bluuebunny/crossref_metadata_2025 huggingface dataset:
Is Elevated Serum IL-10 in B-CLL Associated with IL-10 Promoter Polymorphisms?.
Authors: Chu Charles C. , Reese Stephanie T. , Goodwin Leslie O. , Guzowski Dorothy , Chandrasekaran Alamelu , Gawel Craig , Li Wentian , Gregersen Peter K. , Yan Xiao J. , Sherry Barbara , Centola Michael , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas
Published: April 2020

Interleukin-10 (IL-10), a cytokine that regulates inflammation, may play an important role in B-cell chronic lymphocytic leukemia (B-CLL), because of the reported association of high serum IL-10 levels with a lower prognosis of survival. Using the Bio-Plex protein array system, we confirmed that B-CLL patients exhibit higher median IL-10 levels (3.54 pg/ml, n=50) as compared to controls (1.28 pg/ml, n=33) (p<0.0001). We are in the midst of determining B-CLL VH gene mutation status and IL-10 levels. To determine if elevated IL-10 levels are due to inherent genetic polymorphisms, we examined three single nucleotide polymorphisms (SNPs) in the proximal end of the promoter region of the IL-10 gene (-1082 A/G, −819 T/C, and −592 A/C) that may affect IL-10 transcription levels. DNA from an overlapping set of 54 B-CLL patients and 48 normals was genotyped using the Transgenomic WAVE system. The difference in allele frequencies at a single locus showed no trend towards significance between groups using the Pearsons chi-square test and Odds Ratios (OR) with 95% Confidence Intervals (CI) for each SNP (-1082 (p = 0.686, OR = 1.122, CI = 0.641–1.966), −819 (p = 0.844, OR = 1.062, CI = 0.585–1.926) and −592 (p = 0.720, OR = 1.116, CI = 0.613–2.031)). B-CLL cases and normal subjects showed no significant departure from Hardy-Weinberg equilibrium in single locus genotype frequencies. Differences in single locus genotype frequencies between B-CLL cases and controls showed no significant differences for each SNP (−1082 (p = 0.600, OR = 1.366, CI = 0.425–4.389), −819 (p = 0.638, OR = 0.727, CI = 0.192–2.749), and −592 (p = 0.638, OR = 0.727, CI = 0.192–2.749)). The haplotype frequencies were calculated using maximum likelihood method based on the observed genotypes. The differences in maximum likelihood haplotype frequencies, between the B-CLL cases and controls, were insignificant (p = 0.754). The haplotype pair genotype frequency differences between the B-CLL cases and controls were also insignificant (p = 0.921). In conclusion, the allele, genotype, and haplotype frequencies of IL-10 SNPs −1082, −819, and −592 show no trend towards significance overall. This suggests that these promoter SNPs do not contribute to elevated serum IL-10 in B-CLL. However, a suggestion of association with VH gene mutation status is being further investigated with a larger sample size.
The Multi-Functional Cellular Adhesion Molecule CD44 Is Targeted by the 8;21 Chromosomal Translocation.
Authors: Wang Yang , Peterson Luke , Zhang Dong-Er
Published: April 2020

CD44 is a type I transmembrane protein and functions as the major cellular adhesion molecule for hyaluronic acid, a component of the extracellular matrix. CD44 is expressed in most human cell types and is implicated in a wide variety of physiological and pathological processes, including tumor cell growth and metastasis. Its importance in acute myeloid leukemia is highlighted by evidence of its high expression in all human acute myeloid leukemias. In addition, the treatment of leukemic blasts with anti-CD44 antibodies promotes their maturation, suggesting a potential differentiation therapy for patients and a possible role for CD44 in the growth and maintenance of leukemic blast/stem cells. We have recently identified the naturally occurring leukemogenic splice variant of the t(8;21) associated AML1-ETO gene, AML1-ETO9a. To understand the molecular mechanism of AML1-ETO-9a involved leukemogenesis, we performed micro-array analysis with a multipotential hematopoietic cell line. Out of the 39,000 transcripts, 24 decreased more than 3.5-fold including SPARC, Mac25 that are reportedly involved in proliferation of different cells. We also identified 75 transcripts with more than 3.5 fold increase that included Sca-1, Runx2 and CD44. Specifically we show that the presence of AML1-ETO9a significantly increased the expression of CD44 at both RNA and protein levels. Furthermore, the CD44 promoter region −709 to −280 bp is responsive to both AML1-ETO9a as well as AML1-ETO. Thus our observations suggest that AML1-ETO and its splice variant AML1-ETO9a are able to promote the expression of the CD44 gene, linking the 8;21 translocation directly to the regulation of an important cell adhesion molecule potentially involved in the growth and maintenance of the acute myeloid leukemia blast/stem cells.
Correlation of JAK2 Mutation and PRV-1 and WT1 Transcript Amount in Myeloproliferative Disorders.
Authors: Serra Anna , Carturan Sonia , Gottardi Enrico , Arruga Francesca , Messa Francesca , Sterpone Paola , Defilippi Ilaria , Rosso Valentina , Catalano Renata , Martinelli Giovanni , Iuliano Francesco , Saglio Giuseppe , Cilloni Daniela
Published: April 2020

