bluuebunny/crossref_metadata_2025 huggingface dataset:
Transplantation of Primary Human CD34+CD38 - Hematopoietic Stem Cells Recapitulates Idiopathic Myelofibrosis in the NOD/scid/IL2rgKO Mice.
Authors: Saito Noriyuki , Ishikawa Fumihiko , Shimoda Kazuya , Yoshida Shuro , Saito Yoriko , Fukata Mitsuhiro , Kawano Noriaki , Shulz Leonard D. , Akashi Koichi , Harada Mine
Published: April 2020

Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM leads to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38- cells from six IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Six out of thirteen recipients transplanted with patient HSCs exhibited human hematopoietic engraftment, and CD33+ myeloid cells accounted for 80.5+/−9.41% of all the engrafted CD45 + population (as compared with the recipients transplanted with normal HSCs). BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was < 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. To investigate the origin of BM fibroblast, we performed FISH analysis using human and mouse centromeric probes and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of sixty fibroblasts examined, fifty-four cells were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38- HSC fraction and these cells possess differentiation capacity to fibroblasts. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and the development of novel therapeutic agents for IMF.
A Prospective Clinical Trial of a Novel Epstein-Barr Virus (EBV) PCR Panel in Patients with EBV Associated Malignancies.
Authors: Xavier Marin F. , Frank Dale , Nasta Sunita , Schuster Stephen , Andreadis Charalambos , Arnoldi Sharon , Sherman Eric , Berg Sigrid , Mato Anthony , Musher Benjamin , Downs Lisa , Porter David , Stadtmauer Edward , Goldstein Steven , Kim Ellen , Tsai Donald
Published: April 2020

EBV is known to be detectable within a number of different tumors, though the exact relationship between the malignant cells and the EBV virus is unknown in most cases. In post-transplant lymphoproliferative disorders, which are clearly EBV-driven, EBV PCR of peripheral blood has proven to be a clinically useful tumor marker. The goal of our study is to determine the utility of novel EBV PCR panel in EBV associated malignancies. Subjects with suspected active or untreated EBV-associated malignancies were evaluated with a 6 assay EBV PCR panel targeting LMP-1, EBER-1 and EBNA-1 within both whole blood cells and plasma. EBV positive subjects and EBV negative control tumor samples are tested for LMP and EBER using traditional histochemical staining. LDH, SPEP, and traditional EBV serology are also measured. Of the 40 patients who were evaluated to date, 18 subjects showed evidence of elevated blood EBV viral load: 3/5 nasopharyngeal carcinoma, 3/4 angioimmunoblastic lymphomas, 3/6 Hodgkin’s lymphomas, 4/10 peripheral T-cell lymphomas, 3/3 allogeneic bone marrow transplant lymphoproliferative disorders, 1/4 diffuse large B-cell lymphomas, 1/1 non-langerhans histiocytosis, and 0/7 low-grade B-cell NHL. Median DNA PCR copies/ml and ranges in EBV positive patients: EBNA plasma 500 (0–134,000), EBNA whole blood 700 (0–6,200), EBER plasma 200 (0–19,700), EBER whole blood 200 (0–1,000), LMP plasma 0 (0–600), LMP whole blood 0 (0–100). Patients were monitored with EBV PCR as they proceeded through treatment and 12 subjects are evaluable for response. Changes in viral load appear to be highly correlated with clinical tumor response (p=0.0013). 7/7 patients achieving CR had concurrent resolution of their plasma and whole blood EBV viral load. 5/5 patients with persistent disease had persistent elevated plasma and whole blood viral load. EBV histologic studies on the tumor samples are pending. Our data suggests that EBV PCR may be useful in the management of patients with EBV positive malignancies. Further patient enrollment and evaluation is ongoing.
Downregulation of a Candidate Myeloid Tumor Suppressor Gene Isolated from 7q21 Induces Abnormal Mitosis Due to Insufficient Centrosome Maturation.
Authors: Asou Hiroya , Matsui Hirotaka , Ozaki Yuko , Nagamachi Akiko , Aki Daisuke , Ohsugi Miho , Inaba Toshiya
Published: April 2020

