.34% +/- 0.18% for 24-hour old samples). In contrast, annexin V binding was above the upper limit of normal in 96% of 205 specimens from 17 adult sickle cell and 2 beta-thalassemia patients; the mean percentage of annexin-positive cells was 2.86% +/- 2.00% (range, 0.4% to 12.0%). Values varied substantially over time for some patients, and mean values varied between patients. The percentage of annexin-positive cells always decreased after transfusion (11 events in 6 patients), and out of proportion to the amount of blood transfused. In conclusion, increased exposure of PS on a subpopulation of erythrocytes in vivo is a virtually universal feature of sickle cell disease, and its measurement may be useful to evaluate clinical status and response to therapeutic measures such as blood transfusion.
Antibodies to Transcobalamin II Block In Vitro Proliferation of Leukemic Cells
Authors: McLean Gary R. , Quadros Edward V. , Rothenberg Sheldon P. , Morgan A. Charles , Schrader John W. , Ziltener Hermann J.
Published: October 2019

The plasma protein transcobalamin II (TCII) binds and delivers cobalamin (Cbl; vitamin B12) to all cells, which internalize the TCII/Cbl complex by receptor-mediated endocytosis. Congenital deficiency of TCII results in intracellular Cbl deficiency, one effect of which is to disrupt DNA synthesis, leading to megaloblastic anemia. We report here an in vitro culture system in which cell growth is dependent on delivery of Cbl to cells by TCII. Recombinant human holo-TCII was shown to support in dose-dependent manner the growth of the human erythroleukemic cell line K562 and the murine lymphoma cell line BW5147. Free Cbl also supported cell growth; however, at 100- to 1,000-fold higher concentrations than those effective in the presence of apo-TCII. To determine if cellular depletion of Cbl could be achieved by interfering with interactions between TCII/Cbl and its cell-surface receptor, several monoclonal antibodies raised against human TCII were studied. Three antibodies, found to compete for the same binding site on TCII, proved to be effective inhibitors of TCII/Cbl-dependent cell growth. Our results suggest that monoclonal anti-TCII antibodies that block the function of this protein may prove useful in antitumor therapies.
Monoclonal Origin of Concordant T-Cell Malignancy in Identical Twins
Authors: Ford Anthony M. , Pombo-de-Oliveira Maria S. , McCarthy Keith P. , MacLean James M. , Carrico Kadma C. , Vincent Roberto F. , Greaves Mel
Published: October 2019

Acute leukemia has a high concordance rate in young identical twins and in infants this is known, from molecular analysis, to reflect an in utero origin in one twin followed by prenatal metastasis to the other twin via intraplacental anastomoses. The situation in older twins with leukemia has been less clear. We describe a pair of identical twins who were diagnosed with a T-cell malignancy at 9 and 11 years of age, one with T-cell non-Hodgkin's lymphoma and the other with T-cell acute lymphoblastic leukemia. Leukemic cells from the twins shared the same TCRβ gene rearrangement with an identical 11 bp N region. The most plausible interpretation of this result is that these malignancies were initiated in one twin fetus in utero, in a single T-lineage cell that had stable bi-allelic TCRβ rearrangements. Progeny of this cell then spread to the other twin before birth via shared placental vasculature. This was then followed by a 9- and 11-year preleukemic latent period before clinical disease manifestation as leukemia or lymphoma. This result has considerable implications for the etiology and natural history of pediatric leukemia.
Direct Binding of CRKL to BCR-ABL Is Not Required for BCR-ABL Transformation
Authors: Heaney Conor , Kolibaba Kathryn , Bhat Arun , Oda Tsukasa , Ohno Sayuri , Fanning Shane , Druker Brian J.
Published: October 2019

