bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinctive Roles of Stem Cell Factor and IL-7 in T Cell Development.
Authors: Wang Hongfang , Pierce L. Jeanne , Spangrude Gerald J.
Published: April 2020

Notch signaling plays a critical role in T lineage commitment during lymphoid differentiation. However, Notch signaling alone is not sufficient to support T cell development through the CD4/CD8 double positive (DP) stage in vitro. We here report distinct effects of several cytokines on T cell differentiation in the OP9-DL1 cell culture model. Our studies show that Flt3 ligand enhances the proliferation of progenitors but has no obvious effect on differentiation. In contrast, stem cell factor (SCF) favors the proliferation of CD4/CD8 double negative (DN) lymphoid progenitors and inhibits differentiation to the DP stage in a dose-dependent manner. Differentiation of the NK lineage is promoted under these conditions. Conversely, blocking the function of SCF that is expressed endogenously by OP9-DL1 cells inhibits proliferation of lymphoid progenitors and accelerates T lineage differentiation. IL-7 is necessary for differentiation from the DP to the CD8 single positive (SP) stage, and is also required for γδ T lineage development. We also find a dosage effect of IL-7 during T cell development. OP9 and OP9-DL1 stromal cells produce endogenous levels of IL-7 that are sufficient to support B and DP T cell differentiation. However, the amount of endogenous IL-7 is not sufficient to support T cell differentiation from the DP to the SP stage. Addition of exogenous IL-7 (1–10 ng/ml) to the cultures promotes SP differentiation, while blocking endogenous IL-7 with anti-IL-7 antibody inhibits both B and T cell development. We conclude that activation through the Notch pathway is sufficient to suppress B lineage differentiation and thereby promote T lineage commitment, but is not sufficient to promote the subsequent stages of T cell development. SCF promotes expansion and directs NK lineage differentiation at the expense of T cell development, while IL-7 provides both proliferation as well as T lineage differentiation signals. T cell development from the DN to the DP stage requires a low amount of IL-7, while differentiation from the DP to the SP stage requires a higher level of IL-7. The balance between the effects mediated by these cytokines, along with Notch signaling, plays a critical role in regulating development of the T and NK lineages.
Induction of Transient Cytopenia To Analyze Hematopoiesis in Mutant Mice.
Authors: Stanford William L. , Anderson Nicole M. , Minden Mark D. , Barber Dwayne L.
Published: April 2020

The mouse plays an indispensable role in developing our current understanding of mammalian hematopoiesis. Most hematopoietic phenotyping assays in the mouse are non-viable techniques designed to evaluate homeostatic populations, enumerate progenitor populations, and perform functional analysis. The worldwide effort to generate mouse models of human disease and functionally annotate the mammalian genome using mouse mutagenesis (including dominant ENU screens) requires the development of robust standardized viable phenotyping tools. We have developed a phenotyping assay that induces transient cytopenias using various pharmacological agents (5-fluorouracil, phenylhydrazine, and hydroxyurea), the responses to which are monitored by tracing changes in peripheral blood levels of red blood cells, white blood cells and platelets. We have performed detailed analysis of lineage recovery kinetics, developing lineage recovery curves for various strains of mice for both males and females, which allowed us to identify appropriate testing days to identify phenodeviants. We have compared the recovery data with conventional progenitor assays and analyzed a cohort of well-studied naturally occurring and targeted hematopoietic mutants using the transient anemia assays that has yielded novel phenotypes of hemizygous mutant animals. For example, erythropoietin receptor null embryos die of severe anemia at mid-gestation; however, no defect in erythropoiesis has been reported in EpoR +/− mice. We have found that 5-fluorouracil and phenylhydrazine elicit delayed RBC recovery in EpoR +/− mice, demonstrating a critical dose-dependent role for the erythropoietin receptor in stress erythropoiesis. In addition, Stat5 has been shown to play an important role in erythropoiesis and in the regulation of steady state hematopoiesis. We have found that Stat5a/b+/− mice treated with 5-fluorouracil show altered recovery kinetics in RBC, WBC and platelets. Finally, we have adapted the transient cytopenia assay to develop a sensitized dominant ENU screen, enabling us to identify hematopoietic mutants that do not present abnormal blood cell counts in a homeostatic state. Thus, these standardized cytopenia response assays function as surrogate viable assays to analyze progenitor populations.
Proteolytic Activation of the Chemokine CCL15 by Cathepsin G from Neutrophil Granulocytes Modulates Proliferation of Primitive Hematopoietic Precursor Cells.
Authors: Richter Rudolf , Bistrian Roxana , Henschler Reinhard
Published: April 2020

