bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclin D3 Is Required for Peritoneal B-1a Lymphocyte Proliferation.
Authors: Mataraza Jennifer M. , Tumang Joseph R. , Rothstein Thomas L. , Chiles Thomas C.
Published: April 2020

Cyclins D2 and D3 are coordinately induced in response to mitogenic stimulation of splenic B-2 lymphocytes. It is recognized that the cyclin D2:cdk4:pRb pathway plays an essential role in B-2 cell proliferation. By contrast, little is known about the function of cyclin D3 in B cells. Peritoneal CD5+ B-1 cells represent a distinct subset of B lymphocytes and exhibit several unique phenotypic and functional characteristics, which distinguish them from B-2 cells. B-1 cells are associated with several human disease states characterized by B-cell expansion. Notably, B-1 cells represent the normal counterpart for a subset of human chronic lymphocytic leukemia (CLL), the cells of which express CD5. In mice, monoclonal expansions of CD5+ B-1 cells, that resemble CLL, develop as a function of age. These associations with malignant diseases impel the necessity to understand cell cycle control in B-1 cells. In contrast to splenic B-2 cells, stimulation of ex vivo B-1 cells results in an early and transient induction of cyclin D2; however, cyclin D2 complexes account for a relatively minor amount of the total pRb phosphorylation. Evidence for a second cyclin functioning in this capacity is supported by the finding that stimulated cdk4/6-mediated phosphorylation of pRb increases dramatically in mid-to-late G1-phase coincident with the induction of cyclin D3. Thus, in contrast to B-2 cells, the non-overlapping expression of cyclins D2 and D3 points to distinct roles for these D-type cyclins in B-1 cells. We hypothesize that the timing and duration of cyclin D3 induction suggest a unique role in mediating the G1/S transition in B-1 cells. To investigate this possibility, we transduced TAT-p16 peptidyl mimetics into ex vivo B-1 cells immediately prior to cyclin D3 induction to specifically target and inactivate cyclin D3-cdk complexes. We report here that transduction of TAT-p16 mimetics selectively disrupted cyclin D3-cdk4 complexes, whereas cdk2-containing complexes remained intact. Transduction of wild-type TAT-p16 mimetics, but not mutant TAT-p16 mimetics blocked S-phase entry in response to several B-cell mitogens. In addition, we found that transduction of TAT-p16 peptidyl mimetics inhibited pRb phosphorylation by D-type cyclin-cdk4/6 holoenzymes in B-1 cells stimulated with PMA, LPS or CD40L. These results provide the first evidence of a subset-specific functional role for cyclin D3 in B cells and suggest that cyclin D3, in its capacity as a regulator of cdk4/6-mediated pRb phosphorylation, is required for B-1 cell proliferation.
CUL-4A Targets p27 for Degradation and Regulates Proliferation, Cell Cycle Exit, and Differentiation during Erythropoiesis.
Authors: Chun Kristin T. , Jia Nan , Naaldjik Yahaira M. , Kapur Reuben , Li Binghui
Published: April 2020

As erythroid progenitors differentiate into precursors and finally mature red blood cells, they induce the expression of lineage-specific genes, and their proliferation declines until cell cycle exit. CUL-4A encodes a core subunit of a ubiquitin ligase that targets proteins for ubiquitin-mediated degradation, and CUL-4A haploinsufficient mice display hematopoietic dysregulation with fewer multipotential and erythroid committed progenitors (3-fold and 5-fold, respectively). In this study, stress induced by 5-fluorouracil or phenylhydrazine reveals a delay in the ability of CUL-4A +/− mice to recover erythroid progenitors and precursors and to reestablish normal hematocrits. Conversely, over-expression of CUL-4A in a growth factor-dependent, proerythroblast cell line increases proliferation 34% (tritiated thymidine incorporation) and the proportion of cells in S-phase 5%. When these cells are induced to terminally differentiate, endogenous CUL-4A protein expression declines 3.6-fold. Its enforced expression interferes with erythrocyte maturation (beta-globin induction) and cell cycle exit, and instead promotes proliferation. Furthermore, p27 accumulates during erythroid terminal differentiation, but CUL-4A enforced expression increases p27 protein turnover nearly 7-fold and attenuates its accumulation. CUL-4A and p27 proteins co-immunoprecipitate, indicating that a Cul-4A ubiquitin ligase targets p27 for degradation. These findings suggest that a Cul-4A ubiquitin ligase positively regulates proliferation by targeting p27 for degradation and that CUL-4A down-regulation during erythroid terminal differentiation allows p27 to accumulate and signal cell cycle exit.
Induction of Hematopoietic Stem Cell Senescence by Ionizing Radiation.
Authors: Wang Yong , Schulte Bradley A. , LaRue Amanda C. , Ogawa Makio , Zhou Daohong
Published: April 2020