To isolate myeloid tumor suppressor gene(s) in 7q, we searched microdeletions in a region spanning 21.7 Mb within 7q21.2–7q31.1 using a microarray-based CGH system. By investigation of 21 childhood myeloid leukemia patients with normal karyotype, we identified a common microdeletion cluster spanning approximately 120 Kb in the 7q21.3 subband. Eight (38%) patients shared this microdeletion, which was not detected in normal individuals. Real-time quantitative PCR revealed that this region is also deleted in 9 (29%) out of 31 adult RAEB and AML patients. Database search revealed that this region contains three hypothetical genes. Among them, we chose one previously uncharacterized gene for further investigation and named Miki (mitotic kinetics regulator) for the function of its gene product, described below. Immunoblot analysis revealed high levels of Miki expression in most lymphoid leukemia cell lines, while half of myeloid leukemia cell lines expressed Miki at reduced levels. In six leukemia lines carrying monosomy 7, expression levels were generally low. Miki co-localized with the Golgi apparatus in the interphase and with centrosomes and spindles in the mitotic phase. To test the function of Miki, we used si-RNAs to downregulate Miki expression in HeLa and K562 cells, both of which show basically normal metaphase and nuclear morphology. Cells expressing Miki at reduced levels showed insufficient maturation and disturbed positioning of centrosomes, resulting in unorganized spindles including loss of spindle tension, curled and fragile spindles, or even completely disturbed spindle formation. Time-lapse observation revealed prometaphase and/or metaphase delay with unaligned or even totally scattered chromosomes in prometaphase in virtually all cells in the mitotic phase. As a result, cells underwent pre-anaphase arrest and exited mitosis in the absence of chromosome segregation or terminated mitosis by cell death. In the interphase, there were many cells with chromatid bridges and/or bi- or tri-nuclear or even multinuclear cells with micronuclei that resembled pathological cells routinely observed in the bone marrow pictures of MDS patients. Interestingly, myeloid cell lines with low Miki expression, including those with monosomy 7, generally showed abnormal mitosis such as scattered chromosomes and abnormal nuclear morphologies (multi-nuclear cells with small nuclei) at higher frequency than cell lines expressing Miki at high levels. Moreover, induction of Miki restores normal mitosis in leukemia cells with monosomy 7. Miki was poly(ADP-ribosyl)ated (PARsylated) in late G2 to M phase by tankyrase-1, one of PAR polymerase (PARP), and tankyrase-1 activity was required for the binding of Miki to spindles and centrosomes. These data suggest that loss of Miki gene contributes to the development and progression of MDS by disturbing mitosis.
Identification of SCL and LMO Oncogenes as Transcriptional Repressors of HEB/E2A in the Thymus through Functional Genomic/Genetic Approaches.
Authors: Tremblay Mathieu , Herblot Sabine , Todorova Margarita , Lisi Veronique , Major Francois , Lemieux Sebastien , Hoang Trang
Published: April 2020

