bluuebunny/crossref_metadata_2025 huggingface dataset:
Curcumin Supresess Growth and Induces Apoptosis of Primary Effusion Lymphoma Cells.
Authors: Uddin Shahab , Hussain Azhar R. , Al-Hussein Khaled A. , Gutierrez Marina , Bhatia Kishor G.
Published: October 2019

Kaposi ‘s sarcoma-associated herpesvirus (KSHV or HHV8) has also been implicated in primary effusion lymphoma (PEL). KSHV/HHV8 has been shown to activate a number of growth and survival signaling pathways, including PI3-kinase and JAK/STAT, and protect cells from programmed cell death. Indeed Stat-3 is constitutively phosphorylated/activated in PEL cells and plays a role in their survival and proliferation. Curcumin (diferuloylmethane), a natural compound isolated from the plant Curcuma Ionga had very little or no toxicity in human cells and has been shown to have anti-tumor activity in vitro. We describe here that curcumin at 20 μM concentration inhibits cell proliferation in several PEL cell lines (BC1 64 + 1%, BC3 67 + 2%, BCBL1 62 +3% inhibition of growth) and induces apoptosis (BC1 51+ 5%, BC3 47 + 6%, BCBL1 48 + 4 % apoptosis cells). To characterize better the mechanism of action of curcumin, we studied the JAK/STAT pathway and the apoptotic cascade. Curcumin suppressed the constitutively active Stat3 transcription factor in a dose dependent manner. Curcumin also induced loss of mitochondrial membrane potential, as determined by JC1 staining with subsequent activation of capase-3 followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage. In addition, zVAD-fmk, a universal inhibitor of caspases prevented caspase-3 activation as well as PARP cleavage induced by curcumin treatment. IL-10 is a major autocrine growth factor for PEL cell survival. We quantified by ELISA the secretion of IL-10 in PEL cells and found that curcumin abrogated IL-10 secretion. Taken together, our findings suggest that curcumin suppresses constitutively active Stat-3 in PEL cells, leading to inhibition of proliferation and induction of caspase-dependent apoptosis. These results provide the molecular basis and preliminary data for new treatment strategies that may incorporate curcumin in regimens for PEL.
Motexafin Gadolinium Enhances Rituximab-Induced Cytotoxicity Against B-Cell Lymphoma Cell Lines.
Authors: Chen Jun , Ramos Jason , Sirisawad Mint , Miller Richard A. , Naumovski Louie
Published: October 2019

Motexafin gadolinium (MGd, Xcytrin®) is a tumor selective redox active drug that is cytotoxic to many hematolymphoid cell lines and chronic lymphocytic leukemia cells. Previous studies have shown that MGd oxidizes various cellular reducing metabolites and produces reactive oxygen species (ROS), which can induce apoptosis. Rituximab, an anti-CD20 antibody, is used widely in the treatment of B-cell malignancies; however, its precise mechanism of action is uncertain. Some studies have demonstrated that rituximab induces the generation of ROS in sensitive cells. We evaluated the effects of combining rituximab and MGd in the in vitro treatment of HF-1, a cell line derived from a patient with follicular lymphoma. Increases in both apoptosis and cell growth inhibition were seen with MGd plus rituximab compared to each agent used separately. Apoptosis was demonstrated by both annexin-V positivity and caspase-3 activity. Loss of mitochondrial membrane potential and PARP cleavage were also greater when the two drugs were used together than when used separately. Data analysis with CalcuSyn software showed that, with the combination, the Dose Reduction Index (DRI) was more than 1.5 for MGd and 2 to 6 for rituximab regarding both apoptotic effect and growth inhibition. Consistent with MGd as a redox active agent, peroxiredoxin oxidation was higher when MGd and rituximab were used together. Further experiments revealed no increase in uptake of MGd by rituximab treatment, nor a change in the amount of CD20 antigen on the surface of MGd treated cells. We investigated another B-cell lymphoma cell line, DHL-4 for similar cooperative activity of MGd and rituximab. While MGd alone has no substantial apoptotic effect on DHL-4 cells, it enhanced the activity of rituximab in inducing apoptosis. These in vitro findings support the combined use of MGd and rituximab in the treatment of B cell lymphoma.
HLA-DQ7 Is Associated with Susceptibility to Idiopathic Thrombotic Thrombocytopenic Purpura.
Authors: Scully Marie , Brown Julliette , Patel Rajeshree , Machin Samuel J. , Brown Colin
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is a rare disorder associated with microangiopathic haemolytic anaemia, thrombocytopenia and multisystem microvascular thrombosis. It results from a deficiency of ADAMTS 13, required to cleave Ultra large von Willebrand factor multimers. An underlying autoimmune basis is suggested; indeed in up to 90% of idiopathic cases, antibodies, primarily IgG, to ADAMTS 13 can be demonstrated during an acute episode. Joseph et al( Am J Hematology 1994) reported that HLA-DR53 protects against TTP. In this study, twenty patients with idiopathic TTP, 6 male and 14 female, were typed for HLA-DR and DQ by PCR-SSP. Thirteen had recurrent (>3) episodes of TTP and all patients had ADAMTS 13 activity <5% with evidence of antibody to ADAMTS 13 at acute presentation. The HLA-DR and DQ frequencies in the patient group were compared with those found in 203 local blood donors and selected results are shown in the table below. In this study the reduction of DR53 has been confirmed, however, this may not be the primary HLA frequency altered in patients with TTP. Although HLA-DR53 is associated with DR4, DR7 and DR9, only the frequency of DR4 is reduced suggesting that this HLA type may be protective against TTP. The main finding of this study is that the frequency of HLA-DQ7 is significantly increased in the patient group. The frequency of HLA-DR11 (associated with DQ7), is also increased. However, four patients are DQ7 positive but negative for DR11 suggesting an association of DQ7 and susceptibility to TTP. Five patients are negative for HLA-DQ7; of these, four are HLA-DR15 positive, which may also contribute to susceptibility to TTP. HLA-DR and DQ associations with autoimmune disease, such as SLE and IDDM, have been well documented. The findings in this patient cohort suggest that specific HLA-DR and DQ molecules may have contributory roles in resistance or susceptibility to TTP. The repertoire of peptides presented to the immune system by HLA-DQ7 positive patients may increse their risk of developing TTP.