CRKL has previously been shown to be a major tyrosine phosphorylated protein in neutrophils of patients with BCR-ABL+ chronic myelogenous leukemia and in cell lines expressing BCR-ABL. CRKL and BCR-ABL form a complex as demonstrated by coimmunoprecipitation and are capable of a direct interaction in a yeast two-hybrid assay. We have mapped the site of interaction of CRKL and BCR-ABL to the amino terminal SH3 domain of CRKL with a proline rich region in the C-terminus of ABL. The proline-rich region was mutated and the effect of this deletion on BCR-ABL transforming function was assayed. Our data show that this deletion does not impair the ability of BCR-ABL to render myeloid cells factor independent for growth. In cells expressing the proline deletion mutation of BCR-ABL, CRKL is still tyrosine phosphorylated and forms a complex with BCR-ABL as demonstrated by coimmunoprecipitation. Our data suggest that the interaction between CRKL and the proline deletion mutant of BCR-ABL is an indirect interaction as CRKL does not interact directly with the proline deletion mutant of BCR-ABL in a gel overlay assay or in a yeast two-hybrid assay. Thus, a direct interaction of CRKL and BCR-ABL is not required for CRKL to become tyrosine phosphorylated by BCR-ABL and suggests that CRKL function may still be required for BCR-ABL function through an indirect interaction.
Erythroblastic Inclusions in Dominantly Inherited β Thalassemias
Authors: Ho P. Joy , Wickramasinghe Sunitha N. , Rees David C. , Lee Matthew J. , Eden Ayed , Thein Swee Lay
Published: October 2019

While the precipitation of unstable variant β-globin chains has been implicated as a major pathogenic mechanism in dominantly inherited β thalassemia, their instability and presence in intra-erythroblastic inclusions have not been conclusively shown. We report the investigation of two cases of dominantly inherited β thalassemia due to heterozygosity for the β-codon 121 G-T mutation. In one case, we were able to demonstrate the presence of an abnormal β-globin chain in both peripheral blood reticulocytes and bone marrow erythroblasts, and to assess its stability in relation to the substantial amounts of mutant β mRNA transcript. The serum transferrin receptor (TfR) level was markedly increased, an indication of increased erythropoietic activity. In both cases, we could show by immunoelectron microscopy that the intra-erythroblastic inclusion bodies, a prominent feature of diseases in this category, contained not only precipitated α-globin chains, but also β chains. The data confirm previous suggestions that the cellular pathology underlying this group of β thalassemias is related to the synthesis of highly unstable β-globin chain variants, which fail to form functional tetramers and precipitate intracellularly with the concomitant excess α chains, leading to increased ineffective erythropoiesis.
Polycythemia Vera V. Enhanced Proliferation and Phosphorylation Due to Vanadate Are Diminished in Polycythemia Vera Erythroid Progenitor Cells: A Possible Defect of Phosphatase Activity in Polycythemia Vera
Authors: Dai Chun-Hua , Krantz Sanford B. , Sawyer Stephen T.
Published: October 2019

Erythropoietin (EP) and stem cell factor (SCF ) are essential growth factors for erythroid progenitor cell proliferation and differentiation in serum-free culture. It has been previously shown that burst-forming units-erythroid and colony-forming units-erythroid from patients with polycythemia vera (PV) have enhanced sensitivity to EP and SCF compared with normal erythroid progenitors, but little is known about the mechanism for this difference. In the present investigation, the effect of EP and SCF on protein tyrosine phosphorylation in day-8 normal and PV erythroid colony-forming cells, which give rise to colonies of 2-49 hemoglobinized cells, was studied. EP rapidly induced tyrosine phosphorylation of the EP receptor, whereas the most prominent phosphorylated protein induced by SCF was identified as the SCF receptor. No additional phosphorylated proteins were evident when PV cells were compared with normal cells. Culture of normal erythroid progenitors with orthovanadate, an inhibitor of protein tyrosine phosphatases, resulted in an increased number of erythroid colonies and enhanced protein tyrosine phosphorylation. However, in contrast, little enhancement was evident with PV cells. These results indicate that, although vanadate may be acting in normal erythroid progenitors as a phosphatase inhibitor that potentiates the kinase activity induced by SCF and EP, this function is diminished in PV cells. Because erythropoiesis is regulated by a balance between protein tyrosine kinase activity and protein tyrosine phosphatase activity, PV patients may have an abnormal phosphatase activity allowing increased cell proliferation.
Glanzmann Thrombasthenia Caused by an 11.2-kb Deletion in the Glycoprotein IIIa (β3 ) Is a Second Mutation in Iraqi Jews That Stemmed From a Distinct Founder
Authors: Rosenberg Nurit , Yatuv Rivka , Orion Yael , Zivelin Ariella , Dardik Rima , Peretz Hava , Seligsohn Uri
Published: October 2019