The SCL and LMO oncogenes are frequently activated in childhood T cell acute leukemia (T-ALL). SCL is a bHLH transcription factor that forms heterodimers with other members, specifically HEB and E2A. SCL can either activate or repress transcription but its mechanism of action as an oncogene remains to be clarified. Ectopic expression of SCL and LMO in transgenic mice causes thymocyte differentiation arrest during the preleukemic phase with aberrant differentiation at the DN3–DN4 stage, prior to the acquisition of CD4 and CD8. We therefore took several approaches to define the mechanism underlying this differentiation arrest. Using a CD3e-deficient background to eliminate preTCR signaling effects, an event that is not essential for leukemogenesis, we first analyzed global gene expression of pre-leukemic SCLtg/LMOtg DN3 thymocytes against their non-transgenic littermates via the Affymetrix platform. In this context, SCL/LMO globally affects the expression of both positive and negative targets of E2A/HEB. These results were confirmed by real-time quantitative PCR, showing that SCL/LMO repress E2A/HEB activity. Second, bioinformatic analysis of genes significantly affected by SCL/LMO allowed us to identify phylogenetically conserved HEB/E2A binding sites within the promoter regions of half of these genes. E2A/HEB binding was directly assessed through chromatin immunoprecipitation of primary thymocytes, confirming known target genes and revealing novel ones. This assay also showed that SCL associates with HEB and/or E2A on DNA. Moreover, structure function analysis indicate that transcription inhibition by SCL depends on its HLH domain but not on LMO interaction. We therefore conclude that SCL inhibits thymocyte differentiation by inhibiting E2A/HEB targets that include genes required for thymocyte differentiation, cell signalling, DNA repair and replication. Finally, using a genetic approach we show that SCL/LMO collaborates with HEB haploinsufficiency in inducing leukemia. Our observations therefore reveal that the repression of HEB/E2A by SCL/LMO is a crucial step in T cell transformation.

Support from CIHR and FRSQ.
Bcl11a Causes p21Cip1 Down-Regulation and Transplantable Leukemia in Nf1-Deficient Mice.
Authors: Yin Bin , Delwel Ruud , Valk Peter J. , Loh Mignon L. , Shannon Kevin M. , Largaespada David A.
Published: April 2020

Inactivation of the tumor suppressor gene, NF1, is associated with chronic and acute forms of myeloid diseases including juvenile myelomonocytic leukemia. Nf1-deficiency mouse models have shown some phenotypes reminiscent of those of the human disease. However, Nf1mutation in itself does not lead to leukemia. We reported the identification of 37 common proviral insertion sites, loci containing candidate genes whose mutation could cooperate with inactivation of Nf1 to result in BXH-2 AML. Among these candidate genes, Bcl11a, was able to cause leukemia/lymphoma in bone marrow transduction and transplantation assay using Nf1-deficient cells. In this report, we have found that transplantation of these leukemia cells induced secondary leukemia in syngeneic recipient mice. In patients, we previously reported that BCL11A was expressed in juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia samples. Interestingly, high expression levels of BCL11A are also observed in two specific subsets of samples from a large cohort of patients with acute myelogenous leukemia. Moreover, these subsets of patients tended to have a worse outcome. Regarding the biochemical mechanisms underlying the oncogenicity of Bcl11a, we have recently found that p21Cip1 induction by doxorubicin is severely impaired in Bcl11a-expressing cells, while other cell-cycle regulatory proteins are not affected. Examination of BCL11A’s role in leukemia patients using both over-expression and shRNA knocking-down approaches is underway.
Galiximab Sensitizes Malignant Human B Cell Lines to Apoptosis by Chemotherapeutic Drugs.
Authors: Baritaki Stravroula , Suzuki Eriko , Vega Mario , Ho Steffan , Hariharan Kandasamy , Bonavida Benjamin
Published: April 2020