Recently, a JAK2V617F mutation has been described in the majority of patients with polycythemia vera (PV), and in a subset of patients with essential thromocythemia (ET) and idiopathic myelofibrosis (IMF). Similarly, PRV-1 has been found overexpressed in many of these patients. The aim of the study was to correlate PRV-1 and WT1 expression with the presence of JAK2 mutation in order to establish whether these markers identify the same subset of patients. Using a Real Time PCR we studied the expression level of PRV1 and WT1 in 330 patients including 26 IMF, 156 PV, 122 ET, 16 HES. 10 familiar forms of PV were included. In addition a control group of 50 healthy subject and 60 reactive conditions were studied All the patients were analyzed for the presence of JAK2 mutation using the ABI Prims 3100 genetic analyzer. We found that JAK2 mutation was present in 50 % of IMF, 93% of PV and 55% of ET and 6% of HES. PRV-1 was increased in 84% of IMF, 98% of PV and 90% of ET and it was not increased in HES. WT1 was increased in 99% of IMF, 45% of PV and 42% of ET and 100% of HES. No mutations of JAK2 were detected in normal subjects or in reactive conditions. Both WT1 and PRV1 were expressed at low levels in normal subject (for PRV1 the mean value of 2-Delta Delta Ct was 2,05; range 0,05–5,46; for WT1 mean value WT1 copies/104 ABL copies of 4; range 0–20). The mean value of WT1 copies/104 ABL copies in IMF was 306 (p=0,00004 compared to normal controls), in PV was 156 (p=0,0001), in ET was 202 (p=0,0002) and in HES was 161 (p=0,0004). PRV1 mean value in IMF was 144 expressed as 2−Delta Delta Ct, and it was 423 in PV and 290 in ET. Moreover the correlation between the three markers allowed to establish that both WT1 and PRV1 were higher in JAK2 mutated samples when compared to wild type JAK2 with a p value of 0,001 for WT1 and 0,004 for PRV1. In spite of these we were able to identify a subset of patients affected by IFM characterized by increased values of PRV1 and WT1 but not by JAK2 mutation (34%) and a small subset 15% characterized only by WT1 overexpression. Only 5% of PV and 35% of ET patients expressed only PRV-1 but not JAK2 mutation and WT1 expression. HES patients overexpressed WT1 but not PRV1 and only one patient presented the JAK2 mutation. Finally 10 patients affected by familiar form of PV were studied. All the subjects showed normal PRV-1 expression but high level of WT1 transcript and 9 out of 10 presented the JAK2 mutation. This study demonstrates that the combination of all these markers is probably useful for a correct diagnosis of these patients but further studies are required to stratify these patients and to better classify them according to the presence of molecular lesions.
Tel-PDGFβR Specifically Induces Interferon-Stimulated Genes.
Authors: Kim Hani , Barber Dwayne L.
Published: April 2020

Chromosomal translocations involving tyrosine kinases play a significant role in human leukemia. Chronic myeloid leukemia (CML) is associated with the recurrent chromosomal translocation, BCR-ABL (t(9;22)(q34;q11)). Chronic myelomonocytic leukemia (CMML) is linked to TEL-PDGF-β Receptor (PDGFβR) (t(5;12)(q33;p13)) fusion. Another TEL fusion, TEL-JAK2 (t(9;12)(p24;p13) has been observed in CMML and Acute Lymphoid Leukemia. All three fusion proteins induce leukemia-like diseases in animal models, and this is attributed to the constitutive tyrosine kinase activity, which leads to dysregulation of their respective downstream signaling pathways. The downstream targets include STAT transcription factors, MAP kinases, and PI3 kinase. On the other hand, little is known about the gene transcription regulated by these fusions.

The objective of our study is to determine whether BCR-ABL, TEL-PDGFβR and TEL-JAK2 induce distinct gene expression patterns when expressed in cell lines and retrovirally transduced bone marrow cells. Each fusion was expressed in an IL3-dependent murine myeloid cell line, Ba/F3. The specific inhibitor, Imatinib mesylate, was utilized to control the activation/inhibition of BCR-ABL and TEL-PDGFβR, and an inducible system was utilized for TEL-JAK2. Upon activation of the fusion protein, cells were collected at various time-points for cell cycle and microarray analysis (Affymetrix MOE430A). We utilized 8 hr, 12 hr, 24 hr and 1 wk time points. Our rationale was to monitor gene expression changes through the first cell cycle and then to examine the fingerprint at a steady state point.

Analysis of the 1 wk data reveals that a subset of genes are co-regulated (2-fold, p<0.05) by BCR-ABL, TEL-PDGFβR and TEL-JAK2 (Pim1, Id1b, Podxl, Cxcr4, Gp49b and Scin). Interestingly, analysis of the TEL-PDGFβR induced genes (10-fold, p<0.05) revealed a significant overlap with Interferon-Stimulated Gene (ISG) dataset including Cxcl-10, Gbp1, Gbp2, Isg20, Ccl-5, Stat1, Irf7, Serpine-1 and Mx1. Genes identified in this microarray study have been confirmed by Q-PCR in Ba/F3 cells and confirmatory experiments in primary bone marrow cells transduced with each fusion protein are underway. In addition, we will determine whether the transcription of these targets is dependent on STAT1 by utilizing bone marrow cells from STAT1−/− mice. In conclusion, our data reveals that oncogenic chromosomal translocations activate both distinct and co-regulated gene expression and reveal a novel and specific role of Interferon-Stimulated Genes in signaling pathways downstream of TEL-PDGFβR.
ICSBP Activates Transcription of the Gene Encoding Neurofibromin 1 in Differentiating Myeloid Cells.
Authors: Huang Weiqi , Saberwal Gurveen , Zhu Chunliu , Eklund Elizabeth A.
Published: April 2020