Glanzmann thrombasthenia (GT) is a rare bleeding disorder resulting from mutations in either glycoprotein (GP) IIb or GPIIIa genes. The disease is relatively frequent in highly inbred populations such as Iraqi Jews. The molecular basis of GT in 6 unrelated Iraqi-Jewish patients was previously identified as an 11-bp deletion in exon 12 of the GPIIIa gene. We now describe a second mutation found in 3 unrelated Iraqi-Jewish families that consists of an 11.2-kb deletion between an Alu repeat in intron 9 and exon 13 of the GPIIIa gene. The mutant DNA is transcribed into mRNA in which exons 10 through 13 are absent. Splicing of exon 9 directly to exon 14 leads to a shift in the reading frame resulting in a stop codon. The predicted protein is truncated in the middle of the third cysteine-rich domain before the transmembrane domain. Simple DNA-based methods were devised for identification of both mutations in Iraqi Jews for the purpose of carrier detection and prenatal diagnosis enabling prevention of GT. A survey of the general Iraqi-Jewish population for the first 11-bp deletion and the second 11.2-kb deletion disclosed that the allele frequency of the first mutation was 0.0043, whereas none of 700 individuals examined bore the second mutation (allele frequency <0.0007). Among 40 GT patients of Iraqi-Jewish origin 31 were homozygous for the first mutation, 4 were compound heterozygotes for the first and second mutations, and 2 were homozygous for the second mutation. Haplotype analyses using 4 polymorphic markers in the GPIIIa gene showed that each mutation originated in a distinct founder.
MLL Gene Rearrangement, Cytogenetic 11q23 Abnormalities, and Expression of the NG2 Molecule in Infant Acute Myeloid Leukemia
Authors: Hilden Joanne M. , Smith Franklin O. , Frestedt Joy L. , McGlennen Ronald , Howells William B. , Sorensen Poul H.B. , Arthur Diane C. , Woods William G. , Buckley Jonathan , Bernstein Irwin D. , Kersey John H.
Published: October 2019

To study prognostic factors in infant acute myeloid leukemia (AML), we analyzed 44 children treated on Childrens Cancer Group protocols for MLL gene rearrangement by Southern blot, cytogenetic 11q23 abnormalities, and reactivity with monoclonal antibody 7.1. This antibody detects the human homologue of the rat NG2 chondroitin sulfate proteoglycan molecule, which has previously been reported to be expressed on human melanoma. NG2 has been found to be expressed on human leukemic blasts but not on other hematopoietic cells. In childhood AML, NG2 cell surface expression correlated with poor outcome and with some but not all 11q23 rearrangements. In childhood acute lymphoblastic leukemia, NG2 expression correlated with poor outcome and with balanced 11q23 translocations. In this study, 29 of 44 (66%) of infants with AML showed MLL rearrangement and, as expected, this group had a high incidence of French-American-British M4/M5 morphology (22/29). Of the cases tested, 35.1% (13/37) were NG2 positive. All (13/13) NG2-positive cases were rearranged at MLL, whereas only 46% (11/24) of NG2-negative cases had MLL rearrangement. NG2 expression did not correlate with poor outcome (P = .31); there was a trend towards a worse outcome with MLL rearrangement (P = .13). Thus monoclonal antibody 7.1 does not detect all cases of MLL rearrangement in infant AML.
Cyclosporine or Cyclosporine Plus Methylprednisolone for Prophylaxis of Graft-Versus-Host Disease: A Prospective, Randomized Trial
Authors: Deeg H. Joachim , Lin Danyu , Leisenring Wendy , Boeckh Michael , Anasetti Claudio , Appelbaum Frederick R. , Chauncey Thomas R. , Doney Kristine , Flowers Mary , Martin Paul , Nash Richard , Schoch Gary , Sullivan Keith M. , Witherspoon Robert P. , Storb Rainer
Published: October 2019

Patients with a lymphohematopoietic malignancy considered to be at high risk for posttransplant relapse were enrolled in a study to compare the use of cyclosporine (CSP) as a single agent with a combination of methylprednisolone (MP) and CSP for graft-versus-host disease (GVHD) prophylaxis after marrow transplantation from an HLA-identical sibling donor. Sixty patients were randomized to receive CSP only and 62 were randomized to receive CSP plus MP. Daily CSP was started on day −1 (5 mg/kg/d intravenously) and administered at gradually reduced doses until day 180. MP was started on day 7 at 0.5 mg/kg/d, increased to 1.0 mg/kg/d on day 15, started on a taper schedule on day 29, and discontinued on day 72. All 104 evaluable patients (surviving ≥28 days) had sustained engraftment. The incidence rates of grades II-IV acute GVHD were 73% and 60% for patients receiving CSP and CSP plus MP, respectively (P = .01). No difference was seen for grades III-IV GVHD. However, chronic GVHD occurred somewhat more frequently in patients receiving CSP plus MP (44%) than in patients receiving only CSP (21%; P = .02). The incidence of de novo chronic GVHD was marginally higher in patients receiving CSP plus MP (P = .08). No significant differences in the risk of infections were observed. There was a suggestion that the risk of relapse was lower in patients receiving CSP plus MP (P = .10) and, although the overall survival in the two groups was not different (P = .44), there was a slight advantage in favor of CSP plus MP-treated patients for relapse-free survival (P = .07). These results suggest that prophylactic MP, when combined with CSP, has only limited efficacy in acute GVHD prevention and may increase the probability of chronic GVHD.
In Vivo Efficacy and Toxicity of a Single-Chain Immunotoxin Targeted to CD40
Authors: Francisco Joseph A. , Schreiber George J. , Comereski Chuck R. , Mezza Laurence E. , Warner Garvin L. , Davidson Thomas J. , Ledbetter Jeffrey A. , Siegall Clay B.
Published: October 2019