Galiximab is a primatized anti-CD80 antibody that is being investigated as a treatment for non-Hodgkin’s lymphoma. Results to date from clinical trials indicate that galiximab is well-tolerated and suggest clinical activity both as monotherapy and in combination with rituximab (anti-CD20). CD80 (B7.1), expressed not only by malignant B cells but also by normal B cells, monocytes/macrophages, dendritic cells as well as T cells, plays an important role in the complex and dynamic regulation of adaptive immunity by interacting with either co-stimulatory (e.g. CD28) or co-inhibitory (e.g. CTLA-4, PD-L1) receptors expressed by T lymphocytes. Inhibition of anti-lymphoma immunity that exists within the tumor microenvironment may reflect selection of inhibitory immune regulation mediated through CD80. Galiximab may thus function by directly targeting malignant and possibly non-malignant CD80-expressing cells for antibody-dependent cytotoxicity, or by blocking CD80 interactions involved in suppressing anti-tumor immunity. In preclinical xenograft models, which do not assess the effect of galiximab on non-malignant cells given a lack of cross-reactivity with murine CD80, treatment with galiximab exhibited enhanced tumor growth inhibition when used in combination with either rituximab or with the chemotherapeutic drugs fludarabine and doxorubicin (Hariharan et al., abstract 3040, ASCO, 2007). However, the mechanisms by which galiximab enhances the cytotoxic effects of chemotherapy remain unclear. Rituximab has been shown by us to chemosensitize tumor cells by inhibiting intracellular survival pathways. Thus, we hypothesized that treatment of B lymphoma cells with galiximab facilitates the cytotoxic activity of chemotherapeutic drugs by modifying intracellular anti-apoptotic signaling pathways, resulting in the alteration of pro and anti-apoptotic gene products. The above hypothesis was examined using Raji (Burkitt lymphoma) and IM-9 (multiple myeloma) cell lines as models. Treatment with galiximab resulted in significant inhibition of cell growth and proliferation and modest apoptosis with high concentration. Both Raji and IM-9 are resistant to chemotherapeutic drugs; however, pretreatment with galiximab significantly sensitized the tumor cells to apoptosis induced by CDDP. The synergy achieved was found with subtoxic concentrations of CDDP (5–10 ug/mL) and galiximab. Analysis of the survival pathways and gene products regulating apoptosis following treatment with galiximab, CDDP, and combination will be presented. The present findings demonstrate that galiximab can synergistically enhance cytotoxicity mediated by chemotherapeutic drugs. These findings suggest that in vivo galiximab may have a chemosensitizing effect on resistant B cell malignancies, in addition to its direct cytotoxic effect via ADCC and CDC. The clinincal relevance of these findings will be discussed.
Phase II Pilot Study of Rituximab + CHOP in Patients with Newly Diagnosed Waldenström’s Macroglobulinemia, an Eastren Cooperative Oncology Group Trial (Study E1A02).
Authors: Abonour Rafat , Zhang Lijun A. , Rajkumar Vincent , Srkalovic Gordan , Greipp Philip R. , Fonseca Rafael , Gertz Morie
Published: April 2020

Waldenström’s Macroglobulinemia (WM) is a low-grade lymphoplasmacytic disorder associated with an IgM monoclonal protein that may present with anemia, lymphadenopathy, and hepatosplenomegaly. Since combination chemotherapy and Rituximab are both active in this disease, it is possible the response rate can be enhanced by using a combined modality of Rituximab and CHOP. This approach has been used in indolent lymphoma with remarkable results and limited toxicity.

Objective: To determine the response rate in previously untreated patients with Waldenström’s Macroglobulinemia receiving rituximab and CHOP.

Methods: This was a phase II study of standard R-CHOP in symptomatic WM patients. The study was activated on June 15, 2004 and closed on April 26, 2007 due to poor accrual. The median age of the sixteen patients enrolled was 60 (44–79 years), β2M 3 ug/ml (2.1–7.6), median hemoglobulin was 9 g/dl (7.7–10.9), Viscosity 3.4(1.6–10), and IgM 6389 (3229–13300 mg/dl). The most common symptoms were Fatigue (13/16), visual disturbance (7/16) and parasthesias or painful/numb feet (6/16). Only one patient had bulky adenopathy (6.3%).