The human chemokine HCC-2 (MIP-1δ, small inducible cytokine subfamily A [Cys-Cys], member 15, hmrp-2b, MIP-5, NCC-3) is a potent chemoattractant for monocytes, lymphocytes and endothelial cells, and has been shown to inhibit proliferation of primitive hematopoietic precursor cells (HPC). CCL15 is constitutively expressed in gut and liver, and was identified to circulate in human plasma. The length of the N-terminus of CCL15 was found to control biological activity. We therefore aimed to investigate the processing of CCL15 in the hematopoietic system, and the effect of processed isoforms on HPC proliferation. Incubation of full-length CCL15 with cell-free bone marrow supernatant prepared from normal untreated mice revealed a decrease in molecular weight by approximately 3 kD. In contrast, the molecular weight of CCL15 remained unchanged when cell-free bone marrow supernatant of mice with regenerating hematopoiesis after 5 Gy gamma irradiation was analyzed. Incubation of full-length CCL15 with isolated neutrophils, but not monocytes, lymphocytes or thrombocytes revealed N-terminal deletions of 21 (Δ21), 23 (Δ23) and 26 (Δ26) amino acids, as assessed by Edman sequencing/mass spectrometry. Both (1) inhibition of the neutrophil-derived CCL15 proteolytic activity by protease inhibitors Suc-Ala-Ala-Pro-Phe-pNA, secretory leucoprotease inhibitor or elastatinal, and (2) addition of the recombinant serine proteases cathepsin G or elastase to full-length CCL15 revealed that the Δ23 and Δ26 isoforms are produced by cathepsin G, whereas formation of the Δ21isoform is dependent on elastase. In addition, the Δ23 and Δ26 isoforms of CCL15 was also directly isolated and sequenced from blood filtrate (hemofiltrate) collected from renal insufficiency patients. The Δ26 isoform was synthesized and analyzed in HPC proliferation assays. Compared to full length CCL15, the Δ26 isoform displayed a mean 2.5 fold increased potency to inhibit CFU-A colony formation from post 5-fluorouracil murine bone marrow. In addition, the Δ26 isoform efficiently abrogated cytosine arabinoside-induced suicide of murine spleen colony forming unit (CFU-S) in post 5-fluorouracil murine bone marrow, and inhibited growth of human cord blood CD34+ single cell clones induced by hematopoietic growth factors SCF, IL-3, GM-CSF and EPO after flow-cytometric cell sorting (mean 2.3 fold inhibition). Our findings indicate a role for neutrophil-derived cathepsin G in growth control of colony of primitive hematopoietic progenitor cells through the proteolytic processing of CCL15.
Analysis of the X-Linked F-Cell Production Locus.
Authors: Keefer Jeffrey R. , Purvis Shirley H. , Dover George J. , Smith Kirby D.
Published: April 2020

This laboratory has previously identified a locus on the X chromosome at Xp22.2–22.3 (F-cell production locus or FCP) that is responsible for approximately 40% of the genetic variability in F-cell number in patients with sickle cell disease (SCD). We have re-examined the association of this region with F-cell production by multipoint linkage analysis. We have confirmed linkage to Xp22.2–22.3 and refined the candidate locus to a region of approximately 3 cM, between markers DXS452 and DXS410, with a maximum LOD score of 3.315. Linkage to a more extended region of 11 cM with an average LOD score of 1.5 could not be excluded. In an effort to identify candidate genes within this region that influence F-cell production, we screened known genes within this region for transcription factor binding sites that had relevance to HbF production. We first searched for CREB binding sites given the involvement of cAMP in of HbF induction in CD34 cells recently identified by this laboratory. Of the 15 ESTs containing CREB sequences, several also had binding sites for NFE2 or NFE2-like transcription factors. We examined 4 genes, 3 within the refined candidate locus (KIAA1280, TBL1X, and KAL1) and one in the more extended locus region (EGFL6) for expression levels within pedigrees of known F-cell phenotypes. Results indicate that, within families, a reduced level of KIAA1280 message is associated with high F-cell phenotypes in lymphoblastoid cells from patients with sickle cell anemia. The other genes examined had no such correlation.

KIAA1280, also designated BMO42 is a gene of unknown function that is expressed in bone marrow. The predicted protein contains a region homologous to a conserved protein kinase C domain. While not yet definitive, these studies provide the first suggestive evidence for a candidate gene within the FCP locus. Further studies will attempt to define the function of this gene and explore it’s involvement in F-cell regulation.
Various Drugs with or without an Antisickling Effect in the In Vitro Tests Showed a Strong Antisickling Effect in the In Vivo Studies in Transgenic Sickle Mice.
Authors: Asakura Toshio , Abdulmalik Osheiza , Iyamu Efe , Chen Qiukan , Yang Jisheng , Abraham Donald J. , Safo Martin K. , Brugnara Carlo , Evans Greg
Published: April 2020

Since its inception in 1997, the NHLBI Sickle Cell Disease (SCD) Reference Laboratory (Ref Lab) has received over 150 candidate drugs from over 30 researchers, universities and companies for evaluation. All candidate drugs are evaluated for both their beneficial and adverse effects in vitro using more than 10 sophisticated methods. Specifically, each candidate drug is studied to see whether it inhibits cell sickling, prolongs the delay time prior to deoxy-hemoglobin (Hb) S polymerization, increases the solubility of deoxy-Hb S, hydrates red blood cells (RBCs), prevents dehydration of RBCs, shifts the oxygen equilibrium curve (OEC) toward the left, reduces the adhesion of RBCs to endothelial cells, promotes the synthesis of Hb F, causes hemolysis, promotes formation of met-Hb and increases the denaturation of intracellular Hb S. With this new approach, we have found more than 10 new antisickling agents that showed beneficial effects without significant adverse effects in the in vitro tests. Of these, five drugs were further studied in vivo using transgenic mice that produce human Hb S, and all five drugs inhibited the formation of sickled cells in the blood and significantly prolonged their survival time under acute hypoxic conditions. The five drugs are NIPRISAN (plant extracts) [Brit. J. Haematol. (BJH) 122:1001,2003], MX-1520 (prodrug of vanillin) (BJH125: 788,2004), 5-hydroxymethyl-2-furfural (5HMF) (BJH128:552,2005), NS3623 (Blood 97: 1461,2001, in vivo results not shown), and FLOCOR (surfactant) (Blood94:420a,1999). Of these 5 drugs, FLOCOR and NS3623 did not have an antisickling effect in vitro. It is quite interesting that FLOCOR and NS3623 which showed no direct antisickling effect in vitro, showed a strong antisickling effect in vivo. Some of our results suggest that combined use of some of these drugs may have a synergistic effect in vivo. Studies on the combined use of these drugs are planned. Researchers with candidate drugs that may have beneficial effects for SCD are welcome to contact the SCD Ref Lab for free in vitro evaluation of their agents (for more information, please see our website at www.tatcom.com/sickle-cell).
A Transgenic Mouse Model of Homozygous HbE Disease.
Authors: Chen Qiuying , Kuypers Frans A. , Rybicki Anne C. , Bhagat Amrita , Suzuka Sandra M. , Fabry Mary E. , Nagel Ronald L. , Hirsch Rhoda Elison
Published: April 2020