HLA type and TTP HLA type % patients positive % controls positive P value DR4 5.0 35.0 0.01 DR11 50.0 12.3 0.001 DR15 45.0 27.1 NS DR53 35.0 63.5 0.05 DQ7 75.0 34.5 0.001
Ex Vivo Large Scale Generation of Human Platelets from Cord Blood CD34+ Cells.
Authors: Matsunaga Takuya , Tanaka Ikuta , Kobune Masayoshi , Kawano Yutaka , Tanaka Maki , Akiyama Takehide , Kuribayashi Kageaki , Ninomiya Takafumi , Hamada Hirofumi , Niitsu Yoshiro
Published: October 2019

To obtain a large quantity of platelets (PLTs) from cord blood stem cells (CBSC) in vitro, we employed three-phase culture system. We first expanded CBSC on a monolayer of human telomerase catalytic subunit gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL) and thrombopoietin (TPO) for 14 days (1st phase), and then cultured them to differentiate into megakaryocytes for another 14 days with refreshing medium which contain interleukin-11 (IL-11) in addition to original cytokine cocktail (2nd phase). Subsequently, we transferred the cells to a liquid culture medium containing SCF, FL, TPO and IL-11, and cultured them for 5 days (3rd phase) to recover PLTs in the culture medium. The quantity of PLTs recovered from one CB unit (5 x 106 CD34+ cells) was calculated to be 10.5 units (2 x 1011 PLTs). These CB-derived PLTs exhibited quite similar feature as those from peripheral blood in morphology as revealed by electron micrograph and in functions as revealed by aggregation assay and by FACS detecting expression of P-selectin and activated glycoprotein IIb-IIIa antigens upon fibrinogen/ADP stimulation. Thus our three-phase culture system was considered to be useful for large scale generation of PLTs from CB for clinical usage.
Ionising Irradiation Influences Platelet Adhesion and Aggregation under High Flow Conditions.
Authors: Loncar Robert , Araba Fatima , Schmuling Christof , Wenzel Folker , Scharf Rudiger E.
Published: October 2019

Transfusion-associated graft-versus-host disease (GVHD) is a dramatic immunologic reaction mediated by transfused immunocompetent lymphocytes directed against an immunocompromised host. GVHD shows a rapid onset and is characterized by severe symptoms and a poor response to treatment (mortality rate > 90%). Prophylactic gamma irradiation of cellular blood components is the most efficient and reliable way to prevent GVHD. The currently recommended standard dose of gamma irradiation of blood products is 25 Gy. While irradiated blood components including platelet concentrates have been extensively transfused over the past 25 years, only few studies (with controversy results) evaluated the effect of ionising irradiation on platelet morphology and function. In this study, we examined the effect of a standard irradiation dose of 25Gy on platelet function using a platelet function analyzer (PFA-100). Citrate-anticoagulated blood was obtained in duplicate from twelve healthy blood donors. One sample was immediately irradiated according to a standard protocol (25 Gy), the other specimen was used as control. Platelet function of irradiated and control samples was evaluated using PFA-100. This device applies a high shear flow system to simulate the conditions under which platelets are subjected at the site of a damaged blood vessel wall. Anticoagulated whole blood was passed through the membranes coated with either collagen and epinephrine (Col/Epi) or collagen and ADP (Col/ADP) under hemodynamic conditions similar to those of small capillaries. Platelets adhere and aggregate across the aperture until the flow ceases, evaluated by the closure time (CT, sec). Statistical analysis was performed using a standard software (Sigma Plot v. 8; Student’s paired t test). Analysis of platelet function with Col/Epi cartridges showed that irradiated samples had a prolonged closure time compared to corresponding non irradiated controls (144 ± 21 sec vs. 120 ± 18 sec, p<0.05). The averaged CT prolongation was 19±5%. Analysis of CT using Col/ADP cartridges showed also prolongated CT (averaged prolongation 7%) in seven of nine irradiated samples compared to non irradiated controls (97 ± 14 sec vs. 103 ± 13 sec, p<0.05). Our results suggest that a standard dose of ionising irradiation for prevention of GVHD has a significant influence on platelet function. The biochemical nature by which this platelet dysfunction following irradiation is caused, remains to be assessed in further detail.
Effects of Photopheresis on Mononuclear Cells from Healthy Individuals.
Authors: Kumlien Gunilla , Remberger Mats , Ringdén Olle
Published: October 2019

Background: Extracorporeal photopheresis (ECP) has been proposed as an alternative therapy for immune-mediated disease including graft-versus-host-disease (GVHD) and transplant rejection (Photopheresis for the treatment of refractory renal graft rejection, Transplantation2005;79(1):123–5, Kumlien G et al). The mechanisms of photopheresis are still poorly understood but induction of tolerogenic dendritic cells and regulatory T cells as well as a shift of cytokine profile from Th1 to Th2 have been observed in patients treated with ECP. We wanted to investigate some basic effects of ECP on mononuclear cells from healthy individuals unaffected by pathogenic mechanisms and immunosuppressive drugs.