G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37°C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and monkeys, with the maximum tolerated dose being 0.48, 1.0, and 1.67 mg/kg, respectively. Comparative immunohistology showed that the G28-5 specificity was qualitatively similar between human and monkey tissue. In summary, G28-5 sFv-PE40 was effective at inducing complete antitumor responses in lymphoma xenografted mice at doses that were well tolerated in mice, rats, and monkeys.
Treatment of Hepatic Venocclusive Disease With Recombinant Human Tissue Plasminogen Activator and Heparin in 42 Marrow Transplant Patients
Authors: Bearman Scott I. , Lee Jung Lim , Barón Anna E. , McDonald George B.
Published: October 2019

The purpose of this report is to review the Fred Hutchinson Cancer Research Center experience of treating patients with venocclusive disease of the liver (VOD) after marrow transplantation using recombinant human tissue plasminogen activator (rh-tPA) and heparin. The charts of 42 patients who had received rh-tPA and heparin for the treatment of VOD between February 1991 and December 1995 were reviewed. Response to rh-tPA and heparin was defined as a reduction in total serum bilirubin by 50% within 10 days of starting treatment. Total serum bilirubin, percent weight gain, and serum creatinine before, after, and at the start of rh-tPA and heparin were examined to determine whether these laboratory values distinguished patients who responded to treatment from those who did not. We also evaluated whether evidence of multiorgan failure (requirement for supplemental oxygen, requirement for hemodialysis, requirement for mechanical ventilation) or whether the calculated probability of a fatal outcome from VOD could discriminate responders from nonresponders. In addition, the incidence and outcome of bleeding as a major complication of thrombolytic therapy was examined. Twelve patients responded to rhtPA and heparin and 30 patients did not. There were no statistically significant differences between responders and nonresponders in the day treatment was started, dose of rh-tPA, total serum bilirubin, and percent weight gain before, after, or at the start of treatment, or the calculated probability of dying from VOD on the day treatment with rh-tPA and heparin was begun. More nonresponding patients required dialysis or mechanical ventilation (11 of 30) before or at the start of rh-tPA and heparin than responding patients (0 of 12), P = .0183. Serum creatinine was greater at the start of treatment in nonresponding patients (1.9 ± 1.3 mg/dL) than in responding patients (1.1 ± 0.4 mg/dL), P = .0794. Ten patients had severe bleeding episodes, which resulted in death in three patients and may have contributed to death in an additional three patients. Treatment for VOD using rh-tPA and heparin was successful in 29% of patients but was associated with a significant risk of life-threatening hemorrhage. Requirement for supplemental oxygen, dialysis, or mechanical ventilation before the start of treatment were prognostic indicators of no response to thrombolytic therapy. We do not recommend treatment using tPA and heparin in patients with severe VOD who have already developed multiorgan dysfunction.
Tyrosine Phosphorylation and p72syk Activation by an Anti-Glycoprotein Ib Monoclonal Antibody
Authors: Yanabu Mutsumasa , Ozaki Yukio , Nomura Shosaku , Miyake Tetsuya , Miyazaki Yasuhiko , Kagawa Hideo , Yamanaka Yoshitaka , Asazuma Naoki , Satoh Kaneo , Kume Shoji , Komiyama Yutaka , Fukuhara Shirou
Published: October 2019

NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ibα, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF ). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-FcγII receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab′ fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab′ fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the FcγII receptor. Cross-linking of the receptor-bound Fab′ fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5–induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.
Suppression of Alloantigen-Induced T-Cell Proliferation by CD14+ Cells Derived From Granulocyte Colony-Stimulating Factor–Mobilized Peripheral Blood Mononuclear Cells
Authors: Mielcarek Marco , Martin Paul J. , Torok-Storb Beverly
Published: October 2019

The proliferative responsiveness of granulocyte colony-stimulating factor (G-CSF )–mobilized blood was studied in uni-directional mixed leukocyte cultures. Unfractionated mononuclear cells from mobilized blood obtained by leukapheresis at day 4 after initiation of G-CSF (G-PBMC) were hyporesponsive (31.5% ± 9.2% response, P = .003) compared to mononuclear cells obtained from the peripheral blood before administration of G-CSF (preG-PBMC). There was great variability among donors when purified preG- and G-CD4 cells were compared. In eight of 10 donors, G-CD4 cells were equally responsive or moderately hyporesponsive; in two of 10 donors, G-CD4 cells were more strikingly hyporesponsive. CD14 cells derived from leukapheresis products (G-CD14 cells) suppressed alloantigen-induced proliferation by 48.6% ± 7.5% when added to preG-PBMC or preG-CD4 cells at responder-CD14 ratios of 2:1 (P < .001). Suppression was evident (14.4% ± 5.0%) even at responder-CD14 ratios of 8:1 and was largely contact-independent. PreG- and G-CD14 cells had equivalent potency in suppressing proliferative responses. Given that G-CSF–mobilized blood cell grafts contain 50-fold more CD14 cells and only 10-fold more T cells than marrow, we propose that suppression of donor T cells by the large proportion of monocytes present in leukapheresis products could contribute to the unexpectedly low incidence and severity of graft-versus-host disease after peripheral blood stem cell transplantation.
Detection of the t(2; 5)(p23; q35) and NPM-ALK Fusion in Non-Hodgkin's Lymphoma by Two-Color Fluorescence In Situ Hybridization
Authors: Mathew Prasad , Sanger Warren G. , Weisenburger Dennis D. , Valentine Marcus , Valentine Virginia , Pickering Diane , Higgins Chris , Hess Michelle , Cui Xiaoli , Srivastava Deo Kumar , Morris Stephan W.
Published: October 2019

The non-Hodgkin's lymphoma (NHL) subset commonly referred to as large cell lymphoma (LCL) has historically been characterized by its marked cytological, immunological, and clinical heterogeneity. One potential defining feature of these lymphomas, the t(2; 5)(p23; q35), occurs in 25% to 30% of anaplastic LCLs and is also found in cases with diffuse large cell or immunoblastic morphology. We recently identified nucleophosmin (NPM ) and anaplastic lymphoma kinase (ALK ) as the genes on chromosomes 5 and 2, respectively, that are juxtaposed by this translocation. To provide a complementary approach to the use of classical cytogenetics or polymerase chain reaction-based methods for the detection of this abnormality, we have developed a two-color fluorescent in situ hybridization (FISH) assay for the t(2; 5) that may be used for the analysis of both interphase nuclei and metaphase chromosomes. Three overlapping chromosome 5 cosmid clones located immediately centromeric to the NPM gene locus and an ALK P1 clone located telomeric to the chromosome 2 breakpoint were labeled with digoxigenin or biotin, respectively, and used to visualize the derivative chromosome 5 produced by the t(2; 5), evident as juxtaposed or overlapping red and green fluorescent signals. This NPM-ALK FISH assay was initially validated by analysis of a series of cytogenetically characterized cell lines, with the presence of the der(5) chromosome showed specifically only in those lines known to contain the t(2; 5). The assay was then applied in a blinded fashion to a series of eight cytogenetically t(2; 5)-positive clinical specimens and seven known t(2; 5)-negative cases, including three NHL and four Hodgkin's disease biopsy samples. Whereas the t(2; 5)-negative cases were negative by FISH, all eight t(2; 5)-positive cases were positive. One additional case, initially thought to be positive for the translocation by cytogenetics, was proven to not be a classic t(2; 5) by interphase and metaphase FISH. These data indicate that the FISH assay described is a highly specific and rapid test that should prove to be a useful adjunct to the currently available methods for detection of the t(2; 5).
Genetic Correction of p67phox Deficient Chronic Granulomatous Disease Using Peripheral Blood Progenitor Cells as a Target for Retrovirus Mediated Gene Transfer
Authors: Weil Wayne M. , Linton Gilda F. , Whiting-Theobald Narda , Vowells Sarah J. , Rafferty Steven P. , Li Fei , Malech Harry L.
Published: October 2019