Results: Of 16 patients treated, 5 patients have no response data available (it will be presented at the meeting). Objective response was 91% (90% CI: 63.5% ∼ 99.5%) and 1 patient had a minor response with an overall response rate of 100%. Median time to response was 1.6 months from registration and median time to maximum response was 2.1 months. Median duration of response has not been reached. The median follow up time of the 16 patients was 18.3 months with a range of 3.6–24.8 months. Among the 16 treated patients, there were 8 (50%, 90% exact binomial CI: 27.9%∼72.1%) grade 4 neutropenia. 8 patients experienced grade 3 lymphopenia (50%, 90% exact binomial CI: 27.9%∼72.1%). Only 2 grade 3 febrile neutropenia were noted. As of August 4, 2007, none of the 16 patients has died. R-CHOP is a promising regimen in the treatment of patients with Waldenström’s Macroglobulinemia. To succeed in completing clinical trials in this rare disease better international collaboration and involvement in advocacy groups are required.
Activation of Transforming Growth Factor β1 (TGF-β1) Released by Platelets Is Enhanced by Shear and Occurs in Platelet-Rich Thrombi In Vivo.
Authors: Ahamed Jasimuddin , Burg Nathalie , Janczak Christin , Li Jihong , Coller Barry S.
Published: April 2020

Platelets contain 40–100 times as much transforming growth factor β1 (TGF-β1) as other cells, and release it as an inactive (latent) complex. Although several TGF-β1 activating factors have been identified in various cell types, it is still not known whether and how platelet TGF-β1 becomes activated in vivo. In the present study, cell-free human washed platelet releasates (supernatant after stimulation with thrombin 0.125U/ml for 5 min) or serum samples were subjected to either stirring (1,200 rpm) in an aggregometer or a shear stress of 1800 s−1 in a cone and plate device at 37°C. Activation of TGF-β1 was analyzed by ELISA and selectively confirmed using a cell-based plasminogen activator inhibitor 1 (PAI-1) luciferase reporter assay. TGF-β1 was maximally released within 5 min after stimulation of platelets with thrombin (80 ± 23 ng/ml; n=3), but only 0.2% was active. TGF-β1 activity increased slowly and progressively under shear, reaching a maximum of ∼5% of total TGF-β1 (4.1 ± 1.5 ng/ml) after 1 h. In sharp contrast, in two experiments conducted without shear, active TGF-β1 constituted 0.04 and 0.2% of total at 0 time and remained nearly the same (0.1 and 0.2% of total) after 2 h. Activation of TGF-β1 in serum under shear occurred more slowly, but also reached a maximum of ∼5% of total at 2 h (4.1 ± 1.3 ng/ml active; 76 ± 14 ng/ml total; n=6). Without shear at 0 time, active TGF-β1 constituted 0.02 and 0.06% of total and at 2 h it was still only 0.05 and 0.07% of total. To asses whether TGF-β1 becomes activated in vivo during thrombosis, we induced thrombi in the carotid arteries of C57/BL mice with ferric chloride (8%) for 3 min and then removed the platelet-rich thrombi that formed after 5 or 120 min. The arteries were excised (∼4 mm) and the thrombi were removed and dispersed in buffer (200 μl) on ice for 1 h. Total TGF-β1 recovered from 5 min thrombi was 3.4 and 4.8 ng/ml and total TGF-β1 recovered from 120 min thrombi was 3.7 and 0.7 ng/ml. Active TGF-β1 could be detected in thrombi after 5 min and constituted 1.7 ± 1.1% of the total TGF-β1 recovered (n=3). After 120 min, active TGF-β1 could also be detected in thrombi and it constituted 5.1 ± 3.0% of the total recovered (p=0.14 compared to 5 min value; n=3). In sharp contrast, only 0.05% of TGF-β1 released by thrombin from mouse platelets in vitro was active without stirring or shear (n=3). These data indicate that shear can dramatically enhance TGF-β1 activation after release from platelets. Moreover, TGF-β1 activation occurs in vivo during platelet-rich thrombus formation and thus platelets may be an important source of active TGF-β1 in cardiovascular disease and wound healing.
CalDAG-GEFI and Protein Kinase C (PKC) Represent Alternative Pathways Leading to Activation of Integrin αIIbβ3 in Platelets.
Authors: Bergmeier Wolfgang , Crittenden Jill R. , Piffath Crystal L. , Cifuni Stephen M. , Wagner Denisa D.
Published: April 2020