Previous studies in murine models and human myeloid malignancies indicate that the interferon consensus sequence binding protein (ICSBP) functions as a leukemia tumor suppressor. Previously identified ICSBP target genes include genes encoding gp91phox and p67phox (components of the phagocyte respiratory burst oxidase), the Toll like receptor 4, and other genes involved in mediating the phagocyte inflammatory response. Transcription of these target genes involves interaction of ICSBP with PU.1 and/or interferon regulatory factor 1 (IRF1), and is restricted to mature phagocytic cells. These results predict that ICSBP-deficiency would be associated with terminal differentiation block and immune dysfunction. However, the phenotype of ICSBP −/− mice is dominated by development of a myeloproliferative disorder which evolves to acute myeloid leukemia. Therefore, we used the chromatin immuno-precipitation technique to identify novel ICSBP target genes which might be involved in the ICSBP tumor suppressor effect. We identified the gene encoding neurofibromin 1 (the NF1 gene) as an ICSBP-target-gene. We found that ICSBP activates NF1 transcription during cytokine induced differentiation of myeloid progenitor cells. Because Nf1 protein has ras-GAP activity, increased Nf1-expression down regulates cytokine-induced ras activity, resulting in proliferation arrest during myeloid cell differentiation. In the current studies, we investigate the mechanism by which ICSBP activates NF1-transcription. We find that ICSBP is part of a multi protein complex that binds to and activates a cis element in the NF1 gene. This complex also includes the ets protein PU.1 and interferon regulatory factor 2 (IRF2). We find that tyrosine phosphorylation of ICSBP in response to hematopoietic cytokines is required for assembly of this transcriptional activation complex and therefore NF1-transcription. Identification of NF1 as an ICSBP target gene represents a mechanism for ICSBP-leukemia-suppression. These studies also provide the first indication of the importance of NF1-transcriptional regulation to the control of cytokine-induced proliferation in differentiating myeloid cells.
Enhanced In Vivo Homing of Uncultured and Selectively Amplified Umbilical Cord Blood CD34+ Cells by Co-Transplantation with Cord Blood Derived Unrestricted Somatic Stem Cells.
Authors: Chan Shing Leng , Choi Michael S.K. , Wnendt Stephan , Kraus Morey , Teng Eileen , Leong Hwei Fen , Merchav Shosh
Published: April 2020

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. A new pluripotent population of umbilical cord blood (UCB)-derived Unrestricted Somatic Stem Cells (USSCs), with intrinsic and directable potential to develop into mesodermal, endodermal and ectodermal fates, has recently been identified and characterized (1). In this study we evaluated the capacity of USSCs to influence the homing of UCB -derived CD34+ cells into the marrow and spleen of NOD/SCID mice. Cultured USSCs were co-transplanted with CFDA-labeled cord blood CD34+ cells into sublethally irradiated NOD/SCID mice. Femurs and spleens were harvested 16 hrs thereafter and the percentage of CD34+ cells determined by flow cytometry.

USSCs induced a significant enhancement of CD34+ cell homing to both bone marrow and spleen (2.2 ± 0.3 and 2.4 ± 0.6 -fold, respectively; p<0.05). Similar findings were obtained with frozen USSC samples that had been thawed prior to transplantation. The effect of USSCs was specific, as no homing enhancement could be observed by co-transplantation of CD34+ cells with lineage-positive UCB cells, which contain T, B and mature myelo-erythroid cells. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells, with a similar degree of enhancement observed for both early (p5) and late (p10) passage USSCs (1.7 ± 0.1 and 1.9 ± 0.1 -fold, respectively). Enhanced homing was dose-dependent, detectable at USSC/CD34+ ratios of 1:1 and above.

USSCs were also found to enhance the homing of day 14 cells harvested from cultures of selectively amplifiedTM, ex-vivo expanded UCB lineage-negative (lin-) cells. The relative proportion of homing CD34+ cells within the culture-expanded cell population, was unaffected by USSC co-transplantation. Our findings thus demonstrate that USSCs enhance the homing of UCB hematopoietic stem cells and suggest a clinical potential for these cells in facilitating engraftment under conditions of limiting cord blood stem cell numbers.
Antibodies to CD40 Promote Dendritic Cell Recovery and Anti-Tumor Effects after Syngeneic Bone Marrow Transplant (BMT).
Authors: Welniak Lisbeth A. , Berner Vanessa , Blazar Bruce R. , Wiltrout Robert H. , Murphy William J.
Published: April 2020

Disease relapse remains a principle cause of mortality following autologous hematopoietic transplant. Strategies to enhance immune function in the post-transplant setting are needed to eliminate residual disease. CD40 stimulation through the use of agonistic CD40 antibodies has been shown to promote anti-tumor activity both as a single agent and in combination with IL-2 administration with increases in dendritic cell numbers being observed. We hypothesized that CD40 stimulation in combination with myeloablative syngeneic hematopoietic cell transplantation can act together to promote anti-tumor responses. We developed a model in which anti-CD40 given with syngeneic BMT was assessed in advanced tumor-bearing mice. We have found that delaying administration of anti-CD40 for one week post-BMT could obviate the severe toxicity associated with immediate post-BMT administration. BALB/c were given a syngeneic renal cell carcinoma cell line (Renca) i.v. After 4 days, at which time lung metastases were established, the mice were treated with syngeneic BMT followed one week later with initiation of anti-CD40 treatment (100 μg/dose; daily, 4 days per week for 2 weeks). CD40 stimulation resulted in significant expansion of GR-1+/CD11b+ myeloid and CD11c+/I-A+ dendritic cells in the spleen, similar to the expansion of these populations observed in mice that received anti-CD40 and no BMT. However, while B220+ IgM+ B cells are significantly expanded following anti-CD40 administration in mice that do not receive a transplant, B cell recovery is reduced following transplant in mice that also received anti-CD40. T cell recovery post-transplant did not differ significantly in animals that received anti-CD40 or control antibody. Importantly, significant increases in survival of anti-CD40 treated, tumor-bearing recipients of syngeneic BMT were observed compared to either treatment alone (log-rank test p< 0.001). This promotion of anti-tumor activity by CD40 stimulation was seen in mice that were profoundly T cell deficient due to the transplant. These data suggest that despite severe immune depletion that occurs after lethal TBI, immunomodulation by anti-CD40 can result in significant protection from the residual disease despite the severe lack of T cells at this time period.
CD4+CD25+ T Cells Can Inhibit CD8 T Cell Mediated GVHD: Requirement for In Vivo Recognition of Allogeneic Host MHC Class II Antigens.
Authors: Levy Robert B. , Jones Angela
Published: April 2020