Hemoglobin E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE have a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. However, when HbE coexists with β0-thalassemia, it is the most common severe hemoglobinopathy world-wide. National research priorities in the United States now include HbE related diseases because of the growing number of immigrants with these diseases, as well as the significant world impact of this life-threatening condition. In efforts to understand these diseases, a transgenic mouse model of homozygous HbE Disease was generated (Chen et al. Blood Cells Mol. Dis., 2004). Subsequent breeding has yielded knockout transgenic mice expressing exclusively human HbE: RBC smears show hypochromia, target cells, and stress reticulocytes. Measurement of PS exposure with Annexin V does not show a significantly increased population of PS exposing cells in the blood of these mice. Density gradients showing broader RBC density distributions correlate with increased RDW. RBC parameters determined by the ADVIA show low MCV (31.6±1.5fl vs. 46.9±2.1fl for C57) and MCH (9.8±0.1pg vs. 14.8±0.7pg for C57); and normal MCHC (32.0±2.0g/dl vs. 31.2±1.7 g/dl for C57) [n=5]. These RBC parameters mirror human HbE homozygotes. Increased levels of zinc protoporphyrin (ZPP), free protoporphyrin (PPIX), and fluorescent heme degradation products (FHDP) are found. To see if these porphyrins and FHDP arise from alterations in heme synthesis, real-time PCR technology methods were used. A comparison of the gene expression of enzymes involved in heme synthesis was made from RNA extracted from the spleens (the mouse hematopoietic organ). Upregulation in the rate-limiting enzyme erythroid delta-aminolevulinate synthase (ALAS) and other heme synthesis enzymes is suggested. These observations may be explained by increased erythropoiesis, compatible with the increase in % reticulocytes (7.2%±2.5, n=5); and/or elevated RBC porphyrins observed in these mice. This result is consistent with the effect of elevated porphyrins on ALAS in peripheral blood of erythropoietic protoporphyria-chemically induced mice (Inafuku et al., J Dermatol Sci, 1999). Considering the above, we conclude that the HbE full knockout mice are an excellent model for the benign human homozygous EE disease, and will serve as an important control for the proposed transgenic studies to generate HbE/β-thalassemia mice.
Factor VIII Inhibitors in Haemophilia A Patients after Continuous Infusion of Factor VIII Concentrates.
Authors: von Auer Charis , Oldenburg Johannes , Krause Manuela , Miesbach Wolfgang , Scharrer Inge ,   CI-Study Group in Germany
Published: April 2020

Continuous infusion (CI) of coagulation factor concentrates has been used in haemophilia treatment since the early 1990s. Recent reports of the occurrence of an inhibitor (inh.) after CI have raised concerns about this method of treatment. We conduct a retrospective study to investigate the development of inh. After CI of factor VIII (FVIII) concentrates in Germany and report on further study results. A questionnaire was sent to 100 haemophilia treating colleagues in Germany. So far 42 colleagues have answered, in 19 haemophilia treatment centers CI was used as treatment modality, 5 of them still use it today. Altogether 200 CI were conducted in 128 patients (pat.). In 14 pat. Inh. Development was detected. 5 of these pat. Were suffering from severe, 1 from moderate and 8 from mild haemophilia, their median age was 27 years. Indications for treatment before inh. Detection were major bleeds and surgical procedures. Plasmaderived (9) and recombinant (4) factor concentrates were given in various infusion sets. Data concerning gene mutation, exposure days, inhibitor characteristic, former change of product, concomitant medication, vaccination and blood transfusion were collected. Regarding the genotype, we found 4 missense mutations, 2 intron-22-inversions, 1 small deletion, 1 splice site mutation, 6 were unknown. In our study strikingly the inh. Developed very often in pat. With mild or moderate haemophilia and genotypes that exhibit an inh. Prevalence of < 10 %. Our further data confirm the assumption that pat. With mild haemophilia might exhibit a much higher prevalence of inh. Development when treated with an “intensive FVIII - treatment” such as CI. On the other hand there might be an uncommon inh. pathomechanism in connection with CI, resulting in a peculiar group of pat. developing the inh. after this way of application. A prospective multicenter study to investigate the inhibitor development after CI should be conducted.
Inhibitor Epitopes Identified from Inhibitor Positive Plasma of a Hemophilia B Patient Change during ITT.
Authors: Königs Christoph , Kessel Christoph , Stumpf Susanne , Roland Janti , Klingebiel Thomas , Kreuz Wolfhart
Published: April 2020

Hemophilia B is an X-linked bleeding disorder. Substitution of recombinant (rFIX) or plasma derived (pdFIX) FIX preparations are required to restore coagulation. A rare but severe complication is the development of FIX inhibitors that are often accompanied by allergic reactions. Immune tolerance therapies (ITTs) for inhibitor elimination are difficult and can induce further allergic reactions. ITTs for FIX inhibitors are less established and less successful than for FVIII inhibitors.

In this study, three phage displayed random peptide libraries were screened by biopanning to isolate small peptides that mimic the epitope of FIX inhibitors. In the patient, the inhibitor was eliminated by ITT but reappeared again. Plasma samples before ITT and at reappearance of inhibitor were used to isolate inhibitor specific phage clones. Phage clones showing specific reactivity with inhibitor positive plasma compared to control plasma by ELISA were amplified and DNA was sequenced to determine the peptide sequence displayed on the phage. Before ITT, 31 isolated phage clones specific for FIX inhibitors have been sequenced so far. 10/31 contain a common motif. After ITT, 24 clones specific for patient plasma have been sequenced. 10/24 contain a different common motif. Synthetic peptides have been generated according to the phage displayed sequence to demonstrate binding of inhibitors and test for cross reactivity.