Method: Mononuclear cells (MNCs) were collected from six healthy volunteer apheresis donors using CobeSpectra apheresis machine (Gambro BCT, Lakewood, CO, USA) and peripheral blood was drawn immediately before apheresis. Apheresis products were thoroughly mixed and divided into three equal parts. One part was left untreated, one part was irradiated with UVA 365 nm, 2J/cm2 (BioGenic, Vilber Lourmat, Marne-la-Vallée, France) and one part was treated with psoralen (8-methoxypsoralen 200 ng/ml) plus UVA-irradiation (PUVA). Peripheral MNCs and aliquots of apheresis MNCs were analysed simultaneously regarding incorporation of 3H Thymidine after stimulation with mitogens (PHA, ConA, SpA) and production of cytokines correlated with GVHD pathogenesis (IL-2, IL-6, IL-12, TNFα, IFNγ) after stimulation with antigens (BG, PHA, LPS) using commercial ELISA-kits (Quantikine, R&D Systems, Minneapolis, MN, USA). Statitical analysis was performed using a nonparametric method (one-sided ANOVA).

Results: Mitogenic capacity was reduced to baselevel after PUVA-treatment but not affected by the apheresis procedure or UVA-irradiation alone. Table 1. Statistically significant decrease of cytokine production was seen only after PUVA-treatment but production of IL-2 and IL-6 was reduced after both apheresis and UVA-irradiation alone. Small amounts of IL-2, IL-6, TNFα and IFNγ were produced even after PUVA-treatment.

Conclusion: Significant reduction of mitogenic capacity only after PUVA-treatment was the expected finding. Apheresis can theoretically effect cells through mechanic damage (centrifugation) and/or interaction with apheresis harness and UVA is known to have immunomodulatory effects which can explain the reduction of cytokine production. Cytokines detected after PUVA-treatment may be explained by release from apoptotic cells or by production in cells that escape apoptosis.

Lymphocyte proliferation assay ConA PHA SpA MNCs = mononuclear cells peripheral vs apheresis MNCs ns ns ns peripheral vs UVA-irradiated MNCs ns ns ns apheresis vs UVA-irradiated MNCs ns ns ns peripheral vs PUVA-treated MNCs p<0.05 p<0.05 p<0.05 apheresis vs PUVA-treated MNCs p<0.05 p<0.05 p<0.05 UVA -irradiated vs PUVA-treated MNCs p<0.05 p<0.05 p<0.05
Development of B Cell Lymphoma in Eμ-Cyclin D1 X Mutant p53 Transgenic Mice.
Authors: Smith Mitchell R. , Joshi Indira J. , Jin Fang , Al-Saleem Tahseen
Published: October 2019

Background: Mantle cell lymphoma (MCL) is characterized by t(11;14) which dysregulates cyclin D1 expression. Eμ-cyclinD1 transgenic mice, however, are healthy. Additional genetic events must be necessary for lymphomagenesis, and knowledge of these would enhance understanding and therapy of MCL. In addition, a mouse model of MCL would be helpful in drug development. Alterations in p53 have been described in MCL, often associated with the blastic variant.

Objectives and Methods: To determine whether p53 and cyclin D1 can cooperate in lymphomagenesis, we cross bred Eμ-cyclinD1 transgenic mice (Bodrug et al EMBO J, 1996, courtesy of Alan Harris) with mice transgenic for mutant p53 (Jackson Labs, Jacks et al Curr Biol, 1994). Progeny mice were monitored for presence of the transgenes by PCR of tail vein DNA and observed for development of disease.

Results: Of mice carrying both aberrant genes, 24 of 38 developed B cell lymphoma. Mice did not become visibly ill until at least 12 months of age, with median age at sacrifice 15.5 (range 12–23) months. The lymphoma was generally disseminated, involving spleen, liver, diffuse adenopathy and marrow with occasional extranodal sites. Histology varied between small and large cell, with some having a vaguely follicular growth pattern. T cell lymphomas occurred in 2 other mice, while 5 developed osteosarcoma (1 of these in a mouse that also had B cell lymphoma). The B cell lymphomas were clonal by Cμ-VH PCR. Cyclin D1 expression was documented by Western analysis. A cell line has also been developed from one of the B cell lymphomas and this line rapidly grows into disseminated lymphoma in syngeneic mice. These B cell lymphomas differ from the thymic T cell lymphomas seen in heterozygous p53 mutant mice that do not co-express cyclin D1. The latency period differs from cyclin D1 x myc double transgenic mice.

Conclusions: This model demonstrates cooperation between p53 and cyclin D1 pathways in B cell lymphomagenesis and should prove useful in delineating how these signals interact. The cell line may prove useful in pre-clinical testing of new agents for MCL.
Tissue Factor-Dependent Cellular Responses of Factor VII/VIIa in the Absence and Presence of Factor X.
Authors: Petersen Lars C. , Albrektsen Tatjana , Hjortø Gertrud M. , Kjalke Marianne , Rojkjaer Rasmus , Bjørn Søren E. , Sørensen Brit B.
Published: October 2019

Factor VIIa (FVIIa) when bound to tissue factor (TF) triggers both coagulation and intra-cellular signaling. The dose-response of TF/VIIa-mediated factor X (FX) activation and gene regulation by TF-expressing cells were measured and interpreted in relation to plasma in vivo conditions. EC50s in the range 10–30 pM for FVIIa-mediated FX activation were measured with LPS-stimulated monocytes, vascular smooth muscle cells and MDA-MB-231 cells indicating that saturation of FX activation required less than 100 pM FVIIa. Compared to a plasma concentration for FVII/FVIIa of 10 nM this ensures that TF exposure is the determining factor for the coagulation response. This notion was further substantiated by data showing that FVII is readily activated to FVIIa on the TF-expressing cell surface, and that the FX activation kinetics mediated by FVII was similar to that of FVIIa. In MDA-MB-231 cells FVIIa induced TF-dependent signal transduction as measured by IL-8, CXCL-1 and CSF-2 mRNA. Stimulation reached saturation at about 10 nM FVIIa in the presence or absence of 100 nM FX. Furthermore, the maximal level of FVIIa-induced mRNA was similar with or without FX. FX enhanced TF/FVIIa-stimulated mRNA induction at low FVIIa concentrations, whereas a physiological level of FX or FXa (100 nM) alone was insufficient to induce a significant cellular response. In conclusion our results indicate that optimal conditions for triggering both coagulation and signaling prevail already at normal plasma concentration of FVII/FVIIa and FX, and that TF exposure is the determining factor for both responses.
Age Related Expression Patterns of Mouse Liver Nuclear Proteins.
Authors: Kurachi Sumiko , Yasui Manabu , Kasama Emi , Kurachi Kotoku
Published: October 2019