CD4 regulatory T (Treg) cells have shown promise in the transplantation mileu including the ability to inhibit the development of graft vs host disease (GVHD) following allogeneic hematopoietic stem cell transplants (HCT). The antigen specificity of the Treg population(s) involved is not yet clear nor is the role of their activation following transplant. We are interested in determining the requirement for recognition of host MHC antigens following infusion of CD4+CD25+ T cells in an experimental model of GVHD. To clearly distinguish the requirements of regulatory vs GVH reactive cells, a model of CD8 T cell mediated GVHD was developed using highly purified BALB/c (H2d) donor CD8+ T cells (Miltenyi column, 95-98%). CD8 T cells were transplanted together with T cell depleted (TCD) BALB/c BMC into 12.0 GY (6.0 Gy split dose) TBI conditioned C57BL/6 (B6, H2b) recipients. To support development of GVHD by these cells, resistance was inhibited by treatment of recipients with anti-NK1.1mab (PK136) at Days -1, 0 and +7. BALB/c CD8+ T cells at doses of 5.0x106 but not 2.5x106 induced weight loss and some lethality in B6 recipients. 5x106 CD8+ T cells were then transplanted into B6-MHC class II−/ − recipients. GVHD symptoms including weight loss and lethality were readily apparent in these mice post-transplant. Interestingly, GVHD was consistently more severe with respect to the induction of weight loss and lethality in MHC Class II−/ − vs B6-wt recipients. Highly enriched BALB/c CD4+CD25+ T cells (> 95%) were produced from spleen and lymph node cells following negative (B-cells, CD8 and NK) and positive (CD25) selection using Miltenyi magnetic bead columns. Co-transplant of 1x106 CD4+CD25+ T cells together with BALB/c CD8+ T cells into B6 recipients inhibited GVHD as assessed by the absence of weight loss and lethality compared to B6 recipients of CD8+ T cells alone. In contrast, BALB/c CD4+CD25+ T cells failed to protect B6-MHC class II−/ − recipients from severe CD8+ T cell mediated GVHD. These findings demonstrate that donor CD4+ T regulatory cells can suppress GVHD inducing CD8+ T cells after the former recognize host class II alloantigen following transplant. We hypothesize that activated CD4+CD25+ T regulatory cells inhibit GVH reactive T cells at the host APC interface. Future studies in this model can be designed to examine ex-vivo activated and expanded CD4+CD25+ T regulatory populations. Transplant of such cells will enable us to address questions regarding the importance of in vivo recognition of host class II in the regulation of GVHD by these cells.
Deficient Ribosomal Protein S19 in Diamond-Blackfan Anemia Causes a Reduction in bcl-2 and Bad and Leads to Apoptosis in Erythroid Progenitor Cells.
Authors: Miyake Koichi , Utsugisawa Taiju , Flygare Johan , Kiefer Thomas , Hamaguchi Isao , Richter Johan , Karlsson Stefan
Published: April 2020

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythopoiesis and proliferation of hematopoietic progenitors. We have established an in vitro models for RPS19 deficient DBA using lentiviral vector mediated doxycycline (Dox) inducible small interfering RNA (siRNA) against RPS19 (Mol Ther.11:627–637. 2005). Suppression of cell growth and erythroid colony formation correlated with the suppression level of RPS 19, indicating that these cell lines are useful to determine the mechanisms of RPS19 deficient DBA. To elucidate molecular mechanisms in RPS19 deficient DBA, we analyzed cell cycle of Dox induced RPS19 deficient TF-1 cells. RPS19 deficient TF-1 cells showed G0/G1 arrest (82% vs 58%, p<0.05) together with accumlation of p21 and p27, and apoptotic cells detected by Annexin-V analysis also increased compared to control Dox induced TF-1 cells (13% vs 3.1%, p<0.05). Increase of apoptotic cells in RPS19 deficient cells was confirmed by TUNEL assay. Western blot analysis of apoptotic related protein showed that the level of bcl-2 and Bad was decreased in RPS19 deficient TF1 cells compared to control cells. This down-regulation of apoptotic related protein was improved by transduction with lentiviral vector expressing modified RPS19, which is not affected by siRNA but produce normal functional RPS19 protein and rescues the DBA phenotype. Moreover, primary CD34 positive cells from DBA patients detected by Annexin-V analysis also generate a high number of apoptotic cells compared to normal CD34 positive cells during in vitro culture (38% vs 8.9%, n=5, p<0.001). These findings indicate that erythroid progenitor cells are more sensitive to apoptosis than other hematopoietic progenitors and that RPS19 deficiency causes apotosis and accelerated loss of erythroid progenitors in RPS19 deficient DBA.
Adoptive Infusion of Allogeneic KIR Ligand Mismatched NK Cells Reduce GVHD in an MHC Matched Allogeneic Stem Cell Transplantation Model.
Authors: Lundqvist Andreas , Samsel Leigh , Eckhaus Michael , Srinivasan Ramaprasad , Takahashi Yoshiyuki , Snoeijs Maarten , McCoy Philip J. , Berg Maria , Childs Richard
Published: April 2020