Second messenger-mediated inside-out activation of integrin αIIbβ3 is a key step in platelet aggregation. We have recently shown strongly impaired aggregation in response to various agonists in CalDAG-GEFI-deficient platelets. Thrombin-induced aggregation, however, was affected only at low but not high doses of the agonist, suggesting CD-GEFI-dependent and -independent pathways for integrin activation in platelets. In this study, we further evaluated the roles of CalDAG-GEFI and PKC for integrin activation in platelets activated with a PAR4 receptor-specific agonist, GYPGKF (PAR4p). Compared to wild-type (WT) controls, platelets treated with the PKC inhibitor Ro31–8220 or CalDAG-GEFI-deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. In contrast, aggregation/αIIbβ3 activation was significantly reduced at any tested PAR4p concentration in CalDAG-GEFI-deficient platelets pre-treated with Ro31–8220. PAR4p-induced αIIbβ3 activation in the absence of CalDAG-GEFI required co-signaling through the Gi-coupled receptor for ADP, P2Y12, while this was not the case in WT platelets treated with Ro31–8220. Consistent with these results, release of ADP-containing dense granules was completely inhibited by Ro31–8220 at all PAR4p concentrations tested. In contrast, dense granule release was impaired at low but not high dose PAR4p in CD-GEFI-deficient platelets. Independent roles of CalDAG-GEFI- and PKC/Gi-signaling were also observed for PAR4p-induced activation of Rap1, with CalDAG-GEFI mediating the rapid activation of this small GTPase. In summary, our studies identify CalDAG-GEFI and PKC as independent pathways leading to Rap1 and αIIbβ3 activation in mouse platelets activated through the PAR4 receptor.
PECAM-1 Is a Negative Regulator of Platelet Activation by Laminin.
Authors: Crockett Jimmy , Newman Peter J.
Published: April 2020

Interaction of resting platelets with exposed components of the subendothelial matrix is an important activating event that takes place early on at sites of vascular injury. Platelet responses to collagen are mediated both by the integrin α2β1 and the glycoprotein (GP)VI/Fc receptor (FcR) γ chain complex. Upon collagen binding, Src family kinases phosphorylate tyrosines within Immunoreceptor Tyrosine-based Inhibitory Motifs (ITAMs) that are present within the cytoplasmic domain of the FcRγ chain. These phosphorylated ITAMs serve as docking sites for the protein-tyrosine kinase Syk, which, when recruited and activated, initiates the rapid assembly of a series of adaptor proteins and protein-tyrosine kinases that are crucial for platelet activation responses. Recently, platelet activation by laminin, which binds the related integrin α6β1, was shown to also require signaling through the GPVI/FcRγ chain complex. Because the adhesion and signaling receptor, PECAM-1, serves as a negative regulator of collagen-induced platelet activation, we sought to determine whether PECAM-1 might similarly regulate platelet activation by laminin. We found that PECAM-1 became rapidly tyrosine phosphorylated on its cytoplasmic ITIMs following adhesion of either human or murine platelets to immobilized laminin, suggesting that it may be involved in modulating platelet responses to laminin. PECAM-1-deficient murine platelets exhibited both an increased rate and extent of cell spreading on immobilized laminin compared to their PECAM-1-positive, wild-type (wt) counterparts. Granule secretion, as determined by release of serotonin, was also augmented. Taken together, these data support the notion that PECAM-1 is a negative regulator of platelet responses to laminin.
Anemia in Congestive Heart Failure: The Anemia Is Common and Is Associated with More Severe CHF - Results of a National Survey.
Authors: Oster Howard S. , Hoffman Michael , Cohen Eythan , Mittelman Moshe
Published: April 2020

