bluuebunny/crossref_metadata_2025 huggingface dataset:
Human T-cell leukemia virus type I tax/rex DNA and RNA in cutaneous T- cell lymphoma
Authors: Ghosh SK , Abrams JT , Terunuma H , Vonderheid EC , DeFreitas E
Published: October 2019

Peripheral blood mononuclear cells (PBMCs) and T-cell lines from patients with Sezary syndrome (SS) and skin lesions from patients with mycosis fungoides (MF) were examined by polymerase chain reaction (PCR) for DNA sequences homologous to the human retroviruses human T- lymphotropic virus (HTLV)-I and -II. Results obtained using primers and probes from the tax/rex region of HTLV-I indicate that 72% (18/25) of SS patients PBMCs, 80% (20/25) of T-cell lines established from SS- PBMC, and 30% (3/10) of skin lesions from MF patients were positive for HTLV-I tax/rex region DNA. Sequence analysis of the 127-bp fragment amplified by the tax/rex primers from 4 of these individuals was found to be identical to that in prototypic HTLV-I. Negative results were obtained using primers and probes from the HTLV-I gag region and the HTLV-II gag and tax regions. No PCR products were obtained using all primers and probes using DNA from 9 healthy blood donors and 10 cord bloods. Expression of HTLV-I tax/rex mRNA was found in 4 of 8 Sezary patients, as determined by RNA-PCR, indicating that this viral region is being transcribed in vivo. Exposure to Tax/Rex protein in SS- patients is supported by the fact that serum antibodies against p27rex and p40tax was observed in 43% and 29% of these SS patients, respectively. Although the causal relationship between the HTLV-I tax/rex region and cutaneous T-cell lymphoma (CTCL) remains unclear, these findings support the presence of a truncated HTLV-I retrovirus in CTCL patients.
Transgenic mice expressing antisense interleukin-3 RNA develop a B-cell lymphoproliferative syndrome or neurologic dysfunction
Authors: Cockayne DA , Bodine DM , Cline A , Nienhuis AW , Dunbar CE
Published: October 2019

Transgenic mice that expressed antisense interleukin-3 (AS-IL-3) RNA were generated and exhibited either a B-cell lymphoproliferative syndrome or progressive neurologic dysfunction. Each syndrome occurred in the founder or progeny mice of three separate transgenic lines. The lymphoproliferative process involved the accumulation, within peripheral lymphoid organs, of B220+/slgM- pre-B cells that had immunoglobulin (Ig) genes predominantly in germline configuration and expressed lambda 5 and Rag-1 transcripts. Transgenic animals that developed neurologic dysfunction exhibited circling behavior that progressed to ataxia and terminal inanition. AS-IL-3 transcripts were detected in mature CD3+ T cells of asymptomatic transgenic animals, as well as in B220+/slgM- pre-B cells, and CD3+ T cells from animals with the lymphoproliferative syndrome. AS-IL-3 transcripts were also detected in the brains of both young asymptomatic transgenic animals and older transgenic animals with neurologic dysfunction. Decreased IL- 3 production from ConA-stimulated splenocytes was observed in asymptomatic transgenic animals. These observations suggest that this cytokine may have important roles in B lymphopoiesis and neurologic function.
A special report: bone marrow transplants using volunteer donors-- recommendations and requirements for a standardized practice throughout the world--1994 update. The WMDA Executive Committee
Authors: Goldman JM
Published: October 2019

A primary function of the World Marrow Donor Association is to establish general guidelines covering collaboration between international donor registries and practice in regard to bone marrow transplants (BMTs) in which the donor and recipient reside in different countries. To this end, a special report proposing specific recommendations and requirements was published in 1992. This paper amplifies the previous publication and gives special attention to accreditation of national “hubs” (defined as coordinating centers for each country) and donor, harvest, and transplant centers, details of the marrow harvest procedure, use of peripheral blood (PB) stem cells for allografting, and use of PB lymphoid cells for treatment of leukemia relapsing after BMT.
Histiocytes and histiocytosis [see comments]
Authors: Cline MJ
Published: October 2019

The term histiocyte refers to cells of either the macrophage or Langerhans cell lineages. The histiocytic disorders are characterized by the proliferation of cells of these lineages. With recent advances in knowledge of the developmental biology of histiocytic cells, it is now possible to formulate a reasonable catalogue of histiocytic diseases based on ultra-structural and phenotypic markers of cellular origins and molecular or chromosomal markers of malignancy. The catalogue includes the following groups of diseases. Nonmalignant reactive macrophage disorders include (1) macrophage storage diseases, (2) several benign proliferative macrophage disorders that predominantly involve skin and bone, and (3) several hemophagocytic syndromes that vary from indolent and benign to fulminant and fatal. In some of the latter disorders, viruses have been identified as the inciting stimulus. The malignant macrophage disorders include (1) acute monocytic leukemia and (2) chronic myelomonocytic leukemia. A rare disorder that gave rise to a permanent cell line with an anomaly of chromosomal segment 5q35 may also be an example of a histiocytic malignancy. The existence of a separate category of true histiocytic lymphoma of macrophage type is uncertain. Reactive Langerhans cell disorders include (1) congenital self-healing histiocytosis, (2) the many variants of eosinophilic granuloma, and (3) a related disorder designated as relapsing Langerhans cell histiocytosis that is characterized by a relapsing course and infiltration of bone and soft tissues by Langerhans cells. Presumptively neoplastic diseases of Langerhans and dendritic cells include (1) progressive Langerhans cell histiocytosis, a disease with prominent involvement of blood and BM as well as skin and viscera; (2) Langerhans cell lymphoma, and (3) dendritic cell lymphoma. However, clonality as a marker of malignancy has not been proven in these disorders.
Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector
Authors: von Kalle C , Kiem HP , Goehle S , Darovsky B , Heimfeld S , Torok-Storb B , Storb R , Schuening FG
Published: October 2019

Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)
Transcriptional regulation of the human interleukin-6 gene promoter in human T-cell leukemia virus type I-infected T-cell lines: evidence for the involvement of NF-kappa B
Authors: Mori N , Shirakawa F , Shimizu H , Murakami S , Oda S , Yamamoto K , Eto S
Published: October 2019

Freshly isolated leukemic cells from patients with adult T-cell leukemia (ATL) and human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of interleukin-6 (IL-6) protein and mRNA. To clarify the mechanisms that lead to the activation of IL-6 gene in HTLV-I-infected cells, we first studied the regulatory regions in the IL-6 gene transcription by transfection of chloramphenicol acetyltransferase (CAT) reporter plasmids containing the IL-6 promoter. When transfected into HTLV-I-infected T-cell lines MT-2 and HUT-102, IL-6 promoter/CAT plasmids were strongly activated without any stimulation. By deletion analysis of 52 upstream region of IL-6 promoter, the DNA region between -73 and -59 bp from the transcription start site of IL-6 gene was important in the expression of IL-6/CAT activities in HTLV-I-infected cells. This region contains nuclear factor (NF)-kappa B binding site. The site-directed mutation of the kappa B motif in IL-6/CAT plasmid resulted in the complete abrogation of IL-6 promoter activity in these cells. Furthermore, when IL-6 promoter/CAT plasmid was introduced into an HTLV-I-uninfected T- cell line, Jurkat, IL-6 promoter activity was silent in the basal level, but strongly increased by the cotransfection with an HTLV-I tax expression plasmid. However, tax expression plasmid showed no transactivation activity, when kappa B site was mutated in IL-6 promoter/CAT plasmid. We found that the IL-6 kappa B site specifically formed a complex with NF-kappa B-containing nuclear extracts from MT-2 and HUT-102 cells. Finally, transfection of HTLV-I tax into Jurkat cells resulted in induction of specific binding of nuclear extracts to the NF-kappa B sequence. These results strongly suggest that HTLV-I tax gene may transactivate IL-6 gene through kappa B site in HTLV-I- positive T-cell lines and activation of NF-kappa B may be crucial in HTLV-I-induced IL-6 gene activation in ATL.
All-trans retinoic acid directly inhibits granulocyte colony- stimulating factor-induced proliferation of CD34+ human hematopoietic progenitor cells
Authors: Smeland EB , Rusten L , Jacobsen SE , Skrede B , Blomhoff R , Wang MY , Funderud S , Kvalheim G , Blomhoff HK
Published: October 2019

In this study we examine the effects of retinoids on purified CD34+ human hematopoietic progenitor cells. All-trans retinoic acid inhibited granulocyte colony-stimulating factor (G-CSF)-induced proliferation of CD34+ cells in short-term liquid cultures in a dose-dependent fashion with maximal inhibition of 72% at a concentration of retinoic acid of 1 mumol/L. Although no significant effects were observed on granulocyte- macrophage CSF (GM-CSF)--interleukin-3--or stem cell factor (SCF)- induced proliferation, the combinations of G-CSF and each of these cytokines were all inhibited. Moreover, retinol (3 mumol/L) and chylomicron remnant retinyl esters (0.1 mumol/L) in concentrations normally found in human plasma also had inhibitory effects. Single-cell experiments showed that the effects of retinoic acid were directly mediated. Retinoids also significantly inhibited G-CSF-induced colony formation in semisolid medium, with 88% inhibition observed at a concentration of retinoic acid of 1 mumol/L. However, we did not observe any effects of retinoic acid on G-CSF-induced differentiation as assessed by morphology and flowcytometry. Similar to previous findings using total bone marrow mononuclear cells, we observed a stimulation of GM-CSF-induced colony formation after 14 days. We also observed a stimulatory effect of low doses of retinoic acid (30 nmol/L) on blast-cell colony formation on stromal cell layers. Taken together, the data indicate that vitamin A present in human plasma has inhibitory as well as stimulatory effects on myelopoiesis.
Transcriptional control of the factor IX gene: analysis of five cis- acting elements and the deleterious effects of naturally occurring hemophilia B Leyden mutations
Authors: Picketts DJ , Mueller CR , Lillicrap D
Published: October 2019