Retrospective data suggest NK cells play a role in protecting recipients from graft versus host disease (GVHD) in the setting of killer IgG-like receptor (KIR) ligand incompatibility. In humans, this protective effect is most evident with MHC mismatched transplantation, usually following in vivo or in vitro T-cell depletion. In MHC mismatched murine transplant models, lethal GVHD is reduced following the adoptive infusion of KIR ligand mismatched NK cells; it is unknown whether NK cells can mediate similar protective effects following MHC matched transplantation. Therefore, we investigated the impact of adoptively infusing KIR ligand mismatched NK cells on GVHD in an MHC matched T-cell replete murine model of allogeneic transplantation. Balb/C recipient mice underwent allogeneic bone marrow (8 x 106 cells) and splenocyte (15 x 106 cells) transplantation from B10.d2 donors following 950cGy of irradiation. Allogeneic B10.d2 donor NK cells were first isolated by negative depletion using magnetic beads selecting for CD4, CD5, CD8a, CD19, Gr-1 and Ter-119, and then expanded over 4-6 days in vitro in DMEM media containing 10% FCS and 500U/ml of IL-2. NK cell subsets (KIR ligand matched vs. KIR ligand mismatched) were then isolated by flow cytometry into Ly49I/C+ NK cells (KIR ligand mismatched in the GVHD direction for Balb/C recipients) and Ly49A/G+ NK cells (KIR ligand matched for Balb/C recipients). On day +4, recipient mice received a single tail vein injection with either KIR ligand matched, KIR ligand mismatched or unsorted “bulk” NK cells (0.5–1.0 x 106 NK cells). All (9/9) control transplant recipients (no adoptive NK cell infusion) as well as recipients of Ly49A/G (KIR ligand matched) NK cells (13/13) developed skin GVHD, in contrast to 4/7 (57%, p=0.03) recipients of bulk NK cells and only a minority (13% [1/8], p < 0.01) of animals receiving KIR ligand mismatched NK cells. Using a cumulative clinical GVHD scoring system (total score = 9), overall GVHD was decreased in recipients of KIR ligand mismatched NK cells (median score = 0 at day +45) compared to mice that received KIR ligand matched NK cells (median score = 3; p = 0.15) or no NK cells (median score = 3; p= 0.12); no significant difference in survival was observed between cohorts. This murine model provides the first in vivo evidence that adoptively infused KIR ligand mismatched allogeneic NK cells reduce GVHD following T-cell replete MHC matched allogeneic transplantation. The impact of infusing multiple doses of KIR ligand mismatched NK cells on GVHD and their ability to induce a graft-vs-tumor effect in tumor bearing Balb/c mice is currently being evaluated.
Lymphopenia-Associated CD4+ T Cell-Mediated Colitis Correlates with Homeostatic but Not with Endogenous Proliferation.
Authors: Blank Christian , Schuster Kerstin , Andreesen Reinhard , Mackensen Andreas
Published: April 2020

Transfer of peripheral T cells into lymphopenic hosts results in a burst-like proliferation, called endogenous proliferation (EP) and an antigen-independent proliferation, called homeostatic proliferation (HP). Formerly both were named lymphopenic or homeostatic proliferation. T cells that undergo either endogenous or homeostatic proliferation acquire an activated phenotype, develop the ability to produce Interferon-gamma (IFN-g) and to lyse target cells specifically, but only homeostatic proliferating T cells acquire a CD44bright CD62Lbright phenotype.

We developed a murine model to distinguish effects of both types of lymphopenia-associated proliferation. In this model we were able to induce polyclonal populations of syngenic CD4+ or CD8+ peripheral T cells (derived from C57BL/6 mice) to proliferate homeostatically (after transfer into lymphopenic RAG−/− mice) whereas only EP occurred when transferring into mice that inherit only an additional single CD8+ T cell clone (transfer into the non-lymphopenic P14/RAG−/− mice).

CFSE dilution was observed from CD4+ or CD8+ T cells transferred into RAG−/− mice, whereas HP was inhibited when transferring the same cells into P14/RAG−/− mice, whereas EP occurred in both recipients. Independent of CFSE dilution T cells developed CD44bright phenotype after transfer into RAG−/− as well as in P14/RAG−/− mice as early as day 5, whereas the host P14tg CD8+ clone kept its naïve phenotype.

Applying our model on the CD4+ T-cell-induced colitis model, we found HP to play a pivotal role in induction of colitis. Despite the notion of EP being triggered by antigens from the bowel, colitis was only induced when HP occurred, independent of the occurrence of EP. In detail CD4+ T cells induced colitis 10 weeks after transfer into RAG−/− mice (EP and HP). However neither RAG−/− mice transplanted with CD8+ T cells nor P14/RAG−/− mice transplanted with CD4+ T cells (only EP) developed any signs of colitis. In addition the treatment with or without antibiotics during these 10 weeks did not influence on the induction of colitis.

We therefore conclude that the lymphopenia-mediated colitis after transfer of polyclonal CD4+ T cells correlates only with the occurrence of homeostatic proliferation. Furthermore the absence of regulatory CD4+CD25+ T cells within the suppressing P14 tg CD8+ clone indicates HP-induced colitis to be mediated by competition for cytokines or self-MHC stimulus. These findings might be of important relevance for research concerning chronic inflammatory bowel disease and donor lymphocyte induced colitis.
Graft-Versus-Tumor Activity Against Renal Cell Carcinoma in a Mouse Model of HSCT.
Authors: Ramirez-Montagut Teresa , Chow Andrew , Kochman Adam A. , Hubbard Vanessa M. , Suh David , Smith Odette M. , Sindhi Hamad , Terwey Theis H. , Kim Theo D. , Waldman Elisha , Alpdogan Onder , van den Brink Marcel R.M.
Published: April 2020