Exposure to ionizing radiation (IR) and certain chemotherapeutic agents not only causes acute bone marrow (BM) suppression but also leads to long-term residual hematopoietic injury. This later effect has been attributed to the damage to hematopoietic stem cell (HSC) self-renewal. Using a mouse model, we investigated whether IR induces senescence in HSCs, as induction of HSC senescence can lead to the impairment of HSC self-renewal. The results showed that exposure of C57BL/6 mice to a sublethal dose (6.5 Gy) of total body irradiation (TBI) resulted in a long-lasting quantitative and qualitative reduction in HSCs (Lin− c-kit+ Sca-1+ or LKS+ cells). Compared to control HSCs, HSCs from irradiated BM at 4 weeks after TBI exhibited a significant reduction in day-35 CAFC frequency and deficiency in cell proliferation and colony formation in a single cell culture assay stimulated with SCF/TPO and SCF/TPO/IL-3, respectively. In addition, transplantation of irradiated HSCs (500 LKS+ cells/recipient) produced less than 1% long-term (2-month) engraftment in a competitive repopulation assay while transplantation of the same number of control HSCs resulted in 24.8% engraftment. Furthermore, HSCs from irradiated mice expressed increased levels of p16Ink4a and senescence-associated beta-galactosidase (SA-beta-gal), two commonly used biomarkers of cellular senescence. In contrast, hematopoietic progenitor cells (Lin− c-kit+ Sca-1− or LKS− cells) from irradiated mice did not show significant changes in clonogenesity in a CFU assay and expressed minimal levels of p16Ink4a and SA-beta-gal. These results suggest that exposure to IR can induce senescence selectively in HSCs but not in HPCs. Interestingly, this IR- induced HSC senescence was associated with a prolonged elevation of p21Cip1/Waf1, p16Ink4a and p19ARF mRNA expression, whereas the expression of p27Kip1, p18Ink4c and p19 Ink4d mRNA was not increased. This suggests that p21Cip1/Waf1, p16Ink4a and p19ARF may play an important role in IR-induced senescence in HSCs, since their expression has been implicated in the initiation, establishment and maintenance of cellular senescence. Therefore, these findings provide valuable insights into the mechanisms underlying IR-induced long-term BM damage. This could lead to the discovery of novel molecular targets for intervention to circumvent IR-induced BM toxicity. In addition, understanding how normal HSCs senesce after IR and chemotherapy will help us to elucidate the molecular mechanisms whereby leukemia/cancer stem cells evade these cancer treatments and provide better knowledge of organismal aging.
Bad Phosphorylation Is Reduced in Response to Inhibition of Constitutively Activated FLT3.
Authors: Kim Kyu-Tae , Levis Mark , Small Donald
Published: April 2020

Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 play an important role in leukemogenesis and their presence is associated with poor prognosis in acute myeloid leukemia (AML). Previously we showed that human leukemia-derived myeloid cell lines having constitutive activation of FLT3 undergo apoptotic cell death when treated with FLT3 inhibitors. Examining the anti- and pro-apoptotic proteins in constitutively activated FLT3 signaling in BaF3/ITD, MOLM-14 and MV4-11 cell lines, we found that the level of Bad phosphorylation was greatly decreased by FLT3 inhibitor treatment. Bad is a proapoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death. Survival factors such as IL-3 inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136. Both Ser112 and Ser136 are rapidly dephosphorylated after treatment with FLT3 inhibitors in BaF3/ITD, MOLM-14, and MV4-11 cells. We also found Bad to be highly phosphorylated in both wild-type and mutant FLT3 primary AML samples. In confirmation of the cell line data, Bad was rapidly dephosphorylated after treatment of the primary samples with lestaurtinib (CEP-701). Upstream molecules responsible for phosphorylation of Bad include Akt, Erk/MAPK, Pim-1, and Pim-2. We previously reported Pim-1 as one of the genes whose expression is induced by constitutively activated FLT3. We and other groups have also shown that constitutively activated FLT3 induces multiple signaling pathways, including PI3/Akt, Erk/MAPK, and Jak/STAT. The dephosphorylation of Bad in response to FLT3 inhibition also correlates with deactivation of Stat5, MEK1/2 and Akt kinase activities and decreased expression of Pim-1. Thus, Bad may be one of the points that these multiple signaling pathways converge in FLT3-mediated cell survival. Thus, it appears that activated FLT3 phosphorylates Bad, resulting in the inability of Bad to bind to Bcl-xL, therefore blocking apoptosis. This is the first report of deactivation of a pro-apoptotic Bcl-2 family member induced by constitutively activated FLT3.
Stem Cell Mobilization and Egress of AML Blasts from the Bone Marrow Are Both Regulated by MT-MMP-Mediated Activation of MMP-2.
Authors: Marquez-Curtis Leah A. , Shirvaikar Neeta , Larratt Loree M. , Turner A. Robert , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna
Published: April 2020

The cell-surface proteolytic enzymes membrane-type (MT)-matrix metalloproteinases (MMPs) activate secreted latent forms of MMPs and play a key role in cell migration and tumor cell invasion and metastasis. Previously we reported that mobilized peripheral blood (mPB) CD34+ cells as well as AML blasts secrete inactive (pro)MMP-2, in contrast to normal steady-state bone marrow (BM) CD34+ cells which do not. In this study we hypothesized that the egress from BM of normal hematopoietic stem/progenitor cells during G-CSF-induced mobilization or of AML blasts shares a common mechanism that involves activation of secreted proMMP-2 by MT-MMPs. We evaluated the expression of MT-MMPs (MT1-MMP, MT2-, MT3- MT4-, MT5-, MT6-) in normal and leukemic hematopoietic cells, as well as in BM stromal cells, and determined their role in proMMP-2 activation and migration. We found MT1-MMP expression (mRNA and protein) in mPB CD34+ cells and in the majority of AML samples (from 21 out of 26 patients) and upregulation of MT1-MMP by G-CSF in steady-state BM CD34+ cells. Moreover, we confirmed the secretion of proMMP-2 in media conditioned by hematopoietic (mPB CD34+, AML) cells and stromal cells. However, we detected the active form of MMP-2 in co-cultures of the hematopoietic (mPB CD34+, AML) cells with stromal cells, as well as in co-cultures of steady-state BM CD34+ cells stimulated with G-CSF with stroma. In cultures of stromal cells alone G-CSF had no effect on the expression of either MT1-MMP or MMP-2. To evaluate the role of MT-MMPs in proMMP activation, we examined the leukemic KG-1 cell line, which we found not to express any MT-MMPs, and did not detect active MMP-2 in co-cultures with stroma. On the other hand, primary AML samples that did not express MT1-MMP but expressed MT2-, MT4- or MT5-MMPs activated proMMP-2 in co-cultures with stroma. Moreover, the MT1-MMP inhibitor epigallocatechin-3-gallate significantly reduced trans-Matrigel migration of mPB CD34+ (by 50%) and AML cells (by 70–90%). Hence we suggest that in the BM microenvironment (i) MT1-MMP localized on the surface of hematopoietic cells activates MMP-2, inducing a highly proteolytic microenvironment, and (ii) MT1-MMP upregulation by G-CSF in BM CD34+ cells can result in CD34+ cell mobilization from BM. Similarly, the constitutive high expression of MT1-MMP and other MT-MMPs in AML blasts also contributes to MMP-2 activation in the BM microenvironment and may be conducive to the egress of AML blasts from the BM.
Inhibitory and Proliferation-Inducing Effects of Valproic Acid on Leukemic Stem Cells from Different Acute Myeloid Leukemias.
Authors: Bug Gesine , Zheng Xiaomin , Schoch Claudia , Ruster Brigitte , Schwarz Kerstin , Kampfmann Manuela , Hoelzer Dieter , Ottmann Oliver G. , Henschler Reinhard , Ruthardt Martin
Published: April 2020