Previously we reported the discovery of the ASE/AIE-type age related regulatory mechanism of gene expression, the first molecular mechanism for age dimension homeostasis (Kurachi et al, Science 1999; Zhang et al, J Biol Chem 2002; Kurachi & Kurachi J Thromb Haemost 2005). Here we report a comprehensive analysis of the age associated changes of mouse liver nuclear proteins, an experimental model for studying the dynamic regulatory mechanisms of the age-dimension homeostasis. Liver nuclei obtained from mice (C57BL/6xSJL, male) at 1, 3, 6, 12, 18 and 21 months of age (n=10–20/age) were used for preparing protein extracts. The protein extracts were then subjected to 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (pH range of 4–11) and protein identification by MALDI-TOF/MS. As analyzed by the PDQuest program combined with Mascot Protein Identifier, out of over 5000 protein spots identified by 2D-PAGE, approximately 2800 protein spots were found unique including isomers. Intensity changes of these protein spots along the age axis were determined and analyzed in details with the PDQuest software. Twelve proteins were found to show no significant changes in their intensity throughout the life-span (smaller than 20% fluctuation). Besides some complex patterns, at least 6 unique age-related patterns were found, including those of puberty-onset increase, puberty-onset decrease, puberty-onset increase up to 6 months of age followed by decrease, unique increase only at around 12 months of age, ageing associated increase or decrease. Age related pattern changes were observed for some isomers among many, which were likely generated by differential posttranslational modifications or conformational changes. These findings support that in addition to the ASE/AIE type mechanism, there exist multiple novel age-dimension regulatory mechanisms, and their various combinations are responsible for producing complex and subtle age related regulatory patterns for a large number of genes. This is the first comprehensive analysis of the liver nuclear proteins in relation to age, and would provide a valuable basis for studying many age related phenomena, diseases as well as epigenetic effects.
NMR Structural Analysis and Folding Studies of the Factor XI Apple 4 Domain as a Model for Factor XI Homodimerization.
Authors: Riley Paul W. , Samuel Dharmaraj , Cheng Hong , Roder Heinrich , Walsh Peter N.
Published: October 2019

Factor XI (FXI) is a 160kD homodimeric component of the intrinsic pathway of the blood coagulation system. The FXI Apple 4 domain (A4) mediates homodimer formation even when the cysteine responsible for the intermolecular disulfide is mutated to serine (C321S). Preliminary NMR structural studies of the isolated wild type (wt) A4 domain showed a flattened, planar structure (Samuel D, Cheng H, Riley PW, Walsh PN, Roder H. Blood104, 481a, 2004), compared to a more globular structure consisting of two layers of β-sheet and a flanking α-helix that was observed on further analysis of wt and C321S A4. Hydrogen exchange NMR and surface NMR analysis (SEAHSQC), as well as temperature dependent hydrogen exchange NMR showed that in C321S and wt A4, the loop surrounding C321 or S321 is flexible and exposed to the solvent. Thus, the globular structure is the major species in both wt and C321S A4, but the planar structure is present as well. Fluorescence anisotropy studies of C321S A4 yielded a Kd value of ~64 nM (monomeric protein concentration), compared with ~72 nM for a dissociable mutant full-length FXI (G326C) obtained by analytical ultracentrifugation (Sinha D, Marcinkiewicz M, Lear JD, Walsh PN. Biochemistry, in press, 2005), suggesting that only the A4 domain mediates dimer formation in the intact protein. In addition, fluorescence and circular dichroism (CD) studies of the folding equilibrium of C321S A4 indicate that starting from the unfolded monomeric state, partial folding precedes dimerization. This folding mechanism for FXI A4 is consistent with the observed failure of a naturally occurring mutant (FXI F283L) to dimerize intracellularly (Meijers JC, Davie EW, Chung DW. Blood79, 1435–1440, 1992). To determine the structural consequences of the mutation, F283L was introduced into the noncovalent dimer C321S A4, which increased the Kd of the homodimer interaction to ~5 μM, but did not change the secondary structure (from comparison of the CD spectra), or the high degree of thermostability observed for C321S A4. Since the F283 sidechain has been tentatively assigned to the surface of FXI A4 near the interface, the mutation to Leu may significantly alter the interface, thereby raising the Kd but not changing the overall secondary structure significantly. In summary, there appears to be a mechanism by which a conformational change or productive folding process occurs in the monomer state that activates it for native dimer formation. Also, inserting the F283L (type III) patient mutation does affect the dimer stability, but not the overall secondary structure.
Hematopoietic Repopulation, In Vivo, with Genetically Defined Clones Derived from HOXB4 Expressing ES-Cells.
Authors: Pilat Sandra , Carotta Sebastian , Schiedlmeier Bernhard , Kamino Kenji , Mairhofer Andreas , Schmidt Manfred , von Kalle Christoph , Steinlein Peter , Ostertag Wolfram , Baum Christopher , Beug Hartmut , Klump Hannes
Published: October 2019