The surface of the model of FIX was screened for these motifs with a novel software tool. Different conformational epitopes could be identified on the surface of the molecule before and after ITT.

Small peptides (mimotopes) have been identified that mimic epitopes for FIX inhibitors in a hemophilia B patient. The sequences can be aligned to different conformational epitopes on FIX. It appears that the epitope has changed in the course of the ITT. The mimotopes can also be used to target inhibitor specific B cells for a novel approach in inhibitor elimination in hemophilia B.
Functional Characterization of Aspergillus Fumigatus Specific T-Cells Clones.
Authors: Lehrnbecher Thomas , Koehl Ulrike , Roilides Emmanuel , Simitsopoulou Maria , Hanisch Mitra , Tramsen Lars , Latge Jean-Paul , Topp Max S. , Einsele Hermann , Klingebiel Thomas , Beck Olaf
Published: April 2020

Invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients with hematological malignancies, in particular in patients who have undergone allogeneic hematopoietic stem cell transplantation (SCT). There is a growing body of evidence that T-cells play an important role in the immunological response to Aspergillus fumigatus. Using the Aspergillus fumigatus antigen extract EC SAB and the IFN-γ secretion assay (Miltenyi Biotec, Germany), we generated Aspergillus fumigatus specific T-cell clones by limiting dilution (n=4). Flow cytometry revealed a cell population of CD3+/CD4+ cells (mean±SEM, 98.2±1.2%). Functional assessment by ICC revealed that an average of 8.7% of these cells (range, 6.6%–18.5%) specifically secreted IFN-γ on stimulation with EC SAB, which supports the TH1 response of the generated cells to Aspergillus fumigatus antigens. The antigenic components of EC SAB are one or more proteins, since the addition of proteinase completely suppressed the stimulating effect of this preparation. The percentage of IFN-γ producing CD3+/CD4+ cells was less than 1% upon activation with antigen extracts from Aspergillus flavus, Aspergillus niger, Alternaria alternata, Mucor racemosus, Penicillium notatum and Candida albicans, indicating that the generated T-cell clones are specific for Aspergillus fumigatus. A strong proliferation of the generated Aspergillus fumigatus specific T-cells was seen after re-stimulation with EC SAB, whereas alloreactivity was reduced compared to CD4+ T-cells of the original fraction. Hyphal damage of Aspergillus fumigatus was assessed by means of an XTT assay. Polymorphonuclear leukocytes (PMNs) showed a similar hyphal damage when tested alone (mean±SEM, 14.2±2.1%), in combination with antigen presenting cells (APCs) (15.1±1.4%), or in combination with Aspergillus fumigatus specific T-cells (15.0±2.0%). A comparable hyphal damage was seen when Aspergillus fumigatus specific T-cells were co-incubated with APCs (14.2±1.7%). In contrast, the combination of APCs and Aspergillus fumigatus specific T-cells with PMNs resulted in a significantly higher hyphal damage compared to all other settings (23.3±2.8%; P<.0001). Interestingly, APCs alone or Aspergillus fumigatus specific T-cells alone showed a weak, but significant capacity to induce hyphal damage (7.4±1.1% and 11.3±1.8%, respectively). Before considering a clinical application, however, further studies need to focus on defining the optimal antigen(s) which reproducibly induce a TH1 response and elicit high antifungal activity, as well as to characterize the subpopulation of patients undergoing allogeneic SCT who ultimately benefit from either a prophylactic or a therapeutic adoptive transfer of Aspergillus fumigatus specific T-cells.
CMV Immunity Status in Older (>50 Years Old) Subjects after Non-Myeloablative or Reduced Intensity Regimen for Hematopoietic Cell Transplantation (HCT): A Comparison with a Younger Cohort after Ablative HCT.
Authors: Gallez-Hawkins Ghislaine , Thao Lia , Lacey2 Simon F. , Martinez Joybelle , Franck Anne E. , Li Xiuli , Lomeli Norma A. , Spielberger Ricardo , Nakamura Ryotaro , Forman Stephen J. , Diamond Don J. , Zaia John A.
Published: April 2020

Immunity declines with age as demonstrated by cell-mediated and humoral responses to alloantigens. The susceptibility of these elderly subjects to endogenous virus infection, such as human cytomegalovirus (HCMV) reactivation, is a particular concern during the process of hematopoietic stem cell transplantation (HCT) and immune reconstitution. In this report, the host contribution to stem cell engraftment and differentiation was evaluated by comparing the HCMV immune response in older subjects (> 50 y.o.) to a younger (< 50 y.o.) transplant population. This was a retrospective analysis of a subset of data collected prospectively and with IRB approval for characterization of the CMV immune response of allogeneic transplant patients. Within the dataset, two groups of patients were compared. Group 1 consisted of 10 patients >50 y.o. who had received reduced intensity or non-myeloablative conditioning regimen, and Group 2 consisted of 13 patients <50 y.o., most of whom had received a myeloablative regimen. Because 9 of 10 in Group 1 had had CMV reactivation, Group 2 was selected from the subset of younger patients with known post-transplant CMV infection. CMV infection was defined as either a positive CMV blood culture using shell vial assay or a positive CMV PCR on plasma. Subjects were assessed on days 40, 90, 120, 150, 180, and 360 post-HCT by CMV-specific tetramer-binding assay using CD8 cells, assays for intracellular INF-g response of CD4 and CD8 cells, and a T-cell receptor excision circle (TREC) assay. There were no significant differences observed in the CD4+/IFN-g+ cell responses to CMV antigen nor were the rates of activated CD4+/CD69+/IFN-g+ cells different between the groups. Group 1 was also characterized by a robust CD8+/IFN-g+ response to HLA-specific CMV peptides, and all subjects had ≥ 2cells/μl by day 150 post-HCT. The frequency of CMV tetramer positive cells (≥ 2cells/μl) was 50% in Group 1 by day 90 post-HCT and was not statistically different from Group 2. The T cell renewal in the thymus as measured by the TREC spanned over 0 -- 92 copies/μg of total cellular DNA in Group 1 and from 0 – 129 copies/μg in Group 2 during the first year post-HCT (n.s.). In conclusion, CMV immune reconstitution in older transplant subjects, who undergo a reduced intensity or non-myeloablative regimen, is robust and, in this small sampling, did not differ from that observed in a younger adult group.
Genetic Polymorphisms in Chemokine Receptor 5, Interleukin 10 and Monocyte Chemoattractant Protein 1 Contribute to Cytomegalovirus Reactivation and Disease after Allogeneic Stem Cell Transplantation.
Authors: Loeffler Juergen , Steffens Michael , Arlt Eva-Maria , Toliat Mohammad-Reza , Wienker Thomas , Hebart Holger , Boeckh Michael , Ljungman Per , Trenschel Rudolf , Einsele Hermann
Published: April 2020