Hemophilia B Leyden is a rare form of inherited factor IX deficiency in which patients experience spontaneous postpubertal recovery of factor IX levels. The mutations resulting in this disorder are localized in a 40-nucleotide region encompassing the major transcriptional start site for factor IX. Here we report the further characterization of five cis- acting elements in the factor IX promoter and the effects on protein binding and transcriptional activation of five Leyden mutations (at nucleotides +13, -5, -6, -20, and -26) that occur within the proximal three elements (sites 1 through 3). Bandshift studies using nuclear extracts from four different rat tissues have shown that at least some of the proteins binding to each of the five sites are ubiquitous in nature. The pattern of DNA binding at site 1 suggests that this element plays an important role in mediating the liver-specific expression of factor IX. Additional studies with liver nuclear extracts obtained at several different points in development have shown an increase in DNA binding at sites 1, 4, and 5 between 1 day and 1 week. Using DNase I footprint analysis and competition bandshift studies, we have shown that the binding of nuclear proteins to each of the mutant sites is disrupted to a variable extent. There appears to be some, although reduced, protein binding to all of the mutant oligonucleotides apart from the -26 mutant. In vitro transcription assays have shown that each of the mutations reduces the global proximal promoter activity by approximately 40%. Two double mutant promoters did not show any additional downregulation in the in vitro transcription assay. In experiments designed to assess the relative transcriptional activity mediated from each of the five sites independently, we have tested artificial homopolymer promoters of each site in the in vitro transcription assay. These studies show that sites 4 and 5 are the strongest activators and that transactivation from site 5 is further enhanced by the albumin D site-binding protein. In summary, these investigations show deleterious effects of each of the Leyden mutations tested on the binding of trans-acting factors and also show disruption of transcriptional activation in a functional in vitro transcription assay. Our results also show that cis-acting elements 4 and 5 are the principal activators of this locus.
Inhibitory effect of all-trans retinoic acid on the growth of freshly isolated myeloma cells via interference with interleukin-6 signal transduction [see comments]
Authors: Ogata A , Nishimoto N , Shima Y , Yoshizaki K , Kishimoto T
Published: October 2019

We showed the dose-dependent growth inhibition by alltrans retinoic acid (ATRA) of myeloma cells freshly isolated from patients. ATRA downregulated the cell surface expression of interleukin-6 receptor (IL- 6R) and/or glycoprotein (gp) 130. The growth-inhibitory activity of ATRA was well correlated with that of anti-gp 130 antibody in every sample. Furthermore, ATRA inhibited the production of IL-6 from both myeloma cells and marrow stromal cells, and recombinant IL-6 (rIL-6) could partially recover the myeloma cell growth that had been inhibited by ATRA. These data suggest that ATRA may inhibit the proliferation of myeloma cells both by the downregulation of IL-6R and gp130 expression on myeloma cells and by the inhibition of IL-6 production from myeloma and stromal cells. Prednisolone (PSL) and interferon-gamma (IFN-gamma) also inhibited the myeloma growth, while their effects were different from those of ATRA on IL-6 R and gp130 expression, IL-6 production, and morphological change. The inhibitory effect of ATRA on myeloma cell proliferation was observed in 10 of 14 samples obtained from eight patients, which suggests that ATRA may be a potent new therapeutic agent for some myeloma patients.
WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia
Authors: Inoue K , Sugiyama H , Ogawa H , Nakagawa M , Yamagami T , Miwa H , Kita K , Hiraoka A , Masaoka T , Nasu K
Published: October 2019

The WT1 gene encoding a zinc finger polypeptide is a tumor suppressor gene that plays a key role in the carcinogenesis of Wilms' tumor. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine relative levels of WT1 gene expression (defined in K562 cells as 1.00) in 45 patients with acute myelogenous leukemia (AML), 22 with acute lymphocytic leukemia (ALL), 6 with acute mixed lineage leukemia (AMLL), 23 with chronic myelogenous leukemia (CML), and 24 with non- Hodgkin's lymphoma. Significant levels of WT1 gene were expressed in all leukemia patients and for CML the levels increased as the clinical phase progressed. In striking contrast with acute leukemia, the levels of WT1 gene expression for NHL were significantly lower or even undetectable. Clear correlation was observed between the relative levels of WT1 gene expression (< 0.6 v > or = 0.6) and the prognosis for acute leukemia (AML, ALL, and AMLL). Patients with less than 0.6 levels had significantly higher rates of complete remission (CR), disease-free survival, and overall survival than those with > or = 0.6 levels, whereas CR could not be induced in any of the 7 patients with acute leukemia having greater than 1.0 levels of WT1 gene expression. The quantitation of the WT1 gene expression made it possible to detect minimal residual disease (MRD) in acute leukemia regardless of the presence or absence of tumor-specific DNA markers. Continuous monitoring of the WT1 mRNA was performed for 9 patients with acute leukemia. In 4 patients, MRD was detected 2 to 8 months before clinical relapse became apparent. In 2 other patients, the WT1 mRNA gradually increased after discontinuation of chemotherapy. No MRD was detected in the remaining 3 patients with AML who received intensive induction and consolidation therapy. Simultaneous monitoring of MRD by RT-PCR using primers for specific DNA markers in 3 patients (2 AML-M3 with PML/RAR alpha, and 1 AML-M2 with AML1/ETO) among these 9 patients detected MRD comparable with that obtained from quantitation of WT1 gene expression. In a patient with acute promyelocytic leukemia, the limits of leukemic cell detection by RT-PCR using either WT1 or promyelocytic leukemia/retinoic acid receptor-alpha gene primers were 10(-3) to 10(- 4) and 10(-4) for bone marrow, and 10(-5) and 10(-4) for peripheral blood, respectively. Therefore, we conclude that WT1 is a new prognostic factor and a new marker for the detection of MRD in acute leukemia.
Growth arrest and terminal differentiation of leukemic myelomonocytic cells induced through ligation of surface CD23 antigen
Authors: Ouaaz F , Sola B , Issaly F , Kolb JP , Davi F , Mentz F , Arock M , Paul-Eugene N , Korner M , Dugas B
Published: October 2019