In the context of somatic gene therapy of the hematopoietic system, transplantation of molecularly defined and, hence, “safe” clones would be highly desirable. However, techniques which allow gene targeting, subsequent in vitro selection and clonal expansion are only available for embryonic stem (ES) cells. After in vitro differentiation, some of their progeny cells are capable of mediating long term hematopoietic repopulation after transplantation into immunodeficient recipient mice, in vivo. This is especially efficient when the homeodomain transcription factor HOXB4 is ectopically expressed (1). We have recently shown that HOXB4-ES-cell derivatives behave similar to bone marrow cells also expressing this transcription factor ectopically, both in vitro and in vivo (2). Here we demonstrate that long term repopulation (>6 months) in Rag2(−/−)γ C(−/−) mice can be achieved with ES-cell derived hematopoietic cells (ES-HCs) obtained from single, molecularly characterized ES-clones, in which the insertion sites of the retroviral expression vector had been defined. Clones expressing HOXB4 above a certain level showed a high extent of chimerism in the bone marrow of transplanted mice (average 75%; range 45–95%, n=4) whereas ES-HC clones expressing lower levels only repopulated with very low efficiency (average 2.5% chimerism, range 1–4%, n=6 mice). These results suggest that the capability of long-term repopulation, in vivo, is highly dependent on the expression levels of HOXB4 in the transplanted clones. Only mice reconstituted with ES-HC clones expressing high amounts of HOXB4 and thus showing substantial chimerism, recapitulated the morphohistological phenotype observed in polyclonally reconstituted mice. This included the bias towards myelopoiesis, “benign” myeloid proliferation in spleen and the incompatibility of HOXB4 expression with T-cell poiesis (2). In summary, we demonstrate that repopulation of the hematopoietic system can be achieved with preselected clones of genetically manipulated stem cells in which a) the insertion site of the retroviral (gene therapy) vector has been characterized prior to transplantation and b) in which ectopic HOXB4 has to be expressed above a certain threshold level. Thus, ES cells carry the potential for performing safe somatic gene therapy when using integrating gene therapy vectors. Nevertheless, advanced cell therapy will certainly require the expression of HOXB4 in a regulated manner to avoid unwanted effects such as disturbed lineage differentiation.
AMD3100 + G-CSF Improves Hematopoietic Progenitor Cell (HPC) Collection in Patients with Hodgkin’s Disease (HD).
Authors: Cashen Amanda , Devine Steven , Vij Ravi , DiPersio John
Published: October 2019

Autologous stem cell transplantation is indicated for patients with HD who have primary refractory disease or who relapse after a first remission. For these patients, as for other patients undergoing autologous transplantation, the number of CD34+ cells infused is a reliable predictor of neutrophil and platelet engraftment, and doses ≥ 5 x 106 CD34+ cells/kg are associated with faster count recovery. However, among the 98 patients with HD who have undergone G-CSF-alone mobilization at our institution in the past five years, 22% did not achieve a minimum HPC collection of 2 x 106 CD34+ cells/kg in ≤ 5 apheresis procedures, and only 15% achieved a collection ≥ 5 x 106 CD34+ cells/kg. AMD3100 mobilizes HPCs by reversibly inhibiting the interaction of CXCR4 and SDF-1α. It has been shown to improve HPC mobilization in patients with multiple myeloma and non-Hodgkin’s lymphoma. Here we present results for the first ten HD patients treated with a mobilization regimen of AMD3100 + G-CSF. To date, ten patients with relapsed (8) or refractory (2) HD have been mobilized with G-CSF (10 ug/kg/d) + AMD3100 (240 ug/kg/d) beginning on day 4. Apheresis was performed 11 hours after each AMD3100 dose. The first dose of AMD3100 produced a median (range) 3.0 (1.9–5.1) fold increase in the number of circulating CD34+ cells. Six patients (60%) achieved a collection of ≥ 5 x 106 CD34+ cells/kg, and all patients collected > 2 x 106 CD34+ cells/kg (range, 3.6–9.4 x 106 CD34+ cells/kg). The median (range) number of apheresis procedures performed per patient was 2 (1–4). No grade II-IV adverse events were ascribed to AMD3100. Eight patients have been transplanted with G-CSF + AMD3100 mobilized cells. All have had prompt and stable engraftment, with median neutrophil recovery at day +9 (9–11) and median platelet recovery at day +20 (15–23). Two patients had very early engraftment, with absolute neutrophil count greater than 100 on day +7. We conclude that AMD3100 + G-CSF is a well-tolerated and effective mobilization regimen in patients with HD. All patients (100%, 95% CI 69%-100%) mobilized with AMD3100 + G-CSF achieved the minimum collection of 2 x 106 CD34+ cells/kg, and a significantly higher proportion of patients (60%, 95% CI 26%–88%) achieved the goal collection of ≥ 5 x 106 CD34+ cells/kg than did the historical controls (15%). Importantly, the median collection in the first two days of pheresis was 5.4 x 106 CD34+ cells/kg, which is significantly better than historical controls, who collected a median 3.0 x 106 CD34+ cells/kg in the first two days of pheresis (p=0.014). Our results demonstrate that the mobilization regimen of AMD3100 + G-CSF can improve the number of HPCs collected and decrease the number of days of pheresis in HD patients. This regimen will be pursued further in this patient population.
BCR/ABL Modifies the Kinetics and Fidelity of DNA Double-Strand Breaks Repair in Leukemia Cells.
Authors: Slupianek Artur , Nowicki Michal O. , Skorski Tomasz
Published: October 2019