Chronic granulomatous disease (CGD) can result from any of four single gene defects involving the components of the superoxide (O−2 ) generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We show that transduction of peripheral blood CD34+ hematopoietic progenitors from a p67phox deficient CGD patient with replication defective amphotropic retrovirus encoding p67phox (MFGS-p67phox) significantly corrected the CGD functional defect in phagocyte oxidase activity in vitro. Using a chemiluminescence assay of oxidase activity, we showed that transduced patient CD34+ progenitors differentiating to myeloid cells in culture produced 25% of the total superoxide produced by normal CD34+ progenitors differentiating in culture. A flow cytometric assay of oxidase activity used to assess the oxidase function of individual cells in the cultures indicated that up to 32% of maturing granulocytes derived from transduced CD34+ progenitors from the p67phox CGD patient were oxidase positive with the average level of correction per granulocyte of 85% of that seen with granulocytes in similar cultures of CD34+ progenitors from normal volunteers. Nitroblue tetrazolium dye reduction assays of colonies of transduced progenitors in soft agar indicated that in some studies restoration of oxidase activity occurred in myeloid cells within 44% of granulocyte-erythrocyte-monocyte colonies, and within 28% of the combined group of granulocyte colonies/monocyte colonies/granulocyte-monocyte colonies. These high correction rates were achieved without any selective regimen to enrich for transduced cells. This study provides a basis for development of gene therapy for the p67phox deficient form of CGD.
The HRX Proto-oncogene Product Is Widely Expressed in Human Tissues and Localizes to Nuclear Structures
Authors: Butler Lisa H. , Slany Robert , Cui Xiangmin , Cleary Michael L. , Mason David Y.
Published: October 2019

Chromosomal rearrangement of the HRX (MLL, ALL-1, Htrx) gene situated at chromosome band 11q23 is one of the most frequent genetic changes in infant leukemias of myeloid and lymphoid lineage and in treatment-induced secondary leukemias. The HRX gene codes for a predicted 431-kD protein that shows significant homology to the Drosophila trithorax protein, an Hox epigenetic regulator. Typically, the region encoding the HRX gene is rearranged, mostly in reciprocal translocations with a number of partners, resulting in a range of fusion genes. However, this is not the only abnormality affecting HRX because partial duplication of the gene, as well as interstitial deletions, can occur. Despite extensive studies of HRX at the genetic level, the protein products of the HRX gene and their patterns of expression in normal and leukemic cells remain uncharacterized. In this study we analyzed the distribution and localization of HRX proteins in cell lines and human tissues, using both polyclonal and monoclonal antibodies. The specificity of these reagents was confirmed using cells transfected with the HRX-ENL fusion gene. Western blot analyses of protein extracts from cells carrying the t(11; 19) and t(4; 11) translocations showed HRX chimeric proteins whose migrations corresponded to the sizes predicted from analyses of translocation-induced fusion mRNAs expressed by the derivative 11 chromosomes. Immunocytochemical analysis showed a punctate distribution of wild-type and chimeric HRX proteins within cell nuclei, suggesting that HRX localizes to nuclear structures in cells with and without 11q23 translocations. Nuclear staining was found in the majority of tissues studied with the strongest reactivity in cerebral cortex, kidney, thyroid, and lymphoid tissues. Thus, HRX is widely expressed in most cell types including hematopoietic cells, a finding that precludes an immunocytochemical approach for diagnosis of leukemias bearing 11q23 structural abnormalities.
Short- and Long-Term Multilineage Repopulating Hematopoietic Stem Cells in Late Fetal and Newborn Mice: Models for Human Umbilical Cord Blood
Authors: Harrison David E. , Astle Clinton M.
Published: October 2019