Anemia has been recognized as a risk as well as a precipitating factor in ischemic heart disease, and in congestive heart failure (CHF). However, there are still open questions regarding the prevalence of anemia in CHF, and the severity and associated clinical manifestations of the anemic CHF patient. In order to characterize CHF in Israel, a national survey was conducted during the period March 1–April 30, 2004. Data were collected on all patients with CHF exacerbation admitted to 96 departments of medicine and 26 cardiology units throughout the country. In this study we have focused on anemia in CHF patients with a special emphasis on the differences observed between CHF patients with anemia (A group) and CHF patients with no anemia (NA group).

Results: Of 2388 patients admitted for exacerbation of CHF, 1326 (55.5%) were found to have anemia (<13g/dl for men, <12g/dl women). Both A and NA groups had similar male/female ratio (57.2%/42.8% A vs 53.5%/46.5% NA). The anemic patients (A) tended to be older (74.8 yrs A vs 72.5 yrs NA, p=0.00001), to have more prior hospitalizations (2.0 A vs 1.3 NA, p=0.0001), and to suffer from a more severe CHF. For example, 49.3% of anemic vs only 41% of non-anemic patients were reported to have baseline New York Heart Association (NYHA) class III–IV. Most notably both in-hospital and six-month mortality rates were significantly higher among anemic CHF patients (in-hospital: 6.6% A vs 4.6% NA, p=0.039; six-month: 25% A vs 15% NA, p<0.00001). Interestingly, no difference was observed between the two groups in the percentage of CHF patients taking aspirin prior to admission. However, fewer anemic patients were discharged with aspirin (63.7% A vs 72.7% NA, p=0.00001).

Conclusions: In this large survey of patients with CHF exacerbation, anemia was found to be common and the anemic patients were found to be older, to have more prior hospitalizations, more severe CHF (NYHA class III–IV), and higher in-hospital and six-month mortality rates. Because both groups had similar rates of aspirin usage, it may be suggested that aspirin plays no significant role in the development of anemia in such patients. Thus, the common practice of withholding this medication from anemic CHF patients may deserve reevaluation. Our data call for a prospective clinical trial, which will test the possibility that correction of the anemia (by RBC transfusion or recombinant erythropoietin) can improve the clinical outcome of the anemic CHF patient.
Thrombopoietin Stimulates the Formation of Endothelial Cell-Cell Contacts and Activates the Small GTPase Rap1.
Authors: Tijssen Marloes R. , di Summa Franca , van Stalborch Anne-Marieke , Van der Schoot C. Ellen , van Buul Jaap D. , Hordijk Peter L. , Voermans Carlijn
Published: April 2020

In addition to hematopoietic cells, the receptor for thrombopoietin (Tpo), c-Mpl, is expressed on human umbilical vein endothelial cells (HUVEC). In this study, we demonstrated that Mpl mRNA and protein can also be detected in immortalized human bone marrow endothelial cells (HBMEC). Furthermore, it was investigated whether Tpo affects endothelial monolayer integrity by either cell spreading or cell-cell contacts. By continuously monitoring the electrical resistance of freshly plated HUVEC or HBMEC with ECIS (electric cell-substrate impedance sensing), we observed a significant increase in the electrical resistance when endothelial cells were treated with 100 ng/ml of Tpo just prior to seeding, as shown in the figure below. This increase in electrical resistance was not due to increased cell adhesion or proliferation of the endothelial cells. However, a significant increase in initial spreading could be detected. Besides its effect on spreading, Tpo increased the electrical resistance of a confluent endothelial monolayer when compared to untreated monolayers. This indicates that Tpo promotes the formation of endothelial cell-cell contacts. Indeed, a decrease in immunostaining levels of phosphotyrosine could be observed in monolayers treated with Tpo. Also the junctional protein VE-cadherin showed diminished co-localization with phosphotyrosine staining when cells were treated with Tpo. The small GTPase Rap1 plays an important role in the formation of endothelial cell-cell contacts. In a pull-down experiment, using RalGDS as bait for active Rap1, we could demonstrate that Tpo transiently activates Rap1 in HUVEC. Thus, these results may put Tpo forward as a regulator of endothelial cell-cell contacts, possibly after vascular damage and subsequent release of Tpo by activated platelets. To our knowledge, Tpo is the first cytokine exerting this effect on endothelial cells.