Acute myelogenous leukemia (AML) cells express CD23 surface antigen after in vitro treatment with various cytokines, including interleukin- 4 (IL-4) and interferon gamma. Subsequent ligation of CD23 by specific monoclonal antibody (MoAb) induces substantial morphologic and functional modifications in these cells. In the present study, we investigated the role of CD23 in the proliferation and the maturation of leukemic cells from AML patients or the U937 cell line. CD23+ cell treatment with CD23 MoAb inhibited the proliferation of leukemic cells. This correlated with their terminal differentiation after 7 to 9 days incubation because they (1) definitively lost their growth capacity; (2) adhered to culture flasks and became monocyte/macrophage-like; and (3) expressed mature monocyte markers including nonspecific esterases. Intracellular mechanism of this antitumoral effect was then analyzed in U937 cells. Induction of high-density surface CD23 expression by IL-4 or granulocyte-macrophage colony-stimulating factor coincided with a transient decrease of U937 cell proliferation. CD23 ligation during this low-proliferative phase induced a rapid activation of L-arginine- dependent pathway and the intracellular accumulation of cyclic guanosine monophosphate and cyclic adenosine monophosphate (cAMP). Induction of these early messengers was followed by the activation of nuclear factor-kB transcription factor and the modulation of proto- oncogene expression by U937 cells. Whereas U937 cell treatment with IL- 4 decreased c-fos/c-jun expression, CD23 MoAb reinduced c-fos/c-jun and promoted the expression of cell maturation-associated proto-oncogenes junB and c-fms, during the first 24 hours. Both IL-4 and CD23 MoAb downregulated the expression of c-myb. CD23 ligation also induced the production of TNF alpha by U937 cells. Inhibitors of cAMP and nitric oxide reversed CD23-mediated modification in U937 cells. These data evidence the ability of CD23 surface antigen to mediate terminal differentiation of early leukemic myelomonocytic cells.
Clinical significance of p53 mutations in relapsed T-cell acute lymphoblastic leukemia
Authors: Diccianni MB , Yu J , Hsiao M , Mukherjee S , Shao LE , Yu AL
Published: October 2019

In T-cell acute lymphoblastic leukemia (T-ALL), p53 gene mutations were found in 12 of 51 patients in first relapse (24%). In a retrospective study, bone marrow samples at diagnosis were obtained from 9 of the 12 relapsed patients with p53 mutation; only one patient was found to harbor a p53 mutation at diagnosis. No further p53 mutations were identified in 18 unpaired diagnosis T-ALL samples. This is the first report of a p53 mutation in T-ALL at diagnosis. p53 mutations in relapsed T-ALL were clinically relevant. Patients with p53 mutations experience a shorter duration of survival than those patients without p53 mutations. Additionally, patients with p53 mutations were significantly less likely to have achieved a complete second remission from reinduction therapy than those patients without p53 mutations and experience a shorter duration of survival from relapse even when a second reinduction is obtained. Though primarily identified only at relapse, p53 mutations were also associated with a decreased duration of first remission and overall decrease in survival from diagnosis. Patients with p53 mutations had a 3.8-fold increase in risk of death than those patients without p53 mutations. These findings suggest that p53 mutation is associated with poor clinical outcome that is characterized by (1) a shortened duration of survival after first relapse; (2) a reduced response to reinduction therapy; (3) a shortened duration of first remission; and, hence, (4) an overall decreased duration of survival and increased risk of death.
Pulse cyclophosphamide therapy for refractory autoimmune thrombocytopenic purpura [see comments]
Authors: Reiner A , Gernsheimer T , Slichter SJ
Published: October 2019