Clinical observations and experimental findings indicated that BCR/ABL stimulates genomic instability leading to mutations and chromosomal abnormalities. The accumulation of genetic errors is believed to be responsible for the transition from a relatively benign CML chronic phase (CML-CP) to the aggressive blast crisis phase (CML-BC) and the resistance to imatinib mesylate. BCR/ABL- positive leukemia cells accumulate an excess of potentially lethal DNA double-strand breaks (DSBs) caused by reactive oxygen species (ROS) or genotoxic treatment. However, BCR/ABL tyrosine kinase facilitates the repair of DSBs and promotes survival. Therefore, the infidelity of DSBs repair processes may contribute to genomic instability in leukemia cells exposed to elevated numbers of spontaneous and/or induced DSBs. To test this hypothesis DSBs repair efficiency and fidelity was examined and compared in parental and BCR/ABL-transformed cells. Nuclear foci detected by γ-H2AX (the form of H2AX histone that is quickly phosphorylated on Serine 139 by ATM, ATR and/or DNA-PKcs kinases on megabase-length fragments near DSB sites) immunofluorescence served as indicators of DSBs. We found that BCR/ABL-positive leukemia cells acquire more DSBs after γ-irradiation in comparison to normal cells. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in mammalian cells. HR and NHEJ reaction sites in the nuclei can be visualized by double-immunofluorescence detecting co-localization of γ-H2AX foci with RAD51 or Ku70, respectively. NHEJ and HR appear to work in a time-dependent fashion (NHEJ followed by HR) and be more active in BCR/ABL-transformed cells in comparison to normal counterparts. Time-dependent engagements of NHEJ and HR mechanisms in repair of DSBs after γ-irradiation are accompanied by elevated accumulation of Ku70 and RAD51 proteins in cell lysates obtained from BCR/ABL cells. Specific DSBs repair assays confirmed that BCR/ABL leukemia cells in comparison to normal cells displayed enhanced capability of HR and NHEJ. However, analysis of DSBs repair products revealed that the repair mechanisms were less faithful in former cells generating large deletions and point mutations during NHEJ and HR, respectively. In summary, BCR/ABL leukemia cells display facilitated, but unfaithful HR and NHEJ, which may contribute to accumulation of genetic errors in surviving leukemia cells leading to malignant disease progression.
Graft-Versus-Tumor Effects after Allogeneic Hematopoietic Cell Transplantation for the Treatment of Hematologic Malignancies.
Authors: Usui Eiji , Fukuda Takahiro , Kato Ruri , Yamasaki Satoshi , Maruyama Dai , Morita Yuriko , Kim Sung-Won , Mori Shin-ichiro , Tanosaki Ryuji , Tajima Kinuko , Heike Yuji , Makimoto Atsushi , Tobinai Kensei , Takaue Yoichi
Published: October 2019

Adjustment of immune reaction, balancing between graft-versus-tumor (GVT) effect and graft-versus-host disease (GVHD), is of primary importance for improved therapeutic outcome after allogeneic hematopoietic cell transplantation (HCT). We retrospectively reviewed the medical records of 315 patients with hematologic malignancies who underwent allogeneic HCT at our center between 2000 and 2004. The median age of the patients was 46 (range, 1–68) years. The diagnoses included acute myeloid leukemia or myelodysplastic syndrome (n=153), chronic myelogeneous leukemia (n=30), acute lymphoblastic leukemia (n=36), lymphoma (n=90), and other hematologic malignancies (n=6). Seventy-two patients had low-risk and 243 had high-risk disease. Patients were conditioned with fludarabine-based reduced-intensity (n=160) or conventional myeloablative regimen (n=155). Donors included HLA-matched (n=120) or mismatched (n=53) relatives and unrelated volunteers (n=142). Stem cell source was G-CSF-mobilized peripheral blood stem cell (n=169), bone marrow (n=117), or cord blood (n=29). Of the 187 patients with measurable disease before HCT, 139 (75%) achieved complete remission. In a multivariate analysis, myeloid disease and the development of acute or chronic extensive GVHD were associated with a higher probability of achieving complete remission. Currently, 155 patients (49%) are alive with a median follow-up of 931 (range, 88–1968) days. Multivariate analyses revealed that a higher HCT-specific comorbidity index (HCT-CI) score, disease risk, graft rejection, and transplant from donors other than HLA-matched relatives were associated with increased risks of poor overall survival (OS) and progression-free survival (PFS). A higher HCT-CI score, greater patient age, graft rejection, and transplant from donors other than HLA-matched relatives were associated with an increased risk for non-relapse mortality (NRM). Patients with high-risk disease were associated with an increased risk of relapse or progressive disease (PD). After adjusting for these significant variables, grade III–IV acute GVHD was associated with increased risks of high NRM and poor OS and PFS. Chronic extensive GVHD was associated with an increased risk of NRM, without any significance for PD, OS, and PFS. In a subset analysis of 160 patients who received reduced-intensity conditioning, chronic extensive GVHD was associated with a decreased risk of PD. In conclusion, marginally intensified GVT effect with severe acute or chronic extensive GVHD appears to be offset by increased toxicities and mortality. Future study should focus on identification of narrow window for better control of immune reaction to induce tolerable GVHD with clinically significant GVT effect.
Large Granular Lymphocytosis Following Transplantation: Institutional Experience over a 10-Year Period at City of Hope.
Authors: Popplewell Leslie L. , Chang Karen , Palmer Joycelynn , Forman Stephen J.
Published: October 2019