The antileukemic activity of the histone deacetylase inhibitor valproic acid (VPA) in combination with all-trans retinoic acid and/or low-dose chemotherapy is currently evaluated in clinical trials for the treatment of acute myeloid leukemia (AML). Preliminary data demonstrate that only a small subset of patients responded to these VPA-containing regimens and none of them achieved a molecular remission (Bug et al., Cancer 2005, in press). Recently, it has been shown that the in vitro exposure of normal hematopoietic stem cells (HSC) to VPA significantly increases their proliferation and self-renewal potential. Given the fact that normal and leukemic stem cells are believed to share regulatory mechanisms of proliferation and self-renewal, we investigated whether VPA can also stimulate leukemic stem cells.

First we confirmed the expansion of normal HSC in vivo by treating C57BL/6J mice with VPA for two days. Here we report that orally administered VPA led toa significant increase in the cellularity of both bone marrow (BM) and spleen;a significant increase of Sca1+/c-Kit+ cell numbers in BM and spleen;a slight splenomegaly (0.11–0.42 g vs. 0.06–0.08 g in control mice);a significant acceleration of cell cycle progression in spleen cells.

Next we exposed highly enriched CD34+/CD38− cells (average purity of 84.2+/−1.6%) from the peripheral blood of 6 consecutive AML patients at first diagnosis to VPA (100 μg/ml) in presence of SCF, FL, TPO and IL-3 in liquid culture as well as in colony forming unit (CFU) assay. Chromosomal aberrations were detected in 5 out of 6 AML samples. Here we show that i.) the exposure to VPA inhibited differentiation and maintained a higher level of CD34+/CD38− cells in liquid culture at day 7 and 14 as compared to untreated controls in all samples; ii.) the majority of CD34+ cells were of leukemic origin as revealed by fluorescence in situ hybridization (FISH) analysis of 4 out of 5 AML samples with chromosomal aberrations; iii.) in one t(8;21)-positive AML sample VPA reduced the proportion of leukemic CD34+ cells to 60% and 17% on day 7 and 14, respectively, as compared to controls (92% and 86%); iv.) in one trisomy 8-positive AML sample VPA induced expansion of leukemic CD34+ cells; v.) the exposure to VPA did not only increase the number of CFUs, but also the cellularity of single colonies which exhibited an immature morphology and whose leukemic origin was confirmed by FISH analysis of one AML sample.

In conclusion, VPA was shown to have differential effects on CD34+/CD38- cells from untreated AML patients ranging from a selective inhibition of t(8;21)-positive cells, over a stabilization of the leukemic stem cell compartment, up to the amplification of trisomy 8-positive cells. Therefore, the inclusion of VPA into regimens for AML treatment has to be reconsidered.
Hematopoietic Progenitor Cell Response to SDF-1 alpha and SDF-1 alpha Production Are Impaired in Chronic Phase Chronic Myeloid Leukemia at Diagnosis.
Authors: Prebet Thomas , Chabane Kaddour , Tigaud Isabelle , Wattel Eric , Michallet Mauricette , Nicolini Franck E.
Published: April 2020