Human Cytomegalovirus (HCMV) infection is still one of the most challenging complications after allogeneic stem cell transplantation (alloSCT). Here, we demonstrate that defined single nucleotide polymorphisms (SNPs) are associated with the susceptibility to HCMV infection after alloSCT.

Genotyping of 83 patients (with HCMV reactivation) and 71 controls (no HCMV reactivation) was performed. All donors and SCT recipients were Caucasians. Patients suffered from chronic myeloproliferative disorders (n = 16) and AML /ALL /SAA /NHL /MDS (n = 117). For 21 patients, underlying disease could not be determined retrospectively. Genotyping was performed by PyroseqencingTM on the PSQTM HS96A System (Biotage AB, Uppsala Sweden) or on ABI 7900 ABI PRISM® Sequence Detection System using TaqMan® Assay-By-DesignTM and Assay-On-Demand TM (Applied Biosystems).

By Armitage’s-Trend-Test, no significant association for 78 /87 markers and the occurrence of HCMV reactivation or disease (p > 0.05) was found. However, 9 markers showed a significant association with HCMV reactivation and /or disease. These markers are located in CCR5, MCP 1and IL10. If comparing individuals with HCMV-DNAemia versus no reactivation regardless of disease, 5 markers showed a significant association (in MCP 1and IL10). Two markers of MCP 1were significantly associated with HCMV reactivation, but none of the markers analyzed in CCR5 and IL10. Thus, we presume that HCMV reactivation is associated with polymorphisms in MCP1, whereas disease might be related to polymorphisms in CCR5 and IL10. Analyzing the clinical parameters, we found an association between CD34 selection and HCMV reactivation (p = 0.0378) or HCMV disease (p = 0.001). Furthermore, severe acute GvHD was significantly related to HCMV disease (p = 0.0015), chronic GvHD was significantly associated to HCMV disease only if reactivation versus disease was compared (p = 0.034). Treatment with corticosteroids led to HCMV reactivation (p = 0.036). In conclusion, screening of alloSCT patients for the presence of defined polymorphisms may help to predict the individual risk of HCMV reactivation and disease, and thus justifying additional antiviral prophylaxis.
Complete Eradication of Minimal Residual Disease (MRD) in Patients with Hairy Cell Leukemia (HCL) after Cladribine (2CDA) Followed by an Extended Course of Rituximab.
Authors: Ravandi-Kashani Farhad , O’Brien Susan , Michael Keating , Jones Dan , Faderl Stefan , Ferrajoli Alessandra , Wierda William , Odinga Shirley , Pierce Sherry , Koller Charles , Verstovsek Srdan , Jorgensen Jeffrey , Kantarjian Hagop
Published: April 2020

The nucleoside analogs 2CDA and pentostatin are effective in the initial therapy of patients with HCL, but relapses are common. We have investigated whether an 8 week course of therapy with weekly rituximab after 2CDA can eradicate MRD assessed by flow cytometry as well as by immunoglobulin heavy chain (IgH) polymerase chain reaction (PCR) assay using framework-1, -2 and -3 primer sets. Treatment constituted 2CDA 5.6 mg/m2 IV given over 2 hours daily for 5 days. One month after initiation of 2CDA, bone marrow (BM) MRD was assessed and rituximab 375 mg/m2 IV was given weekly for 8 weeks. BM MRD was reevaluated again after the completion of rituximab. To date, 12 patients have been treated including 10 with newly diagnosed disease and 2 after relapse from prior therapy (2CDA in one patient and chlorambucil in another). The median age of the patients was 55 years (range 31–73). Two patients had variant HCL. Fluorescent in situ hybridization (FISH) was positive for the presence of a clone with p53 deletion/monosomy 17 in 2 patients. One patient with variant HCL had complex cytogenetics. Seven of 10 evaluable patients had mutated IgVH gene whereas IgVH was unmutated in 3 patients. All 12 patients (100%) have achieved CR defined as no hairy cells in BM and blood with normalization of counts (ANC >1.5 x 109/L, Hgb >12.0 g/dL, Plt > 100 x 109/L) after completion of all therapy; 5 patients still had 1% to 50% hairy cells in BM after 2CDA therapy. MRD by flow was positive in 11 patients after 2CDA therapy but became negative in all 12 patients after rituximab. MRD by PCR was positive in 5 of 10 evaluable patients after 2CDA therapy and became negative in 10 of 11 evaluable patients (equivocal in 1) after rituximab. With a median duration of follow-up of 11 months (range 3–13) no patients have relapsed (median response duration 6+ months, range 1–13). One patient has developed a second cancer (pancreatic). We conclude that therapy with an extended course of rituximab is effective in eradicating MRD in HCL. The predictive value of achievement of negative MRD, presence of chromosome 17 abnormalities, or the mutational status of IgVH on the risk of relapse should be evaluated in larger series and with longer follow-up.
Submicroscopic Interstitial Deletions in 13q14 Are Detectable in Metaphase Cells by Fluorescence In Situ Hybridization (FISH) with D13S319 in Chronic Lymphocytic Leukemia (B-CLL).
Authors: Van Dyke Daniel L. , Stockero Kimberly , Fink Stephanie , Smoley Stephanie , Paternoster Sarah , Schwager Susan , Shanafelt Tait , Zent Clive , Call Timothy , Kay Neil , Dewald Gordon
Published: April 2020