Large Granular lymphocyte (LGL) expansion and LGL leukemia have been described in patients following both ablative and non-myeloablative conditioning regimens for hematopoietic cell transplantation (HCT), and have been associated with viral infection and graft-vs.-host disease (GVHD). We describe here an analysis of 41 cases of clonal LGL expansion identified at our institution over a 10-year period. Of these, 18 occurred in patients post autologous HCT (autoHCT), 23 in patients post allogeneic HCT (alloHCT). In the same time period, an additional 23 diagnoses were made in patients who had never undergone HCT. LGL expansion appeared an average of 485 days (range 30 – 1935 d) from alloHCT. In most patients, this occurred as an isolated finding, while in 5 patients, the clonal expansion was detectable on serial evaluations over a period of months or years (75 – 1662 d). LGL expansion was detected an average of 133 days from auto HCT, and only one patient had detectable clonal expansion over a prolonged period of time (70d). AutoHCT patients ranged from 23 to 72 years, average age 48.5 years. AlloHCT patients ranged from 26 to 62 years, average age 47.8 years. Incidence of reactivation of CMV postHCT was 56.5% in the alloHCT and 11% in the autoHCT. Relapse rates were 39% in the alloHCT and 28% in the autoHCT groups. At a median follow-up of 1372 days, 14 alloHCT patients remain in remission, with 9 relapses, and 5 deaths. At a median follow-up of 887 days, autoHCT patients remain in remission, with 5 relapses, and 3 deaths. Deaths were primarily attributed to relapse of disease and infectious complications, while no deaths were attributable to LGL clonal expansion. No patients with LGL clonal expansion have required treatment specifically for this condition.
Detectable Minimal Residual Disease before Haematopoietic Stem Cell Transplantation Predicts Extremely Poor Prognosis in Children with Acute Lymphoblastic Leukaemia.
Authors: Muzikova Katerina , Fronkova Eva , Sramkova Lucie , Reznickova Leona , Formankova Renata , Sedlacek Petr , Stary Jan , Trka Jan
Published: October 2019

The level of minimal residual disease (MRD) prior to allogeneic haematopoietic stem cell transplantation (HSCT) was shown to be an independent prognostic factor for the outcome of paediatric patients with high-risk acute lymphoblastic leukemia (ALL). Retrospective studies which used (semi-)quantitation of clone-specific immunoglobulin/T-cell receptor (Ig/TCR) rearrangements documented feasibility and practicality of such an approach. Recently, this approach was disputed by Imashuku et al (BMT 2003) due to great occurrence of the clonal evolution and generally high MRD levels prior HSCT in their cohort. In our prospective study, MRD before and after HSCT was monitored in a cohort of 36 children with ALL consecutively transplanted in our centre between VIII/2000 and VII/2004. We used a quantitative real-time PCR approach introduced and standardised by European Study Group on MRD in ALL. In 25 of 36 patients MRD level prior HSCT was assessed (9 patients lacked adequately sensitive Ig/TCR target; two lacked analysable DNA prior HSCT). Seventeen patients were MRD-negative prior HSCT (including two with MRD level below the quantitative range 10(−4)) and 8 were MRD-positive up to 9x10(−2). In the MRD-positive subgroup, 7 events (6 relapses) occurred post-transplant in striking contrast to only one relapse in MRD-negative subgroup (EFS log-rank p<0.0001). MRD proved to be the only significant prognostic factor in a multivariate analysis (p<0.0001). Adoptive immunotherapy including donor lymphocyte infusions in patients with adverse dynamics of MRD after HSCT had only limited and/or temporary effect. Clonal evolution did not present a problem precluding MRD monitoring in any of patients suffering a post-transplant relapse. We show that MRD quantitation using clonal Ig/TCR rearrangements represents a feasible approach for the risk assessment in paediatric ALL patients undergoing allogeneic HSCT. However, our ability to respond to detectable MRD levels after HSCT and to avert an impending relapse is very limited. The change of the approach to MRD-positive patients prior HSCT is necessary because of very questionable benefit of HSCT in these children. Supported by grants MSM0021620813, FNM 9735 and GAUK 62/2004.
Incompatible Inhibitory KIR Ligands Contribute To Lower Survival Rates in Leukemia Patients Receiving T-Replete Hematopoietic Cell Transplants (HCTs).
Authors: Sun J.Y. , Dagis A. , Miller M.M. , Longmate J. , Rodriguez R. , Gaidulis L. , Forman S.J. , David D.
Published: October 2019

Natural killer (NK) cells are becoming increasingly recognized as an important part of the immune system. Killer Ig-like receptors (KIRs) are the major form of receptors used by human NK cells. KIRs transmit either inhibitory or activating signals regulating NK cell activity. Some inhibitory KIRs specifically recognize HLA-A, B or -Cw allotypes on target cells. Thus differences in inhibitory ligand (iKIRL) phenotypes between donors and patients could result in NK cell alloreactivity that further results in different HCT outcomes. To test this hypothesis, the present study evaluated 362 patients with either ALL (99), AML/MDS (141), or CML (122). The patients were transplanted (1996~2003) with T-replete bone marrow, or peripheral blood stem cells from unrelated donors. High resolution HLA typing with DNA-based methods had been prospectively performed for donor selection. The KIR genes of patients and donors were retrospectively typed by a multiplex PCR-SSP method. The cohort was divided into three groups by the HLA and KIR profiles: 247 cases with matched HLA at the antigen level of HLA-A, B, Cw, DRB1, and DQB1 (MH), 64 with mismatched HLA (mMH) and 51 with mMH plus mismatched iKIRL (mMH+miKIRL). A significantly different rate (P=0.009) of estimated one year overall survival was found between the MH (61%, 95%CI 55–67%), the mMH (46%, 34–59%), and the mMH+miKIRL (29%, 18–43%) groups. When analyzing the event free survival, a similar difference (P=0.003) was observed between the MH (57%, 51–63%), the mMH (43%, 32–56%) and the mMH+miKIRL (25%, 15–39%) groups. Further analysis demonstrated a different rate (P=0.02) of relapse between the MH (two-year estimation: 21%, 16–28%), the mMH (16%, 7–31%), and the mMH+miKIRL (36% 18–58%) groups. That the MH group experienced more relapses than the mMH is consistent with the GvL effect. The mMH+miKIRL group experienced the most relapses implied miKIRL undermine the GvL effect. We also tested the influence of lacking iKIRL in patients within the MH group, and found a significant survival difference (P=0.05) among the patients lacking two iKIRLs (n=67, 54% 42%–65%), one iKIRL (n=104, 59% 49%–68%), or none (n=76, 70% 58%–79%). This finding contrasts with the previously reported beneficial role of potential NK cell alloreactivity in T-depleted HCT. Additional studies are required to resolve these issues.
Diastolic Dysfunction Is an Independent Risk Factor for Death in Patients with Sickle Cell Disease.
Authors: Sachdev Vandana , Machado Roberto F. , Shizukudu Yukitaka , Rao Yesoda , Sidenko Stanislav , Ernst Inez , St. Peter Marilyn , Coles Wynona A. , Rosing Doug , Blackwelder William C. , Castro Oswaldo , Kato Gregory J. , Gladwin Mark T.
Published: October 2019