SDF-1 alpha and its cognate receptor CXCR-4 are involved in normal Hematopoietic Progenitor Cells (HPC) regulation of adhesion, migration and survival. In Chronic Phase (CP) Chronic Myeloid leukaemia (CML), HPC exhibit a deregulation of all of those phenomena. Here, we investigated by different ways, the involvement of SDF-1 alpha in the pathogenesis of CP CML. Samples of bone marrow (BM) and peripheral blood (PB) from normal donors (ND) (n=10), CP CML (100% Ph1+ metaphases, all selected for Ph1+ LTC-IC) patients at diagnosis (n=35) and CP CML patients in Complete Cytogenetic Response (CCR) (0% Ph1+) (n=10) were explored. First, SDF-1 alpha concentrations were determined by ELISA assays in PB and BM samples, and showed higher yields for CP CML PB (2060±700 pg/ml) compared to CCR PB [1570±300 pg/ml (p=0.002)] and lower concentrations for CP CML BM (3080±1015 pg/ml) compared to CML in CCR BM [3990±680 pg/ml (p=0.01)] and ND BM [4060±1020 pg/ml (p=0.03)]. CCR BM and PB, and ND BM and PB SDF-1 alpha concentrations were not different. Further, we analyzed CD34+Lin− HPC migration in a 4h-Transwell™ assay, in SFM conditions ± SDF-1 alpha or ± one of its specific inhibitors SDF-1(G2) vs control. After migration, median percentages of migrating cells were consistently higher for CML vs ND BM (32±9 vs 20±1, p=0.002) in control arm, but equivalent for CML samples with SDF-1 alpha (39±10) vs control (32±9) indicating a relative insensitivity of CML HPC to this chemokine. In addition, SDF-1(G2) significantly inhibited the migration of CML HPCs (28±7) vs SDF-1 alpha (p=0.01). The proportion of CFC and LTC-IC in the migrating fraction was equivalent in the 3 experimental arms (SDF-1 alpha vs SDF-1(G2) vs control). As assessed by FACS, CD34+Lin− cells CXCR-4 surface expression was equivalent for CP CML (31±2.3%) and ND BM (43±2%) before migration, and there was no modification of the CXCR-4 expression after migration for CP CML. As SDF-1 alpha is involved in the regulation of the proliferation of normal HPC, we assessed CP CML CD34+Lin− cells in 3H-Thymidine cycling assays after 14 days culture on M210B4 murine feeder cell line (producing residual concentrations of SDF-1 alpha). CP CML HPC (CFC and LTC-IC) cycling status was not modified either after SDF-1 alpha or SDF-1(G2) exposure whereas ND BM cycling was paradoxically up-regulated by SDF-1(G2) vs control, suggesting a specific functional defect of CP CML HPCs in response to SDF-1 alpha. Taken together, all these results suggest that SDF-1 alpha production by stromal cells is impaired in CP CML, and that migratory and proliferative responses of CP CML HPCs to SDF-1 alpha are likely to be perturbed. This results might illustrate some pathologic interactions between BCR-ABL and signal transduction pathways activated through CXCR-4 ligation to its sole ligand SDF-1 alpha.
Cell Adhesion and Migration Regulated by EphB6 Expressed on Human Hematopoietic Progenitors.
Authors: Matsui Toshimitsu , Matsuoka Hiroshi , Tamekane Akira , Inoue Ryuichi , Shimoyama Manabu , Okamura Atsushi , Obama Hiroya , Kelly Meghan L. , Nakamoto Masaru
Published: April 2020

Normal human hematopoietic progenitors as well as leukemia/lymphoma cells express kinase-defective EphB6 receptors. The only unique high affinity ligand for EphB6 among eight known mammalian ephrins, ephrins-B2 is expressed not only on hematopoietic malignancies, but also on mesenchymal stem cells. However, the biological functions of the receptor and its ligand in hematopoietic cells are largely unknown.

In the present study, we showed that the interaction between EphB6 and ephrinB2 could initiate forward as well as reverse signaling in vitro. Both pre-clustered and unclustered ligands could trigger the signal transduction, but pre-clustered ones more rapidly down-regulated the signaling. We also examined the EphB6/ephrinB2 function in cell adhesion and migration.

Figure Figure

HEK-EphB6 cells placed in the upper chamber of a Transwell apparatus, in which the lower side of filter was coated with different concentrations of ephrin-B2-Fc or Fc, were allowed to migrate to the lower side at 37°C overnight. Vector-transfected cells were used as controls. The cells that had migrated to the lower side of filter were stained, photographed. A BSA-coated filter is shown as a control. EphB6 exerted biphasic effects in response to different concentrations of the ephrin-B2. EphB6 promoted cell adhesion and migration when stimulated with low concentrations of ephrin-B2, whereas it induced repulsion and inhibited migration upon stimulation with high concentrations of ephrin-B2. A truncated EphB6 receptor lacking the cytoplasmic domain showed monophasic positive effects on cell adhesion and migration, indicating that the cytoplasmic domain is essential for the negative effects. We further explored the signal transduction of the biphasic effects.

Figure Figure

The Src family kinase, Fyn was co-immunoprecipitated with anti-EphB6 antibody in the absence or presence of ephrin-B2 stimulation. High concentrations of ephrin-B2 induced tyrosine phosphorylation of EphB6 through a Src family kinase activity. These results indicate that EphB6 can both positively and negatively regulate cell adhesion and migration, and suggest that tyrosine phosphorylation of the kinase-defective EphB6 receptor by a Src family kinase acts as the molecular switch for the functional transition.

Thus, EphB6 expressed on hematopoietic cells may play an important role in the regulation of cell homing to hematopoietic tissues as well as leukemia cell infiltration.
Abnormal Bone Marrow-Derived Mesenchymal Stem Cell: Another Clone in Hematological Malignancy?.
Authors: Yeh Su-Peng , Chiu Chang-Fang , Liao Yu-Min , Lin Chiao-Lin , Lo Wen-Jyi , Lee Cheng-Chun
Published: April 2020

The growth, differentiation, and survival of normal hematopoietic stem cells as well as bone marrow (BM) cancer cells are regulated closely by the BM microenvironment. In case of lymphoma, recent study has demonstrated that the endothelial cells (not lymphoma cells) in the lymphoma microenvironment have the same chromosomal abnormality to that of lymphoma cells themselves. Besides, in our prior study, the mesenchymal stem cells (MSCs) isolated from BM of patients with various hematological malignancies may have different surface antigens expression as comparing to normal control (Leukemia, 2005, doi:10.1038/sj.leu.2403795, published online 19 May 2005). We therefore hypothesize that the MSCs isolated from BM of patients with hematological malignancies are “different” to those of normal and may probably has the same chromosomal abnormality as to that of the hematological cancer cells. In this study, cytogenetic studies were performed on both BM-blood cells and BM-derived MSCs at the same time by using method of conventional G-banding. At submission of this abstract, we have just examined 4 specimens obtained from patients of various diseases as well as normal adult (see table). Surprisingly, we identified a new chromosomal translocation, t(3;9)(q25;q31), on the BM-derived MSCs of a patient (Case 1 of the table, Figure 1) with acute promyelocytic leukemia harboring t(15;17) on the BM blood cells. Besides, a complex chromosomal abnormalities was found in a male patient of AML (Case 2 of the table, Figure 2). Though he has received a successful allotransplant 2 years ago and the BM blood cells had completely converted to donor type (normal 46 XX), the BM-derived MSCs were still recipient in origin and having a chromosomal abnormality that was different to the BM blood cells (45, -Y) at initial diagnosis of AML. BM-derived MSCs from a patient of CML and normal adult were normal. It is still too early to making a solid conclusion at present and we are now analyzing more specimens to look into the phenomenon we found. However, this is a new field in leukemic research that deserved further investigation and our novel finding indicates that abnormal cells (another clone?) may present in the BM stroma of patients with hematological malignancies.