Background: Results of conventional cytogenetic studies on specimens processed without B-cell mitogens are usually normal in B-CLL: this observation is often attributed to lack of mitotic B-CLL cells. Using metaphase FISH with D13S319, we now have evidence that these apparently normal mitotic B-CLL cells often exhibit submicroscopic interstitial deletions in 13q14. This observation indicates B-CLL cells do divide in unstimulated cultures, but the abnormal metaphases are not detected because the abnormalities are submicroscopic and represent a low percentage of proliferating cells.

Methods: We studied 12 patients with B-CLL who had normal metaphases by conventional cytogenetics, but abnormal interphase nuclei by FISH with D13S319. This cohort consisted of 7 females and 5 males who ranged in age from 50 to 87 years. Clinical information was available for 11 patients: Rai stages were 0, I, II, III, or IV for 6, 1, 1, 2, and 1 patients, respectively; 8 patients received treatment and 3 have died. Conventional cytogenetic studies and metaphase FISH were done on bone marrow specimens that were processed by standard 24 and 48-hour unstimulated cultures with Chang Medium BMC. Interphase FISH studies were done on blood or bone marrow (collected within 3 days of specimens for conventional cytogenetic studies) using probes to detect anomalies of chromosomes 6, 11, 12, 13, 14 and 17. For each patient, metaphase FISH was done with commercial probes from Vysis Inc., Downers Grove, IL for D13S319/13q34 using leftover fixed cells from conventional cytogenetics studies.

Results: Metaphase FISH results were normal for 3 bone marrow specimens: the remaining 9 specimens had 1.9 to 13.8% metaphases with interstitial deletions of 13q14. One patient had a cytogenetically visible chromosome 13 deletion in 2.2% of metaphases. In the remaining 8 specimens, the size of the abnormal 13 was similar to the normal 13 homolog and other acrocentric chromosomes. By comparison, interphase FISH revealed 13 to 93% nuclei with 13q-x1 or 13q-x2. Seven patients had additonal anomalies in their interphase nuclei; 3 had 11q-, 2 had 17p-, 1 had +12 and 1 had +IGH.

Conclusions: This investigation indicates that metaphase FISH detects submicroscopic deletions in chromosome 13 as well as infrequent mitotic B-CLL cells in patients who have normal conventional cytogenetic results. Therefore, metaphase FISH is a novel and useful technique to detect abnormal mitotic cells in B-CLL in cultures without special B-cell mitogens. The prognostic significance of abnormal metaphase FISH results is still under investigation. We speculate that this method will allow for identification of more aggressive proliferating disease even in so-called “favorable” prognostic groups based on interphase FISH results.
Transient Response to Imatinib in an Atypical Chronic Myeloproliferative Disease Associated with ins(9;4)(q34;q21q31) and a CDK5RAP2-PDGFRA Fusion Gene.
Authors: Walz Christoph , Schultheis Beate , Metzgeroth Georgia , Schoch Claudia , Curtis Claire , Lengfelder Eva , Haferlach Torsten , Berger Ute , Hochhaus Andreas , Hehlmann Rüdiger , Cross Nicholas C.P. , Reiter Andreas
Published: April 2020

Chronic myeloproliferative disorders (CMPD) associated with rearrangements of the ‘platelet-derived growth factor receptor A’ (PDGFRA) at chromosome 4q12 are excellent candidates for targeted therapy with imatinib. To date, two fusion genes involving PDGFRA have been described: FIP1L1-PDGFRA and BCR-PDGFRA. Here we report a female patient who presented in advanced phase of an atypical myeloproliferative disease. Routine cytogenetic analysis revealed an ins(9;4)(q34;q21q31). Although no break was visible at 4q12, FISH analysis with flanking BAC probes indicated that PDGFRA was disrupted. To identify the partner gene we employed 5′-RACE PCR, exploiting the fact that all known PDGFRA fusions involve exon 12 of this gene. The resulting PCR-products consisted of 5′-sequences derived from CDK5RAP2 (CDK5 regulatory subunit associated protein 2) located on 9q33 and 3′-sequences derived from PDGFRA. CDK5RAP2 encodes a protein that is believed to be involved in centrosomal regulation. FISH analysis confirmed the co-localization of 5′ CDK5RAP2 and 3′ PDGFRA sequences. RT-PCR confirmed the in-frame mRNA fusion between exon 13 of CDK5RAP2, a 40-bp insert which was partially derived from PDGFRA intron 11 and a truncated PDGFRA exon 12. No reciprocal fusion transcript could be amplified by RT-PCR. The predicted CDK5RAP2-PDGFRA protein consists of 1003 amino acids and retains both tyrosine kinase domains of PDGFRA and several potential dimerisation domains of CDK5RAP2 suggesting a mechanism of tyrosine kinase activation similar to BCR-ABL. Treatment with 400 mg imatinib was initiated and the patient achieved a complete cytogenetic and molecular remission. However, hematological response was only partial with residual blasts repeatedly detectable in the blood and marrow. The patient rapidly developed acute leukemia while still remaining in complete cytogenetic and molecular remission suggesting the outgrowth of a second imatinib-resistant leukemic clone. These findings and the fact that the ins(9;4) was only seen in 24% of metaphases at diagnosis suggests that CDK5RAP2-PDGFRA may have been a secondary abnormality. In summary, we have identified a third fusion gene involving PDGFRA, underlining the fundamental role of activated tyrosine kinases in CMPD’s and their possible response to targeted therapy with imatinib.
Predictable Prognostic Factor of CD56 Expression in Acute Myeloid Leukemia with t(8:21) Including Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Yang Deok-Hwan , Mun Yeung-Chul , Shin Ho-Jin , Kim Yeo-Kyeoung , Lee Je-Jung , Jin Choi-Young , Seong Chu-Myong , Kim Hyeoung-Joon
Published: April 2020