Patients undergoing hematopoietic stem cell transplantation (HSCT) have demonstrated responses against several solid tumors including renal cell cancer (RCC) (Childs et al. NEJM 2000). Currently, there is no murine model available to study the role of graft-versus-tumor (GVT) activity in RCC rejection. Here we demonstrate GVT activity against the renal cell carcinoma line RENCA in a MHC class I/II disparate model of C57BL/6 into BALB/c. This line has been transduced with the luciferase gene (RENCA-TGL) that allows for bioluminescence imaging (BLI) of tumor burden. Lethally irradiated recipients of T-cell depleted marrow (TCD) recipients challenged with RENCA succumb rapidly to tumor death while addition of 0.3x10^6 donor CD8+ T cells significantly prolongs survival (median survival of 34 vs 72 days, P < 0.0001) and delays tumor progression (measured by BLI). Mortality in mice challenged with RENCA compared to RENCA-TGL is similar, demonstrating that rejection is not dependent on the immunogenicity of the transduced genes. Recipients of TCD-BM + CD8+ T cells without tumor cells survive and develop minimal graft-versus-host disease (GVHD), demonstrating that CD8+ T cells can mediate potent GVT activity without lethal GVHD activity. We used T cells from donor mice deficient in cytotoxic molecules including IFNγ, TNFα, perforin, and Fas ligand (FasL) to determine the mechanism of GVT activity and found that GVT activity against RENCA activity does not require TNFα or perforin but is dependent on IFNγ and FasL. We determined the clinical significance of our findings in a minimal residual disease model. Recipient mice were challenged with RENCA-TGL one week prior to HSCT to allow for seeding of lung micrometastases. Hosts receiving bone marrow and CD8+ T cells have significantly increased survival and delay in tumor progression compared to recipients of TCD-BM without T cells. These results demonstrate GVT activity in a model similar to the clinical presentation of patients diagnosed with RCC. In conclusion, CD8+ GVT activity against RCC is dependent on IFNγ and FasL and significantly increases host survival without lethal GVHD. Our results provide the first experimental mouse model to study strategies to enhance allogeneic HSCT in the treatment of RCC.
PedsQL™ and EuroQol Most Responsive Measures To Change in Health-Related Quality of Life in Children with Hodgkin’s Disease.
Authors: Klaassen Robert J. , Barrowman Nicholas J. , Barr Ronald D. , Krahn Murray , Hughes Joanna , Rogers Paul , Anderson Ronald , Grundy Paul , Ali S. Kaiser , Yanofsky Rochelle , Jardin Lawrence , Abla Oussama , Silva Mariana , Carret Anne-Sophie , Barnard Dorothy , Cappelli Mario
Published: April 2020

We evaluated four different health related quality of life measures to determine their ability to detect change over time. The four measures included the Health Utilities Index Mark 2 and 3 (HUI 2/3), the PedsQLTM 4.0 Generic Core and Cancer Module, the EuroQol and the Lansky Play - Performance Scale. Children with Hodgkin’s disease, their parents and the clinic nurse were all asked to complete the four measures at four time points: 2 weeks after the 1st course of chemotherapy, on the 3rd day of the 2nd course of chemotherapy, during the 3rd week of radiation and 1 year after diagnosis. At each follow up time point the respondents were asked to indicate whether the child’s HRQL had improved, stayed the same or became worse since the last measurement. 51 adolescents from 12 centres across Canada were enrolled in the study between May 1, 2002 and March 31, 2005. Two patients were excluded: one patient died shortly after the first time point, and the other patient failed to complete any of the questionnaires. The 49 patients included in the analysis had an average age of 14.7 years (8.9 - 17.9), with the two most common stages being IIA (39%) and IVA (18%). Complete data was available on 92% of patients at time 2, 89% at time 3 and 76% at time 4. Four of the 36 patients with complete follow up had relapsed (11%). The summary scores from the patients responses can be seen in the graph below.

Figure Figure

All measures showed a significant change between time 1 and time 4 (<0.05). When the change in child scores was analysed between the time points using the child’s self-reported change in HRQL, the PedsQL and the EuroQol showed significant change at all time points.

Change in Measures According to Reported Global Rating of Change by Patient Time Period HUI2 HUI3 PedsQL PedsQL cancer EuroQol Lansky Improved 1 to 2, n = 7 0.022 0.011 0.015 0.046 0.001 NS 2 to 3, n = 15 NS NS 0.028 0.039 0.017 NS 3 to 4, n = 20 NS NS 0.011 NS <0.001 <0.001 Same 1 to 2, n = 16 NS NS 0.033 NS NS NS 2 to 3, n = 9 NS NS 0.039 0.006 NS 0.028 3 to 4, n = 1 NS NS NS NS NS NS Worse 1 to 2, n = 6 NS NS NS NS 0.033 NS 2 to 3, n = 0 NS NS NS NS NS NS 3 to 4, n = 1 NS NS NS NS NS NS

All of the measures were able to detect change in a diverse group of children with Hodgkin’s Disease. The PedsQL and the EuroQol appeared to be the most sensitive to change.
Quality of Life (QOL) in Sickle Cell Disease (SCD).
Authors: Pakbaz Zahra , Richards Leah , Treadwell Marsha , Hackney-Stephens Ekua , Quirolo Keith , Vichinsky Elliott
Published: April 2020