Chromosomes of BM-blood cells and MSCs Case No. Diseases Chromosomes of BM-blood cells Chromosomes of BM-MSCs Case 1 APL, at diagnosis 46 XX, t(15;17)(q21;q11) 46 XX, t(3;9)(q25;q31). (see figure 1) Case 2 AML (45, -Y at diagnosis), after allotransplant Normal 46 XX (male patient, female donor) 46 XY, t(7;22)(p22;q11); del(13)(q12q22); del(15)(q15?). (see figure 2) Case 3 CML, at diagnosis 46 XY, t(9;22)(q34;q11) Normal 46 XY Case 4 Normal adult Normal 46 XY Normal 46 XY

Figure Figure
Molecular Characterization of Unique Junctional Complexes as Communication Pathways among Mesenchymal Stem Cells.
Authors: Wuchter Patrick , Boda-Heggemann Judit , Straub Beate , Krause Ulf , Seckinger Anja , Kuhn Caecilia , Grund Christine , Gottschling Sandra , Franke Werner W. , Ho Anthony D.
Published: April 2020

The interaction between stem cells and their niche is essential for the balance between self-renewal and differentiation. Mesenchymal stem cells (MSC) from bone marrow are able to differentiate into various tissues, such as bone, cartilage, muscle and fat. We have characterized the intercellular contacts among MSC and previously demonstrated the occurrence of a novel kind of adhering junction, consisting of villiform-to-vermiform cell projections (processus adhaerentes). The molecular compositions of these junctions have now been elucidated. Using a panel of antibodies specific for various components of tight, gap and adhering junctions (the latter comprising desmosomes and adhering junctions), we systematically analyzed the junctional complexes of MSC obtained from bone marrow aspirates from healthy voluntary donors and compared the data to bone marrow tissue in situ. Light and electron microscopy was used and several biochemical analyses were applied, including immunoprecipitation and RT-PCR. We could show that intercellular connections in MSC are realized in vitro by occasional gap junctions and frequent adhering junctions. Intercellular projections are noted, often deeply intruding into the neighbouring cell. These findings have been confirmed in situ in bone marrow biopsy material. Furthermore, we analyzed the molecular composition of these junctions and cell-projections. In most of them, we found the transmembrane glycoproteins cadherin 11 and N-cadherin, together with the cytoplasmic plaque proteins alpha- and beta-catenin and protein p120ctn. Interestingly, similar junctions had been already noted in primary mesenchymal stem cells of day 7 to 8.5 mouse embryos. Our hypothesis is that this junction type might be a prerequisite for early mesenchymal cells. Our findings indicate that intercellular communication in MSC in human marrow is realized through a unique type of junctional complexes not described before. These structures are crucial for the long-term fate and differentiation of the cells and might have significant impact on the interaction with hematopoietic stem cells.
Two Cellular Components of Bone Marrow Origin Contribute to Pulmonary Irradiation Fibrosis.
Authors: Greenberger Joel S. , Niu Yunyun , Zhang Xichen , Cao Shaonan , Carlos Timothy , Epperly Michael W.
Published: April 2020

Following lung irradiation of C57BL/6NHsd mice, migration of marrow origin macrophages into the lungs is detectable at 100 days followed by migration of marrow origin fibroblasts into areas of developing organizing alveolitis/fibrosis. To demonstrate the importance of each cellular migration, mice were irradiated to 20 Gy to the pulmonary cavity. Beginning at day 80, the mice were injected (daily for 15 days I.P.) with antibodies to either mouse macrophages or to a pan-T-cell antigen (M1/70 or TIB-207, respectively). Groups of mice were injected at day 80 with DS-red labeled Smad3+/+ clonal bone marrow stromal cell line cells or a GFP+ Smad3−/− bone marrow stromal cell line. The mice were then followed for survival. Mice injected with the M1/70 antibody had increased survival compared to those receiving TIB-207 or control irradiated mice (percent survival at 180 days 90% and 60% compared to 40%, p=0.0225 and 0.8263). There was a decrease in the number of macrophages in the lungs of mice injected with M1/70 compared to TIB-207 injected or control irradiated mice. Mice injected with bone marrow stromal cells from the Smad3−/− line, which have abrogation of the TGF-β signaling pathway, showed decreased numbers of labeled cells in areas of organizing alveolitis/fibrosis compared to mice that were injected with Smad3+/+ cells at 120 days (5.4 ± 1.6% vs. 21.0 ± 3.3% cells per 100x high powered field in areas of organizing alveolitis/fibrosis, respectively, p = 0.0003). These results demonstrate a critical importance of both marrow hematopoietic (macrophage) and stromal fibroblasts to the development of irradiation-induced pulmonary fibrosis.
A ComParison of Retroviral Integration Sites in Donor T-Lymphocytes In Vivo and In Vitro.
Authors: Giordano Frank A. , Laufs Stephanie , Fehse Boris , Nagy K. Zsuzsanna , Hotz-Wagenblatt Agnes , Lauterborn Daniel , Fellenberg Kurt , Kuehlcke Klaus , Naundorf Sonja , Zander Axel R. , Zeller W. Jens , Fruehauf Stefan
Published: April 2020

Genetically modified T-Lymphocytes (TLCs) have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused TLCs have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD).