Autoimmune thrombocytopenic purpura (AITP) is generally a chronic disorder in affected adults. Twenty-five percent of these patients will become refractory to routine therapy (corticosteroids and splenectomy), as well as most other available agents. Intravenous pulse cyclophosphamide therapy was used to treat 20 patients with severe refractory AITP who had previously failed to achieve a sustained remission with a mean of 4.8 agents (range 2 to 8). Patients received 1 to 4 doses (mean 2.0) of 1.0 to 1.5 g/m2 intravenous cyclophosphamide per course. Of the 20 patients treated with pulse cyclophosphamide therapy, 13 patients (65%) achieved a complete response (CR), four (20%) a partial response (PR), and three patients (15%) failed to respond. Of the 13 complete responders, eight have remained in remission with stable platelet counts during followup intervals of 7 months to 7 years (median 2.5 years). Five patients developed recurrent AITP 4 months to 3 years following a CR. Of these, two patients responded to subsequent courses of pulse cyclophosphamide therapy with current remissions of 1 and 4 years. Of the four patients who obtained a PR, two remain in partial remission after 10 months and 4 years; one relapsed after 18 months and, after retreatment, is still in remission at 6 months. Of the patient characteristics examined, duration of disease was most strongly associated with response to pulse cyclophosphamide. Side-effects of treatment included neutropenia (three patients, one of whom developed staphylococcal sepsis), acute deep venous thrombosis (two patients), and psoas abscess (one patient). Intravenous pulse cyclophosphamide should be strongly considered in the treatment of patients with refractory AITP. There is a relatively low incidence of side-effects, and it can be administered easily on an out- patient basis.
The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets
Authors: Debili N , Wendling F , Cosman D , Titeux M , Florindo C , Dusanter-Fourt I , Schooley K , Methia N , Charon M , Nador R
Published: October 2019

The Mpl receptor (Mpl-R) is a cytokine receptor belonging to the hematopoietin receptor superfamily for which a ligand has been recently characterized. To study the lineage distribution of Mpl-R in normal hematopoietic cells, we developed a monoclonal antibody (designated M1 MoAb) by immunizing mice with a soluble form of the human Mpl-R protein. With few exceptions, Mpl-R was detected by indirect immunofluorescent analysis on all human leukemic hematopoietic cell lines with pluripotential and megakaryocytic phenotypes, but not on other cell lines. By immunoprecipitation and immunoblotting, M1 MoAb recognized a band at 82 to 84 kD corresponding to the expected size of the glycosylated receptor. Among normal hematopoietic cells, M1 MoAb strongly stained megakaryocytes (MK) and Mpl-R was detected on platelets by indirect immunofluorescence staining or immunoblotting. On purified CD34+ cells, less than 2% of the population was stained, but the labeling was weak and just above the threshold of detection. However, dual-labeling with the M1 and antiplatelet glycoprotein MoAbs showed that most Mpl-R+/CD34+ cells coexpressed CD41a, CD61, or CD42a, suggesting that cell surface appearance of Mpl-R and platelet glycoproteins could be coordinated. M1-positive and M1-negative subsets were sorted from purified CD34+ cell populations. Colony assays showed that the absolute number of hematopoietic progenitors was extremely low and no primitive progenitors were present in the CD34+/Mpl-R+ fraction. However, this cell fraction was significantly enriched in low proliferative colony-forming units-MK. When the CD34+/Mpl-R+ fraction was grown in liquid culture containing human aplastic serum and a combination of growth factors, mature MK were seen as early as day 4, whereas the predominant cell population was erythroblasts on day 8. Similar data were also obtained with the CD34+/Mpl-R- fraction with, however, a delay in the time of appearance of both MK and erythroblasts. In conclusion, Mpl-R is a cytokine receptor restricted to the MK cell lineage. Its expression is low on CD34+ cells and these cells mainly correspond to late MK progenitors and transitional cells. These data indicate that the action of the Mpl-R ligand might predominate during the late stages of human MK differentiation.
Glanzmann's thrombasthenia associated with deletion-insertion and alternative splicing in the glycoprotein IIb gene
Authors: Peretz H , Rosenberg N , Usher S , Graff E , Newman PJ , Coller BS , Seligsohn U
Published: October 2019

Glanzmann's thrombasthenia is a bleeding disorder characterized by a decrease or absence of the functional platelet membrane glycoprotein (GP) complex, GPIIb/IIIa (alpha IIb beta 3). We describe a new deletion- insertion mutation in the GPIIb gene causing type I Glanzmann's thrombasthenia in two siblings of a consanguineous Iranian-Jewish family. The proband's platelets bound more antibodies against the vitronectin receptor-alpha V beta 3 than normal platelets, suggesting a normal GPIIIa (beta 3) gene and a defect in the GPIIb gene. Sequencing of amplified cDNA and genomic DNA fragments showed a 6-bp deletion and 31-bp insertion in exon 25 of the GPIIb gene. The predominant platelet GPIIb mRNA of the proband was a product of the splicing of exon 24 to a cryptic AG acceptor site in the insertion and encoded for deletion of amino acids Leu817-Asn826 and insertion of eight different amino acids. Cotransfection of COS-7 cells with expression vectors containing wild- type GPIIIa cDNA and the mutated GPIIb cDNA failed to produce detectable amounts of GPIIb/IIIa on the surface of the cells. Allele- specific restriction analysis of genomic DNA of family members showed homozygosity for the mutation in the affected siblings, heterozygosity in the parents, and homozygosity for the normal allele in an unaffected sibling. The observed mutation is in a region that is conserved from rodents to humans and has been suggested to be involved in the interaction between GPIIb and GPIIIa when these GPs are complexed in solution.
Inhibition of cellular differentiation by the SCL/tal oncoprotein: transcriptional repression by an Id-like mechanism
Authors: Goldfarb AN , Lewandowska K
Published: October 2019