Blood from late fetal and newborn mice is similar to umbilical cord blood obtained at birth in human beings, an important source of stem cells for clinical transplantation. The mouse model is useful because long-term functions can be readily assayed in vivo. To evaluate the functions of hematopoietic precursors in the blood and other tissues of late fetal and newborn mice, short- and long-term multilineage repopulating abilities were measured in vivo by competitive repopulation. Manipulations that might affect cell function, such as enrichment, tissue culture, or retroviral marking, were avoided. Hematopoietic stem cell functions of late fetal or newborn blood, liver, and spleen, were assayed as myeloid and lymphoid repopulating abilities relative to standard adult marrow cells. Donor cells from these tissues as well as adult control donor marrow cells were all of the same genotype. Cells from each donor tissue were mixed with portions from a pool of standard adult “competitor” marrow distinguished from the donors by genetic differences in hemoglobin and glucosephosphate isomerase. After 21 to 413 days, percentages of donor type myeloid and lymphoid cells in recipient blood were measured to assay the functional abilities of donor precursors relative to the standard. These relative measures are expressed as repopulating units, where each unit is equivalent to the repopulating ability found in 100,000 standard adult marrow cells. Thus, measures of repopulating units do not compare single cells but overall repopulating abilities of donor cell populations. Relative functional abilities in 1 million nucleated cells from late fetal or newborn blood were several times less than those found in adult marrow, but far more than in normal adult blood, and appeared to include long-term functional primitive hematopoietic stem cells (PHSC) similar to those in marrow. To estimate functional abilities of individual PHSC, variances among large groups of identical recipients were analyzed using both the binomial model and competitive dilution, a new model based on the Poisson distribution. The data best fit the hypothesis that individual PHSC from adult marrow, late fetal blood, or newborn blood each produce similar fractions of the total lymphoid and erythroid cells found in the recipient for many months.
Human Plasminogen Activator Inhibitor-1 (PAI-1) Deficiency: Characterization of a Large Kindred With a Null Mutation in the PAI-1 Gene
Authors: Fay William P. , Parker Andrew C. , Condrey Lorraine R. , Shapiro Amy D.
Published: October 2019

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue- and urokinase-type plasminogen activators, is considered a critical regulator of the fibrinolytic system. We previously reported a child with abnormal bleeding and complete PAI-1 deficiency caused by a frame-shift mutation in exon 4 of the PAI-1 gene. The purpose of this study was to provide genetic and clinical data on the extended pedigree of the original proband to better define the phenotype associated with PAI-1 deficiency. Allele-specific oligonucleotide hybridization was used to genotype individuals, and serum PAI-1 antigen was measured by enzyme-linked immunosorbent assay. By this approach we have identified 19 individuals who are heterozygous for the PAI-1 null allele and 7 homozygous individuals with complete PAI-1 deficiency. Clinical manifestations of PAI-1 deficiency were restricted to abnormal bleeding, which was observed only after trauma or surgery in homozygous affected individuals. A spectrum of bleeding patterns was observed, including intracranial and joint bleeding after mild trauma, delayed surgical bleeding, severe menstrual bleeding, and frequent bruising. Fibrinolysis inhibitors, including ε-aminocaproic acid and tranexamic acid, were effective in treating and preventing bleeding episodes. Other than abnormal bleeding, no significant developmental or other abnormalities were observed in homozygous PAI-1–deficient individuals. Heterozygous PAI-1 deficiency was not associated with abnormal bleeding, even after trauma or surgery. These observations define the clinical spectrum of PAI-1 deficiency and provide additional evidence to support the hypothesis that the primary function of plasminogen activator inhibitor-1 in vivo is to regulate vascular fibrinolysis.
Death of Bystander Cells by a Novel Pathway Involving Early Mitochondrial Damage in Human Immunodeficiency Virus–Related Lymphadenopathy
Authors: Carbonari Maurizio , Pesce Anna Maria , Cibati Marina , Modica Alessandro , Dell'Anna Lucia , D'Offizi Giampiero , Angelici Alberto , Uccini Stefania , Modesti Andrea , Fiorilli Massimo
Published: October 2019

Destruction of immune cells in peripheral lymphoid tissues plays presumably a pivotal role in acquired immune deficiency syndrome pathogenesis. We found that cell suspensions obtained from lymph nodes of eight human immunodeficiency virus (HIV)-infected individuals contained variable proportions (2.1% to 18.3%, median 11.2%) of dead lymphocytes permeable to supravital dyes, represented by CD4+, CD8+, and B cells. The frequency of dead cells correlated directly (R = 0.847) with the amount of HIV provirus in the cell populations, and HIV provirus was enriched in the dead cell fractions. Similar proportions of dead cells were observed in cell suspensions from lymphadenopathic lymph nodes of HIV− donors, but not from small resting HIV− lymph nodes. Electron microscopic and flow cytometric analyses revealed that most dead cells from HIV+ lymph nodes lacked internucleosomal DNA fragmentation but displayed combined features of apoptosis and necrosis, eg, chromatin condensation and mitochondrial swelling. Cells with similar morphology were readily identified in lymph node tissue sections, and marked mitochondrial swelling could be occasionally observed in cells with otherwise normal morphology. Our findings have two major implications. One is that the in vivo cell death in HIV-infected lymph nodes occurs predominantly through a novel pathway, related to but distinct from classical apoptosis and characterised by early and severe mitochondrial damage. The second implication is that HIV-related lymphadenopathy is accompanied in vivo by massive destruction of uninfected lymph node cells. Comparable levels of cell death were observed in other inflammatory lymphadenopathies not related to HIV; however, the uniquely endless and generalized nature of HIV lymphadenopathy might render this “inflammatory” cell destruction a powerful pathogenetic mechanism, accounting for the progressive disruption and depletion of lymphoid tissues seen in HIV infection.
p100: A Novel Proliferation-Associated Nuclear Protein Specifically Restricted to Cell Cycle Phases S, G2 , and M
Authors: Heidebrecht H.J. , Buck F. , Steinmann J. , Sprenger R. , Wacker H.H. , Parwaresch R.
Published: October 2019