CD56 expression in acute myeloid leukemia (AML) has been associated with extramedullary leukemia and multi-drug resistance, but the clinical and prognostic significances are not yet clearly defined. Recently, some investigators reported that AML patients with t(8;21) showed more frequent CD56 expression rate and the expression of CD56 antigen adversely affected disease-free survival (DFS). It could explain a diverse clinical outcome in AML patients with favorable cytogenetics. This study investigated CD56 expression in 37 adult de novo AML patients with t(8:21) between November 1996 and June 2005 at three institutions. Immunophenotyping was performed with flow cytometry (Coulter EPICS XL) and considered positive if at least 20% of blasts expressed. CD56 was expressed in 25 cases (67.6%). There was no statistically significant differences in age, sex, leukocyte count, the percentage of bone marrow blasts or the presence of additional cytogenetic abnormalities between the CD56+ and the CD56- group. The complete remission (CR) rate to standard dose cytarabine or N4-behenoyl-1-D-arabinofuranosylcytosine (BH-AC) and idarubicin was similar in both groups (91.7% v 88.7%; P=0.73), but the relapse rate to high-dose cytarabine or allogeneic hematopoietic stem cell transplantation (HST) was quite different (60% v 25%; P=0.08). Allogeneic HST was performed from siblings in 15 patients (40.5%) who achieved CR, 8 patients (32.0%) in CD56+ and 7 patients (58.3%) in CD56- group (P=0.16). The median durations of DFS were significantly shorter in CD56+ (median, 12.2 months) than in the CD56- group (median, not reached) (P =0.02). Also, the median durations of survival showed the same results in the CD56+ group (median, 14.9 months) compared with the CD56- group (median, not reached) (P=0.01). Within fifteen transplanted patients, the median durations of DFS in eight CD56+ patients was significantly shorter than seven CD56- patients (median, 24.4 months v not reached; P=0.02)(Fig.1 and 2).We concluded that CD56 expression was associated with reduced DFS and survival for AML patients with t(8:21) including transplanted patients. Although further larger studies are needed, we suggested that CD56 expression at diagnosis is a predictable prognostic factor in AML with t(8:21).

Fig. 1 Disease-free survival (DFS) and Overall survival (OS) for patients with t(8:21) with CD56+ (n−25) and CD56− (n−12) group. Fig. 1. Disease-free survival (DFS) and Overall survival (OS) for patients with t(8:21) with CD56+ (n−25) and CD56− (n−12) group. Fig. 2 Within transplanted patients, decreases from survival (DFS) for patients with t(8:21) with and without CD56 expression. Fig. 2. Within transplanted patients, decreases from survival (DFS) for patients with t(8:21) with and without CD56 expression.
Understanding the Membrane Proteome of Developing Natural Killer Cells.
Authors: Lund Troy , McCullar Valarie , Anderson Lorraine B. , Miller Jeffrey S.
Published: April 2020

We are interested in the biological as well as molecular processes involved in natural killer cell differentiation and function based on the growing interest in these cells for cancer therapy and allogeneic transplantation. Determining the proteomic complement is a useful tool in predicting cellular function and fate. iTRAQ is a new tandem mass-spec method which not only allows identification of proteins in the picomole to femtomole range, but also allows one to quantify differences in specific protein amount between two samples. Using this method one can potentially get reliable proteomic information from cells and also compare expression differences between cell populations. For the first time shown here, we have applied the new method of iTRAQ to identify and quantify the expression of membrane localized proteins in two previously characterized distinct but related NK cells populations. One population was derived in vitro from umbilical cord blood stem cells (CD34+Lin−CD38−) differentiated with murine stroma and human cytokines (IL-3, Flt3L, SCF, IL-7. IL-15) and the other from NK cells expanded from CD3-depleted adult peripheral blood and IL-2, both resulting in cultured populations of >90% NK cells. iTRAQ was employed for multiplex peptide labeling followed by 1- and 2-dimensional high performance liquid chromatography (1D- and 2D-HPLC) and then tandem mass spectroscopy was used to analyze the proteomic contingent of membrane associated proteins. We were able to identify well as quantify differences in expression levels of 35–133 proteins in a typical 1D-HPLC experiment. This increased to >400 proteins when 2D-HPLC was used. Over 75% of the proteins were identified with more than 2 peptide fragments. Gene ontogeny analysis showed the majority of proteins to be involved in cell signaling, nucleic acid binding, or cell adhesion. Nearly all proteins were associated with the plasma membrane, membrane bound organelle (lysosome or peroxisome), or nucleus. Several unique proteins were found which previously have not been known to be expressed by NK cells: BASP1, nmb, and AIF-1. More important, we were able to confirm several of our iTRAQ results by RT-PCR, Western blot, and FACS analysis. This is the first demonstration, and verification of using iTRAQ to determine the “membrane proteome” in NK cells and represents a valid and powerful new tool in the science of proteomics. These novel differences between NK cells and their precursors will be studied further to understand NK cell interactions and novel mechanisms which govern NK cell development.
CyclinA2 as a Proliferation Marker for Peripheral Blood T Cell Subsets Measured by Flow Cytometry.
Authors: Smith Cecilia U. , D’Costa Sybil , Rabellino Enrique M. , Shankey Vincent , Wilkinson Julie
Published: April 2020