In cases of T-cell acute lymphoblastic leukemia (T-ALL), the basic helix-loop-helix (bHLH) oncogene SCL/tal undergoes frequent rearrangements activating ectopic expression. Despite the compelling epidemiological association of SCL/tal expression with T-ALL, no specific transforming function has been attributable to the protein product. However, investigators have recently demonstrated that forced overexpression of SCL/tal can block monocytic differentiation of M1 murine myeloid leukemia cells. Thus, inappropriate expression of wild- type SCL/tal protein may in part account for the maturation arrest phenotype observed in T-ALL cells. In this study, ectopic expression of the SCL/tal gene blocked the differentiation of C2C12 muscle precursor cells. Characterization of the mechanism of differentiation blockade showed that the SCL/tal protein repressed transcriptional activation by the myogenic bHLH factor MyoD. Protein interaction analysis showed that SCL/tal and MyoD compete for common partners (E bHLH proteins) but do not directly bind one other. A model is thus proposed in which ectopic SCL/tal protein, by its ability to titrate out E proteins, prevents the formation of bHLH complexes that drive cellular differentiation: the “Id-like” mechanism.
Striking inverse correlation between IgG anti-F(ab')2 and autoantibody production in patients with cold agglutination
Authors: Terness P , Kirschfink M , Navolan D , Dufter C , Kohl I , Opelz G , Roelcke D
Published: October 2019

Previous experiments showed that the physiologic IgG anti-F(ab')2 antibody suppresses the response of human autoreactive B cells. In the present study, we analyzed the IgG anti-F(ab')2 antibody in 293 patients with cold agglutination (CA). Their average IgG anti-F(ab')2 titer was not much different (211 +/- 8.3) from that of 279 healthy persons (195 +/- 6.7). However, CA patients with high anti-F(ab')2 titers had low CA autoantibody titers and vice versa (P = .0028; rho = - 0.175). The stratification of patients according to the auto-antibody's specificity (anti-I, anti-i, anti-Pr) showed an inverse correlation between anti-F(ab')2 and CA in the anti-I group (P = .0057; rho = - 0.180). Interestingly, the association was present only in patients whose disease was caused by noninfectious agents (P < .0001; rho = - 0.423). The inverse correlation argues for an important role of the IgG anti-F(ab')2 in the regulation of autoantibody production in CA patients.
Contribution of interleukin-1 to activation of coagulation and fibrinolysis, neutrophil degranulation, and the release of secretory- type phospholipase A2 in sepsis: studies in nonhuman primates after interleukin-1 alpha administration and during lethal bacteremia
Authors: Jansen PM , Boermeester MA , Fischer E , de Jong IW , van der Poll T , Moldawer LL , Hack CE , Lowry SF
Published: October 2019

Although studies with interleukin-1 receptor antagonist (IL-1ra) in animal models have shown that IL-1 contributes to mortality in sepsis, the mechanisms whereby IL-1 mediates lethal effects are not well established. A possible mechanism is that IL-1 enhances the activation and release of other inflammatory mediator systems such as coagulation, fibrinolysis, neutrophils, and secretory-type phospholipase A2 (sPLA2). We investigated this possibility by assessing the effect of intravenously injected recombinant human IL-1 alpha (rhIL-1 alpha) on these plasma parameters in baboons. In addition, we examined the course of these inflammatory parameters in baboons after a challenge with a lethal dose of Escherichia coli and while receiving a 24-hour constant infusion of IL-1ra or placebo. Intravenous administration of IL-1 alpha (10 micrograms/kg) induced the formation of thrombin, as evidenced by the appearance of thrombin-antithrombin III (TAT) complexes into the circulation (peak levels, 188 +/- 92 ng/mL at 2 hours), as well as the activation of fibrinolysis, assessed by circulating plasmin-alpha 2- antiplasmin complexes (PAP complexes; peak levels, 0.4% +/- 0.03% of fully activated plasma at 1 hour), the release of tissue-type plasminogen activator (t-PA; peak levels, 6 +/- 2 ng/mL at 2 hours), and its inhibitor, plasminogen activator inhibitor (PAI; peak levels, 724 +/- 246 ng/mL at 4 hours). Il-1 alpha administration also induced the release of sPLA2 (maximal levels, 336 +/- 185 ng/mL at 8 hours), but not degranulation of neutrophils. In the septic baboons, a significant reduction of the formation of thrombin (peak TAT levels decreased from 582 +/- 78 ng/mL to 219 +/- 106 ng/mL; P < .005), the release of t-PA (peak levels decreased from 37 +/- 11 ng/mL to 17 +/- 2 ng/mL; P < .001), and its inhibitor, PAI (peak levels decreased from 2,639 +/- 974 ng/mL to 1,110 +/- 153 ng/mL; P <.001), was observed in the group receiving IL-1ra compared to that receiving placebo. The release of neutrophilic elastase was also significantly attenuated in IL-1a-treated animals (peak levels, 1,024 +/- 393 and 655 +/- 104 ng/mL in control and treatment groups, respectively; P < .05). The difference between sPLA2 levels in both groups, although higher in the controls (maximal levels, 3,140 +/- 1,435 ng/mL in control v 2,217 +/- 1,375 ng/mL in IL-1ra-treated group), was not significant. Thus, IL-1 contributes to activation of various other mediator systems in severe sepsis in nonhuman primates. We propose that these effects may explain the lethal actions of IL-1 in this sepsis model and suggest a similar role for IL-1 in severe human sepsis.
Role of the glycoprotein Ib-binding A1 repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor
Authors: Lankhof H , Wu YP , Vink T , Schiphorst ME , Zerwes HG , de Groot PG , Sixma JJ
Published: October 2019