By immunization with nuclear lysates of L428 cells, we raised a monoclonal mouse antibody, Ki-S2 (IgG1 ). In Western blots, this antibody recognizes a nuclear antigen with an apparent molecular mass of 100 kD, termed p100. Protein sequencing of p100 showed that this is a hitherto unknown protein. Immunohistochemical examination of cryostat and paraffin sections of nearly all human tissue types and neoplasms showed that p100 was exclusively expressed in the nuclei of a fraction of proliferating cells. Cell sorting and fluorescence-activated cell sorting analysis of stimulated peripheral blood mononuclear cells showed that p100 was exclusively expressed in proliferating cells from the transition G1/S until the end of cytokinesis. During mitosis, this protein is strictly associated with the spindle pole and with the mitotic spindle, whereas during S and G2 , p100 is diffusely distributed throughout the cell nucleus. Immediately after completion of cytokinesis, p100 was rapidly degraded. In L428 cells, p100 is phosphorylated at least during mitosis. It has a turnover time of about 1 hour. Studies on routinely processed paraffin sections of specimens of malignant lymphoma, benign and malignant nevocellular tumors, and breast cancer showed that in all cases less than 40% of the Ki-67–positive growth fraction expressed p100. Thus, p100 might prove to be a more reliable measure of cellular proliferation and one that is more closely correlated to cancer prognosis, beyond its general biologic relevance as a cell cycle protein.
Inhibition of Apoptosis in a Human Pre-B–Cell Line by CD23 Is Mediated Via a Novel Receptor
Authors: White Lindsey J. , Ozanne Bradford W. , Graber Pierre , Aubry Jean-Pierre , Bonnefoy Jean-Yves , Cushley William
Published: October 2019

Human CD23 is a 45-kD type II membrane glycoprotein, which functions as a low-affinity receptor for IgE and as a ligand for the CD21 and CD11b/CD11c differentiation antigens. CD23 is released from the surface of cells as soluble fragments, and a 25-kD species of soluble CD23 (sCD23) appears to act as a multifunctional cytokine. In this report, sCD23 is shown to sustain the growth of low cell density cultures of a human pre-B–acute lymphocytic leukemia cell line, SMS-SB: no other cytokine tested was able to induce this effect. Flow cytometric analysis indicates that sCD23 acts to prevent apoptosis of SMS-SB cells. SMS-SB cells cultured at low cell density possess low levels of bcl-2 protein. Addition of sCD23 to cells at low cell density maintained bcl-2 expression at levels equivalent to those observed in SMS-SB cells cultured at higher cell densities. No CD23 mRNA was found in SMS-SB cells, ruling out an autocrine function for CD23 in this cell line model. Although SMS-SB cells do not express the known receptors for CD23, namely CD21, CD11b-CD18, or CD11c-CD18, the cells specifically bind CD23-containing liposomes, but not glycophorin-containing liposomes. Binding of CD23-containing liposomes is inhibited by anti-CD23 but not by anti-CD21 or anti-CD11b/c monoclonal antibodies. The data show that sCD23 prevents apoptosis of the SMS-SB cell line by acting through a novel receptor.
Alternative Splicing of a Novel Glycophorin Allele GPHe(GL) Generates Two Protein Isoforms in the Human Erythrocyte Membrane
Authors: Huang Cheng-Han , Blumenfeld Olga O. , Reid Marion E. , Chen Ying , Daniels Geoff L. , Smart Elizabeth
Published: October 2019

The Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at −6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.