Proliferation is regarded as a sensitive parameter for assessing immune response to infectious agents, autoimmune conditions, and organ engraftment in transplantation. Proliferation of peripheral blood mononuclear (PBMC) cells has been measured by a number of flow cytometric methods including propidium iodide (PI), incorporation of Bromo-deoxy-uridine (BrdUr), and dilution of Carboxy-fluorescein diacetate, succinimidyl ester (CFSE) intensity. Standardization of these methods however, has been hampered by their technical complexity. The restricted expression of CyclinA2 protein in the cytoplasm during the S and G2 cell cycle phases has provided the basis for a simplified intracellular flow-based proliferation assay. In these studies, ex vivo stimulated human PBMC were tested for the validation of CyclinA2 as a proliferation marker for T cell subsets. Freshly isolated or cryopreserved PBMCs were incubated in vitro with SEB/CD28 for periods up to 72 hours and processed using different methodologies for lymphocyte cell cycle measurement. These included BrdUr /7AAD, PI/CyclinA2; cylinA2 with additional T cell phenotyping markers and a combined CyclinA2 /BrdUr proliferation method. Results for each cell subset were scored for the percentage of positive proliferating cells by each methodology. PBMC co-stained for CyclinA2 and DNA content by PI demonstrated that all cells expressing Cyclin A2 corresponded to cells at S or G2 phase. Furthermore, the time course assays combining BrdUr labeling with CvclinA2 confirmed that CyclinA2 expression corresponded selectively to cells incorporating BrdUr. When PMBC were surface phenotyped for CD3 and CD4/CD8, reproducible populations of CD4 and CD8 T cell subsets incorporating BrdUr were measured for expression of Cyclin A2. In our assay model 11 to 15 % positively stained for Cyclin A2 corresponded to approximately 10% and 5% of CD3/8 and CD3/4 CyclinA2 cells, respectively. The percentage of total BrdUr and total CyclinA2 positive cells that were detected in the dual-labeled specimens corresponded to the percentage detected by each individual method. In conclusion, measurement of in vitro lymphocytes proliferation using expression of CyclinA2 protein expression is highly correlated with BrdUr uptake demonstrating that CyclinA2 is an accurate marker of cell proliferation. Preparation of cells for measuring Cyclin A2 takes only 60 minutes as compared with a total 12 to 16 hours for BrdUr pulsing and staining time. The simple CyclinA2 preparation method described here makes it a practical, fast, and reliable proliferation flow assay that can be easily standardized for enumeration of proliferating lymphocyte subsets.
Time to Failure and CD3-Expression Are Strong Prognostic Factors in Relapsed Anaplastic Large Cell Lymphoma of Childhood and Adolescence.
Authors: Woessmann Wilhelm , Lenhard Meike , Peters Christina , Burkhardt Birgit , Ebell Wolgang , Klingebiel Thomas , Mann Georg , Greil Johann , Sykora Karl-Walter , Janssen Dirk , Zimmermann Martin , Schrappe Martin , Reiter Alfred
Published: April 2020

We examined the feasibility and efficacy of a salvage strategy of intensive re-induction chemotherapy followed by autologous or allogeneic hematopoetic stem cell transplantation (HSCT) for children and adolescents suffering from relapsed anaplastic large cell lymphoma after BFM front-line therapy.

From 4/1990 to 2/2003, 75 evaluable patients, median age at relapse 9.8 (range 0.4–22.2) years, were enrolled. The median time from the start of first therapy to failure was 7.1 (range 1–76) months. 17 pts suffered from progressive disease during front-line chemotherapy. ALK staining was positive in 57 pts, negative in 5 pts and not available in 13 pts. CD3 reactivity was positive in 24, negative in 42 pts and not available in 7 pts. 2 pts had B-cell ALCL. Salvage therapy consisted of 2 or 3, 5-day chemotherapy courses based upon dexa, vindesine, cytarabine, etoposide, MTX 5g/m2, cyclophosphamide/ifosfamide, vincristine, doxorubicine, intrathecal therapy followed by HSCT. Conditioning regimen: total body irradiation (2x2 Gy day −7,−6,−5), etoposide 40 mg/kg day -4, cyclophosphamide 60 mg/kg/d days −3,−2. Data were updated as of April 1st, 2004. With a median follow-up of 6.9 [0.8–13.2] years, the probability of survival at 3 years after first relapse is 54+6%. 28 pts died of lymphoma, 6 pts of toxicity. 41 pts are alive in ≥CR2. 21 pts received no salvage or chemotherapy only (2 TRM, 18 death of disease, 1 CR). 39 pts received autologous HSCT (21 CR, 2 TRM, 16 re-relapses [6 >CR2]). 15 pts received allogeneic HSCT (10 CR, 3 TRM, 2 relapses). 3/17 pts with relapse during front-line survived compared to 37/57 pts who relapsed later (p<.001). pSurvival at 3-years was 62+8% for CD3-neg. pts and 45+10% for CD3-pos pts (ns). However, pEFS after autologous HSCT was dependent on the CD3 status: 69+10% for 23 CD3-neg pts (16 CR, 1 TRM, 6 relapse), 9+9% for 11 CD3-pos pts (1 CR, 1 TRM, 9 relapse), p=.002. In contrast, all 7 CD3-pos patients who received allogeneic HSCT (4 of them after relapse subsequent to autoHSCT) remained free of disease thereafter. 5/5 ALK-negative patients relapsed after front-line therapy, were CD3-positive and progressed again, two of them after autologous HSCT.

We conclude that this strategy proved feasible and efficacious for pts who suffered a relapse after front-line therapy while outcome for pts with progressive disease during front-line treatment was poor. Autologous HSCT is an efficient consolidation for pts with relapses of CD3 negative ALCL but not efficacious for those with CD3 positive tumors. These patients may benefit from allogeneic HSCT.