Yet, reports on insertional mutagenesis in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development.

We investigated retroviral integration sites in vitro and in vivo. Therefore, donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN for a clinical HSV-Tk study were examined. TLCs of four different donors as well as whole blood samples of two patients transplanted with donor TLCs were analyzed either using highly sensitive and specific ligation-mediated PCR (LM-PCR).

A total of 114 retroviral integration sites were detected in vitro. 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31).

Current analyses are focusing at the in vivo pattern of retroviral integration in DNA of TLCs obtained from transplanted patient’s TLCs. Therefore we developed a new high sensitive PCR method (HS-PCR), an improved LM-PCR to even detect minimal quantities of transduced DNA.
Impact of Dendritic Cell CD16+ Recovery on Outcome after Reduced-Intensity Conditioning Allogeneic Stem Cell Transplantation.
Authors: Talarn Carme , Urbano-Ispizua Alvaro , Martino Rodrigo , Batlle Montse , Herrera Concha , Perez-Simon Jose Antonio , Torrebadell Montse , Fernandez-Aviles Francesc , Gaya Anna , Granell Miquel , Marin Pedro , Sierra Jordi , Montserrat Emili
Published: April 2020

Dendritic cells (DCs) play a critical role in the regulation of alloimmune responses. Therefore, the number and function of these cells after allogeneic stem cell transplantation (allo-SCT) might influence patients’ outcome, an aspect scarcely investigated, particularly in the setting of reduced-intensity conditioning transplants (allo-RIC). Against this background, we studied DCs recovery in 92 patients (median age 50, range 17–67; male 57%), undergoing allo-RIC from HLA identical donors in five Spanish institutions between October 2002 and December 2004. Median follow up was 250 days (range, 25–708). Conditioning regimen consisted on fludarabine 150 mg/m2 + melphalan 140 mg/m2 for lymphoid malignancies (n=54), and fludarabine 150 mg/m2 + busulphan 10 mg/kg for myeloid malignancies (n=38); 71 (90%) patients had advanced disease. Peripheral blood samples were obtained at 1, 3, 6 and 12 months after transplant. DCs were identified as positive for HLA-DR and negative for lineage markers (CD3, CD14, CD19, and CD56). The expression of CD33, CD123 and CD16 was used to identify DC1, DC2 and DC CD16+ subsets, respectively; the immunophenotypic analysis being centralized at a single institution. The most significant association with clinical outcome was found with DC CD16+ levels at three months after transplantantation. Thus, at that time point, patients with a DC CD16+ count lower than the median (<1.5×104/mL) had a significantly higher incidence of relapse (59% vs. 45%; p=0.04), more transplant related mortality (30% vs. 0%; p=0.01), worse event free survival (EFS) (24% vs. 51%; p=0.002) and overall survival (0% vs. 55%; p=0.002). Only two factors were associated with worse EFS in the multivariate analysis: autologous transplantation before allo-RIC (RR 6.5, 95%CI 1.6–26.2; p=0.008), and DC CD16+ count lower than the median at +3m (RR 13.1; 95% CI 1.7–100.5; p=0.01). In conclusion, this study shows that besides a well-known poor prognostic factor (i.e., prior autologous transplantation) a low DC CD16+ count at three months after allo-RIC is associated with a poorer outcome, this probably reflecting impaired immunosurveillance mechanisms.
An Analysis of Angiotensin Converting Enzyme Gene Polymorphisms and Non-Infectious Pulmonary Dysfunction after Hematopoietic Stem Cell Transplantation.
Authors: Onizuka Makoto , Ando Kiyoshi , Kishi Kengi , Kawada Hiroshi , Morishima Yasuo , Hotta Tomomitsu , Inoko Hidetoshi
Published: April 2020

Non-infectious pulmonary dysfunction (NIPD) is a common but often fatal complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). An insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene has extensively been studied in relation to pulmonary fibrosis, and the D-allele frequency was reported to be significantly higher in patients with pulmonary fibrosis than in the control. We have previously found that increased D-allele frequency is also associated with NIPD after HSCT from HLA haplo-identical sibling donor. In this study, we performed a retrospective analysis to assess the relation between ACE I/D polymorphism and unrelated donor HSCT. 772 patients were enrolled in this study (461 males and 311 females, median age 23; range 0 to 52). The underlying diseases were 229 ALL, 188 ANLL, 168 CML, 66 MDS, 74 aplastic anemia, 24 lymphoma, 12 inherited disease and an other disease. DNA samples and clinical information were obtained from the Japan Marrow Donor Program registry. Determination of the ACE genotype was accomplished by polymerase chain reaction. NIPD occurred in 73 patients (9.5%). The D allele frequency and the D/D genotype frequency were not significantly different between the patients with NIPD and the patients without NIPD. However, the D/D genotype patients, whose donor also had D/D were frequently observed in the NIPD population (23.8% vs 9.1%; p = 0.05). Furthermore, an investigation of the relation between ACE genotype and onset of NIPD revealed that NIPD patients with the D/D genotype tended to be diagnosed faster than patients with the I/D or I/I (mean onset day 48.5, 95.2 and 110.6; p = 0.037). Our results suggest the involvement of ACE gene polymorphism in early onset NIPD, which may be different from late onset NIPD in respect to the pathogenesis.
Haploidentical Stem Cell Transplantation in Children: A Comparison between Positive Selection of Stem Cells and Depletion of T- and B-Cells.
Authors: Lang Peter , Bader Peter , Greil Johann , Klingebiel Thomas , Niethammer Dietrich , Handgretinger Rupert
Published: April 2020