To assess the relative importance of the glycoprotein (GP) Ib binding domain and the RGDS binding site in platelet adhesion to isolated von Willebrand factor (vWF) and to collagen preincubated with vWF, we deleted the A1 domain yielding delta A1-vWF and introduced an aspartate- to-glycine substitution in the RGDS sequence by site-directed mutagenesis (RGGS-vWF). Recombinant delta A1-vWF and RGGS-vWF, purified from transfected baby hamster kidney cells, were compared with recombinant wild-type vWF (WT-vWF) in platelet adhesion under static and flow conditions. Purified mutants were coated on glass or on a collagen type III surface and exposed to circulating blood in a perfusion system. Platelet adhesion under static condition, under flow conditions, and in vWF-dependent adhesion to collagen has an absolute requirement for GPIb-vWF interaction. The GPIIb/IIIa-vWF interaction is required for adhesion to coated vWF under flow conditions. Under static condition and vWF-dependent adhesion to collagen, platelet adhesion to RGGS-vWF is similar as to WT-vWF, but platelet spreading and aggregation are abolished.
In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody
Authors: Michon J , Moutel S , Barbet J , Romet-Lemonne JL , Deo YM , Fridman WH , Teillaud JL
Published: October 2019

Neutrophils isolated from cancer patients treated with granulocyte colony-stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G-CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo.
Molecular analysis of clonality in Castleman's disease
Authors: Soulier J , Grollet L , Oksenhendler E , Miclea JM , Cacoub P , Baruchel A , Brice P , Clauvel JP , d'Agay MF , Raphael M , Sigaux J
Published: October 2019

Castleman's disease (CD) is a rare atypical lymphoproliferative disorder that is morphologically and clinically heterogenous and is associated with a risk of developing malignant lymphoma. We report the clonality status of CD tissues in 34 patients, including 14 patients infected by the human immunodeficiency virus (HIV). Four patients presented a localized form and 30 presented a multicentric form. Two cases were associated with B-cell lymphoma, 3 cases with Hodgkin's disease, and 9 cases (8 HIV+) with Kaposi's sarcoma. Histologically, 8 cases were of the hyaline-vascular type and 26 were of the plasma cell or mixed types. The Ig and T-cell receptor (TCR) V(D)J rearrangements were analyzed using polymerase chain reaction and Southern blot. Clonal IgH rearrangements were detected in only 4 cases, ie, 2 associated with B-cell lymphoma, 1 with Hodgkin's disease, and 1 case without malignancy. A TCR gamma rearrangement of restricted junctional size was amplified in 1 HIV+ case. Finally, polyclonal VH-JH and V gamma-J gamma rearrangements were detected in the large majority of the cases, irrespective of pathologic subtypes, clinical forms, and HIV status. The lymphoid component in CD is therefore commonly reactive, and the rare occurrence of detectable monoclonal lymphoid contingents may be caused by secondary molecular events.
Proteinaceous (angiocentric sclerosing) lymphadenopathy: a polyclonal systemic, nonamyloid deposition disorder
Authors: Michaeli J , Niesvizky R , Siegel D , Ladanyi M , Lieberman PH , Filippa DA
Published: October 2019

Proteinaceous lymphadenopathy with hypergammaglobulinemia (PLWH) is an exceedingly rare disease of unknown etiology. Described primarily as a pathologic entity, relatively little is known about its clinical manifestations or its response to therapy. The disease is often referred to and treated as an unusual form of plasma cell dyscrasia or light chain deposition disease. We have recently encountered a young patient with PLWH who presented with generalized lymphadenopathy, marked liver function abnormalities, hypocomplementemia, cryoglobulinemia, decreased T4/T8 ratio, and ophthalmopathy. Contrary to the notion that PLWH is a clonal disorder, we found no evidence of clonality in this patient. The most characteristic finding in this and in another patient, previously seen at our institution, was marked angiocentric hyaline sclerosis of the small and mid-sized blood vessels of involved lymph nodes and organs. Based on these findings, we propose the term angiocentric sclerosing lymphadenopathy, which more accurately defines this clinicopathologic entity that appears to be distinct from light chain deposition disease and other plasma cell dyscrasias.
Targeted inactivation of the major positive regulatory element (HS-40) of the human alpha-globin gene locus
Authors: Bernet A , Sabatier S , Picketts DJ , Ouazana R , Morle F , Higgs DR , Godet J
Published: October 2019