Background. Pulmonary hypertension, defined by an elevated pulmonary artery systolic pressure measured by Doppler-echocardiogram, has been identified as the major predictor of death in the adult sickle cell disease population. While diastolic dysfunction is also observed in this population, the prevalence in unselected patients, the association with high pulmonary artery systolic pressure, and the attributable mortality remain unknown.

Methods. Diastolic function parameters, pulmonary artery systolic pressures and right and left ventricular size and function were measured prospectively in 215 subjects with sickle cell disease. Associations between these parameters, lab and echocardiographic variables and prospective mortality were determined.

Results. Diastolic dysfunction, measured by conventional and tissue-Doppler echocardiography, was present in 19% of patients with sickle cell disease. Diastolic dysfunction and pulmonary hypertension were both present in approximately 11% of patients and diastolic dysfunction accounted for approximately 10–20% of the variability in tricuspid regurgitant jet velocity. Importantly, Cox Proportional Hazards analysis revealed that diastolic dysfunction, as reflected by low E/A ratio, was associated with prospective mortality with a risk ratio of 1.9 (95%CI 1.0 to 3.7; p=0.028), even after adjustment for tricuspid regurgitant jet velocity. While pulmonary hypertension remained the dominant determinant of mortality risk, even after adjustment for measures of diastolic function (adjusted rate ratio of 5.3; 95% CI= 1.9 to15.0; p<0.001), the risk was additive such that patients with both risk factors had an odds ratio for death of 10.1 (95% CI= 3.2 to 31.9; p<0.001).

Conclusions. Diastolic dysfunction and pulmonary hypertension due to other causes each contribute independently to prospective mortality in patients with sickle cell disease. Patients with both risk factors have an extremely poor prognosis. These data support the implementation of Doppler-echocardiographic screening of adult patients with sickle cell disease to identify individuals at high risk of death for intensified therapy.

Figure Figure
Impact of HLA Class I Typing-Resolution and Mismatches on Survival and Graft-Versus-Host Disease in Patients after Hematopoietic Stem Cell Transplantations from Unrelated Donors.
Authors: Markiewicz Miroslaw , Wojnar Jerzy , Krawczyk-Kulis Malgorzata , Wylezol Iwona , Giebel Sebastian , Holowiecki Jerzy
Published: October 2019

We analyzed 150 consecutive patients (pts) transplanted from unrelated donors (URD) at single institution- Silesian Medical Academy in Katowice- with use of the same standard operating procedure from February 1997 until December 2004 for CML (74 pts), AML (28), ALL (27), SAA (9), MDS (7), MM (1), NHL (1), PNH (1), OMF (1), bi-phenotypic AL (1). 92 pts were transplanted from matched donors, including 59 with complete 10 alleles (HLA-A,B,C,DRB1,DQB1) high resolution (HR) DNA-typing; and 33 with first class match established on base of low-resolution (LR) (13), serological (7) or un-complete typing without HLA-C (13) and second class HR typing. 58 pts were transplanted from mismatched donors (first class typing was HR in 43, LR in 14 and serological in 1 pt): 22 with single allelic mismatch (2 HLA-A, 7-B, 6-C, 7-DQB1), 33 with single HLA-C antigen mismatch and 3 with double mismatches (2 B+C, 1 C+C). Survival advantage at 4 years, although without statistical significance (p=0.16), was observed in the group of pts transplanted from 10/10 alleles matched donors (36+/− 11%) over those with mismatched donors (24+/− 11%). Oppositely, pts transplanted from matched donors who were not completely typed in HR did not achieve better survival (23+/− 17%). Poorest survival (13+/− 12%, p=0.007) was observed in patients transplanted from mismatched homozygous donors (n=10). The risk of acute GVHD grade 3–4 was increased (p=0.007) in pts with mismatched donors (31+/− 6%) when compared to matched completely typed in HR (10+/− 4%). Also the rate of graft failure tended to be lower in pts with matched than mismatched donors (5.1% versus 10.2%, p=0.25). In contrast, relapse rate was lower in mismatched (23+/− 10%) than in HR matched pts (34+/− 12%, p=0.55) what may reflect better GVL effect in mismatched transplant recipients. Unexpectedly, the rate of chronic GVHD was similar in pts with 10/10 alleles matched and mismatched donors (40+/− 10% versus 42+/− 9%, p=0.75). These results indicate that complete high resolution HLA class I typing is necessary for adequate selection of unrelated donors. Class I HLA-B and -C mismatches influence both survival and serious a-GVHD incidence.

10/10 alleles matched mismatched p survival at 4 years 36+/− 11% 24+/− 11% 0.16 aGVHD grade 3–4 10+/− 4% 31+/− 6% 0.007 graft failure rate 5.1% 10.2% 0.25 relapse rate 34+/− 12% 23+/− 10% 0.55 cGVHD 40+/− 10% 42+/− 9% 0.75 10/10 alleles matched mismatched homozygous donors p survival at 4 years 36+/− 11% 13+/− 12% 0.007