Profound depletion of T- and B-cells is a fundamental prerequisite for haploidentical 1–3 loci mismatched stem cell transplantation. Here, we compare hematopoietic engraftment and occurrence of GvHD after positive selection procedures (with anti-CD34 coated or anti-CD133 coated beads, n=64 patients) and after a direct depletion procedure with anti-CD3/anti-CD19 coated microbeads (n=12) in children with leukemias (ALL, AML, CML), lymphomas and nonmalignant diseases. Median purity of stem cells was comparable after CD34+ selection and CD133+ selection, whereas stem cells were only slightly enriched after CD3+/CD19+ depletion (97.5%, 93.4% and 1.02%). Indirect depletion of T-cells by positive selection methods resulted in 10 000 residual CD3+ cells/kg (7000–30 000). Patients with grafts depleted with anti-CD3/anti-CD19 coated microbeads, received 32 000 (7000–160 000) residual T-cells/kg. Those grafts comprised also remarkable amounts of effector cells such as NK-cells (median number: 86 × 106/kg), dendritic cells and monocytes/granulocytes (median number: 700 × 106/kg) with antileukemic activity in vitro. Primary engraftment occurred in 83% of patients with positive selected stem cells. After reconditioning, sustained engraftment was achieved in 98%. In patients with CD3+/CD19+ depleted grafts, primary engraftment was observed in 92% (11/12) and sustained engraftment (after reconditioning) in 100% (12/12). GvHD grade II occurred in 6% (positive selection) and 25% (CD3/CD19 depletion). Only one patient had severe GvHD grade III-IV (CD34+). T-cell recovery was significantly faster in patients with CD3/CD19 depleted grafts. In the group with positive selected grafts, 5 year EFS for patients with ALL CR1-3, myeloic leukemias and nonmalignant diseases was 45%, 22% and 60%, respectively. CD3+/CD19+ depleted grafts were given to patients with refractory disease (AML/MDS, ALL) or with high risk of graft failure. 4/10 patients with leukemias and 2/2 patients with SAA/PNH are disease free (median follow-up: 8 months). Conclusions: Haploidentical transplantation with standard CD34+ or CD133+ selected grafts provides stable long term survival rates. CD3+/CD19+ depletion resulted in better and more reliable engraftment but produced slightly more GvHD than positive selection methods. Our preliminary results indicate, that CD3/CD19 selected grafts may be advantageous regarding engraftment and immunoreconstitution. Since effector cell with potential antileukemic activity are cotransfused, such grafts may be suited in particular for patients with insufficient remission.
Subclinical EBV and CMV Reactivations Commonly Occur Following Antibody-Based Immunosuppressive Regimens for Patients with Severe Aplastic Anemia.
Authors: Scheinberg Phillip , Fischer Stephen H. , Li Li , Nunez Olga , Wu Colin O. , Young Neal S. , Barrett A. John
Published: April 2020

Although reactivation of Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) during periods of immune suppression following solid organ and allogeneic hematopoietic stem cell transplantation (HSCT) can cause fatal complications, the natural history of viral reactivation and the potential for serious complications following antibody-based immunosuppressive (I/S) treatments for bone marrow failure syndromes is not defined. We monitored blood for EBV and CMV reactivation by polymerase chain reaction (PCR) weekly in 39 consecutive patients (total of 44 I/S courses) with aplastic anemia (AA). During the observation period of 3–6 months per patient, none developed clinical features of EBV or CMV reactivation, nor received antiviral therapy. Four regimens were studied: (1) HC: horse ATG + cyclosporine (CsA) (18 pts); (2) HCS: horse ATG + CsA + sirolimus (15 pts); (3) RC: rabbit ATG (Thymoglobulin®) + CsA (5 pts); and (4) CP: alemtuzumab (Campath®) (6 pts). Overall EBV reactivation occurred in 32/39 (82%) seropositive patients at a median of 14 days in the HC arm, 11 days in the HCS arm, 15 days in the RC arm and 6 days in the CP arm from starting I/S. The median peak EBV copies for HC recipients was 4,340 (0–54,250) copies/106 mononuclear cells (MNCs); for HCS 1,420 (0–466,000) copies/106 MNCs; for RC 370,000 (97,000–1,025,000) copies/106 MNCs, and for CP 965 copies/106 MNCs (0–655,000) (P= 0.026). The median duration of PCR positivity for EBV was higher in the RC group (18 weeks) compared to HC (6 weeks), HCS (4 weeks) and CP (1 week) (P=0.002). Higher peak values were associated with a longer duration of viremia following initial I/S (P<0.001)(Figure). CMV reactivation occurred only in 12/29 (41%) seropositive patients, 10 CMV seronegative patients showed no reactivation by PCR. The EBV reactivations in the alemtuzumab arm were less frequent and short lived, presumably because the antibody eliminates the B cell population which is the source for reactivating virus. A more prolonged lymphocyte depletion was seen with RC and CP treated patients when compared to HC and HCS. Our results indicate that subclinical reactivation of both EBV and CMV is common in patients with AA receiving I/S and that different regimens are associated with different intensity of I/S as measured by viral load and lymphocyte count. Our data suggests that higher and longer EBV reactivations are seen following rabbit ATG compared to horse ATG or alemtuzumab, and for the whole cohort, the duration of positivity for EBV PCR correlated to the initial degree of EBV reactivation. While all reactivations were subclinical, our findings suggest that viral reactivation should be monitored when developing new and more potent I/S regimens for treatment of hematologic and non-hematologic disorders as the threshold to the development of CMV or EBV disease is not well understood.