Introduction Assessment of QOL is important for clinicians, as it will allow them to provide the patients with better care. The objective of this study is to assess the QOL in SCD patients. Methods A convenience sample of 34 patients with SCD (50% female) with a mean age of 21 years (SD= 9.4 y), completed a Dartmouth Care Cooperative Chart System (COOP) questionnaire (Table 1) during a comprehensive clinic visit at Children’s Hospital & Research Center in Oakland. High scores (patient ratings of 4 or 5) represent unfavorable levels of health or major limitations. A score of 3 represents moderate impairment while scores of 1 or 2 indicate no impairment. A brief chart review was completed to obtain medical history. Results 60% of the transfused patients (Tx) had severe impairment in at least one domain and this was the case in 71% of non-transfused patients (Non-Tx). As shown in table 2, the most affected domains were different between the two groups of Tx and non-Tx. In non-Tx patients the most affected domains were those of pain, overall health, and social support. In Tx patients, the most affected domains were pain, daily activities, and change in health. Although both groups had a similar amount of pain (non-Tx=42% and Tx=50%), Tx patients reported a better overall QOL (question 9). The most common complications that non-Tx patients suffered from were AVN (71%), vaso-occlusive episodes (46%), acute chest syndrome (46%), pneumonia (17%), reactive airways disease (17%), chest pain (13%), headaches (13%), and sepsis episodes (13%). Data showed that 30% of the Tx patients suffered from strokes previous to start of transfusion. The most common complications found in these patients were acute chest syndrome (30%), reactive airways disease (50%), urinary tract infections (20%), and depression (20%). Conclusions Both transfused and non-transfused SCD patients suffer from serious impairments in QOL. We recommend all SCD patients undergo QOL assessments and intervention which focuses on affected domains. Studies are needed to determine the impact of complications on QOL.

Table 1. COOP Questionnaire Q1 Physical Fitness Q2 Feelings Q3 Daily Activities Q4 Social Activities Q5 Pain Q6 Change in Health Q7 Overall Health Q8 Social Support Q9 Quality of Life Table 2. Frequency of moderate to severe impairment of domains Non-Tx Tx Score 3 (%) Score 4/5 (%) Score 3 (%) Score 4/5 (%) Q1 13 25 10 10 Q2 33 25 11 11 Q3 27 1 22 22 Q4 1 1 0 20 Q5 21 42 10 50 Q6 21 17 38 25 Q7 29 33 10 30 Q8 26 30 13 13 Q9 33 17 20 0
The Likelihood of Hematopoietic Stem Cell Transplantation (HCT) in the United States: Implications for Umbilical Cord Blood Storage.
Authors: Pasquini Marcelo C. , Logan Brent R. , Verter Frances , Horowitz Mary M. , Nietfeld J.J.
Published: April 2020

HCT effectively treats a variety of malignant diseases. Availability of suitable grafts limits application in some cases, prompting the establishment of banks to store umbilical cord blood for later personal or family use. The likelihood of utilizing stored cells depends, in part, on the likelihood of developing a condition for which HCT is indicated. This study estimates the latter likelihood based on data for current HCT use as reported to the CIBMTR from 2001 through 2003. First, age-related incidences of HCT were calculated; then, using the cumulative incidence function, we calculated the lifetime likelihood under two scenarios. In scenario 1, we calculated the likelihood of receiving an autologous HCT by age, in decades, from birth to 70 years. In scenario 2, we calculated the likelihood of being a candidate for either an autologous or an allogeneic HCT. This likelihood was derived from the number of HLA-identical sibling HCTs multiplied by three (to account for patients considered acceptable candidates but lacking an HLA-matched sibling donor) in addition to the number of autologous HCT. The number of HCT performed represented fewer than 20% of the malignant diagnoses most commonly treated with HCT, according to Surveillance, Epidemiology and End Results (SEER) data. After the second decade of life, the age-related incidences of HCT under both scenarios steadily increased with age. The cumulative incidences for scenario 1 ranged from 0.02% (at age 20) to 0.23% (at age 70); for scenario 2 they ranged from 0.06% (at age 20) to 0.46% (at age 70) (Figure). Given the current indications for HCT, the lifetime likelihood of undergoing an autologous HCT or being a candidate for HCT is about 1 in 400 and 1 in 200, respectively. How closely these estimates correspond to the likelihood that a stored cord blood unit is used depends upon several conditions including a) sufficient number of stem cells; b) satisfactory stem cell quality after the storage period; and, c) relative attractiveness, in particular clinical situations, of cord blood cells compared to other graft sources (e.g. peripheral blood or bone marrow).

Figure Figure
Reporting of Randomised Controlled Trials in Hodgkin’s Lymphoma from 1966 to 2002. A Pre- and after CONSORT Evaluation.
Authors: Kober Thilo , Trelle Sven , Greb Alex , Skoetz Nicole , Engert Andreas
Published: April 2020

Introduction/Background: Health professionals base treatment decisions primarily on scientific findings in the medical literature. Randomised controlled trials (RCTs) are considered the best tool to evaluate the applicability of clinical research but there is overwhelming evidence to indicate that the quality of reporting RCTs is less than optimal. The Consolidated Standards for Reporting Trials (CONSORT) statement was developed to help to improve the quality of reports of RCTs and published in 1996.

Methods and Material: Retrospective survey of reports of RCTs in Hodgkin’s lymphoma published in English and German between 1966 and 2002. Evaluation tool was a 14-item questionnaire based on the validated CONSORT checklist. Statistical analysis was performed by chi-square method and Fisher’s exact test where appropriate.

Results: The analysis of 243 published full-text articles in 59 journals from early 1966 to the end of 2002 indicates an improvement in the quality of reporting RCTs after CONSORT but this enrichment is not consistent. A highly significant enhancement post CONSORT was observed in 5 out of 14 reporting items (i.e. 35.7%; description of statistics methods, reporting of power calculation, ITT analysis, primary outcomes and 95% confidence interval (P=<.0005 – .002)). A sixth reporting item (adverse events) achieved borderline significance (P=.055). Intention-to-treat was explicitly mentioned in 11.9% (29/243), summary results for each comparative group was 98.4% (239/243) and precision of estimated effect size (95% confidence interval) were provided in 21.8% (53/243) respectively. Adverse events were reported in 77% (187/243), and conflict of interest was declared in 4.5% (11/243). Deviations in the reported versus performed randomised allocation in a subset of reports specifically mentioning ITT analysis (n=29) ranged from 2.5%–15%.