We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible enhancer of human alpha- globin gene expression.
Human embryonic zeta-globin gene expression in mouse-human hybrid erythroid cell lines
Authors: Luo HY , Deisseroth AB , Chui DH
Published: October 2019

The human alpha-globin-like embryonic zeta-globin chains are present in abundance during the first 5 to 6 weeks of gestation. Subsequently, zeta-globin chains are present in fetal blood at a very low level, which is supplanted by the expression of alpha-globin chains. Adult individuals who are carriers of the (--SEA/) alpha-thalassemia deletion, in contrast to normal adults, have low levels of embryonic zeta-globin chains in their circulating erythrocytes. In this investigation, we constructed stable mouse-human hybrid cells with murine erythroleukemia cells bearing human chromosome 16, with either the normal alpha-globin gene cluster (alpha alpha/) or the (--SEA/) type of alpha-thalassemia deletion. The results on the human zeta- globin gene expression in these hybrid cells indicate that murine adult erythroid transcription factors can induce the expression of human embryonic zeta-globin gene is cis to the (--SEA/) deletion, in parallel with the endogenous mouse alpha-globin gene expression. These data also show the importance of the DNA sequences within the (--SEA) deletion in regulating the expression of zeta-globin gene in cis during normal human hemoglobin ontogeny.
New drug therapy of amyloidoses: resorption of AL-type deposits with 4'- iodo-4'-deoxydoxorubicin
Authors: Gianni L , Bellotti V , Gianni AM , Merlini G
Published: October 2019

Amyloidosis caused by monoclonal Ig light chains (AL) is characterized by the tissue deposition of paraproteins as insoluble fibrils that leads to organ dysfunction and death. After serendipitous observation of its efficacy, the new anthracycline 4′-iodo-4′-deoxydoxorubicin (I- DOX) was evaluated in eight patients with biopsy-proven AL and symptomatic organ involvement who received 1 to 6 administrations of I- DOX at dosages of 15 to 100 mg/m2. Five patients showed substantial clinical improvement concomitant with instrumental and physical evidence of response, and three patients presented objective evidence of amyloid resorption. The effects of I-DOX on amyloid deposits were not associated with cytotoxicity to the amyloidogenic clone. Five patients died of disease-related complications at 4 to 36 months; the remaining three are alive 29, 35, and 44 months after starting treatment. I-DOX caused short-lived granulocytopenia and minimal extra- hematologic side effects. The pharmacokinetics of I-DOX presented features exploitable for diagnosis in amyloidotic patients and documented the active metabolite in the cerebrospinal fluid. We conclude that I-DOX represents an important treatment option for subjects with AL amyloidosis and could be the prototype of a new class of drugs that interfere with and reverse the process of all types of amyloid deposition.
Modulation of acute graft-versus-host-disease after allogeneic bone marrow transplantation by tumor necrosis factor alpha (TNF alpha) release in the course of pretransplant conditioning: role of conditioning regimens and prophylactic application of a monoclonal antibody neutralizing human TNF alpha (MAK 195F)
Authors: Holler E , Kolb HJ , Mittermuller J , Kaul M , Ledderose G , Duell T , Seeber B , Schleuning M , Hintermeier-Knabe R , Ertl B
Published: October 2019

Contribution of host-related cytokine release in the course of pretransplant conditioning to early tissue damage and induction of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) has been shown in experimental models. We performed a clinical phase I/II trial applying a monoclonal antibody neutralizing human tumor necrosis alpha (TNF alpha) during pretransplant conditioning as additional prophylaxis in high-risk patients admitted to allogeneic BMT; TNF alpha serum levels and clinical courses in 21 patients receiving anti-TNF alpha prophylaxis were compared with data from 22 historical controls. Absence of significant release of TNF alpha in the period of busulphan (BUS) treatment, but significant induction of TNF alpha by total body irradiation (TBI) and cyclophosphamide (CY) conditioning were correlated with significantly earlier onset of acute GVHD in patients receiving TBI/CY regimens as compared with BUS/CY-treated patients. Prophylactic application of monoclonal anti-TNF alpha seemed to postpone onset of acute GVHD from day 15 to day 25 (P < .05) after TBI/CY and from day 33 to day 53 after BUS/CY (P < .10) conditioning. Application of monoclonal anti-TNF alpha in low and intermediate doses was safe and not associated with an increased incidence of infectious or hematologic complications. Thus, our data provide indirect and direct evidence for involvement of conditioning-related cytokine release in induction of early acute GVHD in the clinical setting and support further investigation of this novel approach in randomized trials.