Figure Figure
Mesenchymal Stem Cells for Treatment of Severe Acute and Extensive Chronic Graft-Versus-Host Disease.
Authors: Le Blanc Katarina , Frassoni Francesco , Ball Lynne , Uzunel Mehmet , Lanini Edoardo , Sundberg Berit , Lonnies Helena , Dini Giorgio , Bacigalupo Andrea , Locatelli Franco , Fibbe Willem E. , Ringden Olle T.H.
Published: April 2020

Mesenchymal stem cells (MSC) from adult bone marrow have the capacity to differentiate to several mesenchymal tissues and inhibit T-cell alloreactivity in vitro. We have used MSC to treat grades III-IV acute graft-versus-host disease (GVHD) in 14 patients and extensive chronic GVHD in two patients. The MSC dose was median 1.0 (range 0.4–9) x 10E6 cells/kg body weight of the recipient. No side-effects were seen after MSC infusions. Nine patients received one dose, six patients received two doses and one patient received three doses. MSC donors were in two cases HLA-identical sibling donors, twelve haploidentical donors and ten third-party HLA-mismatched donors. Among the 14 patients treated for severe acute GVHD, six had complete responses, four showed improvement and one had stable disease. Three were not evaluable: two due to early death and one due to short follow-up. MSC donor DNA was detected in lymph node and colon in one of the patients who died. Among the 14 patients treated for severe acute GVHD, nine are alive between two months up to three years after transplantation. Four of these patients have extensive chronic GVHD. One patient transplanted for AML in relapse has recurrent leukaemia. The two patients treated for extensive chronic GVHD had transient responses. One of them died of Epstein-Barr virus lymphoma. We conclude that MSC have immune-modulatory and tissue repairing effects and therefore may be used for treatment of severe GVHD.
HIV-Related Burkitt’s NHL (BL) vs. Diffuse Large-Cell NHL (DLCL) in the Pre- and HAART Eras: Significant Differences in Survival with Standard Chemotherapy.
Authors: Spina Michele , Simonelli Cecilia , Talamini Renato , Vaccher Emanuela , Tirelli Umberto
Published: April 2020

Background: The survival of patients with HIV-related NHL has significantly improved by the widespread use of HAART. However, the survival of patients with HIV-BL treated with standard chemotherapy regimens remains poor.

Methods: In order to evaluate the outcome of patients with HIV-BL in comparison to that of patients with HIV-DLCL, we reviewed our series of 253 HIV-NHL diagnosed and treated at the National Cancer Institute of Aviano, Italy from 1984 to 2003, including 125 cases in the pre-HAART era (77 HIV-DLCL and 48 HIV-BL) and in the HAART era (93 HIV-DLCL and 35 HIV-BL).

Results: All patients both with HIV-DLCL and HIV-BL were treated with the same doxorubicin containing chemotherapy regimens. The median OS of all patients was significant longer in the HAART era in comparison to that of the pre-HAART era (15 vs 8 months, p ≤ 0.0001). In addition, in the pre-HAART era, the median OS was similar in patients with HIV-BL vs patients with HIV-DLCL (7 vs 10 months, p = 0.11) whereas in the HAART era the median OS of patients with HIV-BL was significantly shorter than that of patients with HIV-DLCL (8 vs 22 months).

Conclusions: Our data confirm that the significant improvement of the outcome of patients with HIV-NHL is related to the positive impact of HAART on the survival of patients with HIV-DLCL whereas the prognosis of HIV-BL remains poor despite the use of HAART. Taking into consideration that in the HAART era patients with HIV infection are likely to tolerate intensive chemotherapy regimens, a more intensive approach similar to that employed in the general population should be employed also in patients with HIV-BL.
Activating NOTCH1 Mutations Are Associated with Good Treatment Response in Childhood T-Cell Acute Lymphoblastic Leukemia.
Authors: Breit Stephen , Stanulla Martin , Flohr Thomas , Schrappe Martin , Ludwig Wolf-Dieter , Tolle Gabriele , Happich Margit , Muckenthaler Martina M. , Kulozik Andreas E.
Published: April 2020

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10–15 % of pediatric ALL. Very rare cases of T-ALL (< 1 %) harbor the chromosomal translocation t(7;9) that involves NOTCH1, a gene encoding a single-pass, heterodimeric transmembrane receptor. NOTCH1 has an essential function in early intrathymic T-cell development. Recently, it has been demonstrated that more than 50 % of childhood T-ALLs carry activating mutations within the NOTCH1 gene (Weng et al., Science 2004). In the present study, we systematically analyzed the impact of activating NOTCH1 mutations on treatment response in 108 pediatric T-ALLs, registered in the ongoing ALL-BFM 2000 trial. In 56 cases (51.8%) activating NOTCH1 mutations were identified, located either in the heterodimerization (38/56 mutations; 65.5%), in the PEST (10/56; 17.9%) or in both domains (8/56; 14.3%). The presence of activating NOTCH1 mutations was significantly correlated with good prednisolone (p = 0.001, c2 or Fisher’s exact test) and MRD response (p = 0.002). T-ALLs with NOTCH1 mutations were 3.7 times more likely to show a good prednisolone response (95% confidence interval = 1.64–8.33; p = 0.002) and 4.8 times more likely to show a favorable MRD response (95% confidence interval = 2.04–11.11; p = 0.0003) when compared to patients with wild type NOTCH1. Patients with mutated NOTCH1 were thus underrepresented in the high risk group of the ALL-BFM 2000 protocol. This influence of NOTCH1 mutational status on risk stratification was independent from other commonly used criteria, like age and initial white blood cell count (WBC) at the time of diagnosis. Considering the impact of NOTCH1 mutations on long term prognosis, we analyzed those 49 patients of this cohort with a median follow-up of > 4 years. Eight patients relapsed within this follow-up period, 2 patients with mutated and 6 with wild type NOTCH1. With this small number of relapses, this trend towards a favorable influence of activating NOTCH1 mutations on EFS did not reach statistical significance. In conclusion, T-ALLs with NOTCH1 mutations are demonstrated to be more sensitive than those without to the ALL-BFM 2000 treatment strategy and may show a lower rate of relapse.