Conclusions: Despite years of educational efforts, reporting of randomised clinical trials for Hodgkin’s lymphoma in the scientific literature remains unsatisfactory and requires further concerted action by publishers, journal editors, learned societies and medical schools.
IL-31 Receptor and IL-31 in the Positive Regulation of Myeloid Progenitor Cell Proliferation/Survival.
Authors: Broxmeyer Hal E. , Ghilardi Nico , Hangoc Giao , Cooper Scott , Tao Wen , Mantel Charlie , Graham-Evans Barbara , deSauvage Frederic J.
Published: April 2020

Interleukin (IL)-31 receptor (R), also called gp130-like monocyte-receptor (GLM-R; Ghilardi et al. J. Biol. Chem.277:16831, 2002) is related to gp130 (~25% homology), and G-CSF-R (~24%). Its signaling activates STAT3 and STAT5. IL-31 is a four helix bundle cytokine preferentially produced by T helper 2 cells. Nothing is known of the possible hematopoietic effects of IL-31R and IL-31. However, since: IL-31 signals through a receptor composed of IL-31RA and Oncostatin M R (Dillon et al. Nature Immunol. 5:752,2004), Oncostatin M is implicated in homeostasis of myeloid progenitor cells (Broxmeyer et al. Immunity.16:815, 2002), and STAT3 and 5 are implicated by a number of groups in cytokine regulation of hematopoiesis, we hypothesized that the IL-31/IL-31R axis would be involved in regulation of hematopoiesis. We first compared myeloid progenitor cell (MPC: CFU-GM, BFU-E, and CFU-GEMM) numbers and cycling status in marrow and spleen of IL-31R −/− vs. littermate control mice (+/+) using a combination of Epo, SCF and PWMSCM to stimulate in vitro the cells taken from these mice. IL-31R −/− mice had significantly decreased numbers of MPC per femur (~51%) and spleen (~36–45%) with significantly decreased MPC cycling status in marrow (% MPC in S-phase: 0–3% in IL-31R −/− vs. 41–53% in +/+ mice). MPC in spleen of IL-31 −/− and +/+ were both in a slow or non cycling state (0–3% in S-phase). In contrast to CFU-GM from +/+ mice, CFU-GM from IL-31R −/− mice demonstrated little or no synergistic response to combined stimulation in vitro with GM-CSF or IL-3 with either SCF or Flt3-ligand (FL). This translated to decreased absolute numbers per femur of GM-CSF+SCF-, GM-CSF+FL-, IL-3+SCF-, and IL-3+FL- responsive CFU-GM in IL-31R −/− mice. However, there were no significant differences between GM-CSF- or IL-3- responsive CFU-GM per femur between IL-31R −/− vs. +/+ mice suggesting effects on immature subsets of MPC. Recombinant IL-31 was assessed for effects in vitro. IL-31, at concentrations up to 100ng/ml, did not stimulate colony formation by marrow MPC, nor did it enhance or suppress colony formation stimulated by GM-CSF, Epo, Epo+SCF, or Epo+SCF+GM-CSF. However, IL-31 did enhance survival of MPC subjected to delayed growth factor addition in a manner similar to, but not as potent as, that of SDF-1/CXCL12. IL-31 manifested no chemotaxis activity for +/+ MPC, and IL-31R −/− and +/+ MPC were equally responsive to the chemotactic effects of SDF-1/CXCL12. These results suggest that the IL-31R in vivo acts to positively regulate numbers and cycling of immature subsets of MPC in the marrow, and that IL-31 has survival enhancing effects on MPC in vitro.
Cell Cycle Regulation and Cell Fate Decisions in Hematopoietic Stem Cells.
Authors: Passegue Emmanuelle , Wagers Amy J. , Giuriato Sylvie , Anderson Wade C. , Weissman Irving L.
Published: April 2020

The blood is a perpetually renewing tissue seeded by a rare population of adult bone marrow hematopoietic stem cells (HSC). During steady-state hematopoiesis, the HSC population is relatively quiescent but constantly maintains a low numbers of cycling cells that differentiate to produce the various lineage of mature blood cells. However, in response to hematological stress, the entire HSC population can be recruited into cycle to self-renew and regenerate the blood-forming system. HSC proliferation is therefore highly adaptative and requires appropriate regulation of cell cycle progression to drive both differentiation-associated and self-renewal-associated proliferation, without depletion of the stem cell pool. Although the molecular events controlling HSC proliferation are still poorly understood, they are likely determined, at least in part, by regulated expression and/or function of components and regulators of the cell cycle machinery.

Here, we demonstrate that the long-term self-renewing HSC (defined as Lin−/c-Kit+/Sca-1+/Thy1.1int/Flk2−) exists in two distinct states that are both equally important for their in vivo functions as stem cells: a numerically dominant quiescent state, which is critical for HSC function in hematopoietic reconstitution; and a proliferative state, which represents almost a fourth of this population and is essential for HSC functions in differentiation and self-renewal. We show that when HSC exit quiescence and enter G1 as a prelude to cell division, at least two critical events occur: first, during the G1 and subsequent S-G2/M phases, they temporarily lose efficient in vivo engraftment activity, while retaining in vitro differentiation potential; and second, they select the particular cell cycle proteins that are associated with specific developmental outcomes (self-renewal vs. differentiation) and developmental fates (myeloid vs. lymphoid). Together, these findings provide a direct link between HSC proliferation, cell cycle regulation and cell fate decisions that have critical implications for both the therapeutic use of HSC and the understanding of leukemic transformation.