Figure Figure
The GATA-1/FOG-1/NuRD Axis Is Essential for Normal Hematopoiesis.
Authors: Hong Wei , Wang Hongxin , Wang Yuhuan , Choi John K. , Shelat Suresh G. , Poncz Mortimer , Blobel Gerd A.
Published: April 2020

GATA-1 controls the development of erythroid cells and megakaryocytes through its ability to activate and repress gene transcription. GATA-1 binds many nuclear proteins, but only a few of these associations have been examined in vivo. One important example is FOG-1, a critical cofactor that contributes to both gene activation and repression by GATA-1. Loss of FOG-1 generally phenocopies GATA-1 deficiency, impairing both erythroid and megakaryocytic differentiation. We reported previously that FOG-1 directly binds the NuRD protein complex, which contains histone deacetylase and chromatin remodeling activities. This provides one mechanism for GATA-1/FOG-1-mediated gene repression. Accordingly, ChIP profiling of the NuRD proteins MTA-2, RbAp46 and Mi-2β revealed the presence of these molecules at the Kit and Gata2 genes both of which are directly repressed by GATA-1 in a FOG-1-dependent manner. NuRD proteins were spread broadly across the Kit and Gata2 genes but were further enriched at sites occupied by GATA-1 and FOG-1 in vivo. Unexpectedly, we also observed NuRD components at GATA-1-activated genes including β-globin and Ahsp. Moreover, the ability of FOG-1 to augment GATA-1-induced transcription in transient transfection assays required NuRD binding. Hence, NuRD may be bi-functional, contributing to either gene activation or repression, depending on the transcriptional and cellular context. To study the role of the FOG-1/NuRD interaction in vivo we generated mice bearing missense mutations in the Fog-1(Zfpm1) gene that disrupt NuRD binding in the FOG-1 protein. Homozygous mutant mice are born at reduced Mendelian ratios. Surviving animals display ineffective erythropoiesis marked by splenomegaly and impaired erythroid maturation. In addition, homozygous mutant animals display macrothrombocytopenia with impaired platelet function. Thus, recruitment of NuRD by GATA-1 and FOG-1 is essential for both erythropoiesis and megakaryocytopoiesis. Ongoing studies include further phenotypic analysis of the mutant mice, including comparative gene expression analysis in stage-matched wild-type and mutant erythroid cells to identify critical NuRD-dependent GATA target genes, and to resolve whether NuRD is essential for both activation and repression by GATA-1 and FOG-1 in vivo. An important open question under investigation is how recruitment of the NURD complex can lead to suppression of some genes and the enhanced expression of others. The FOG-1/NuRD mutant mice provide useful tools to dissect transcription pathways initiated by GATA-1. Moreover, given the role of GATA-1 mutations in congenital anemias and megakaryoblastic leukemias, enzymatic components of the NuRD complex may provide novel targets for pharmacologic manipulation to treat these disorders.
A Four Protein Plasma Fingerprint of Acute Graft Versus Host Disease (GVHD) Predicts Long Term Survival.
Authors: Paczesny Sophie , Choi Sung , Braun Thomas , Kitko Carrie , Oleg Krijanovski , Clouthier Shawn G. , Weyand Angela , Bickley Dan , Jones Dawn , Whitfield Joel , Levine John E. , Ferrara James L.
Published: April 2020

There are no independent laboratory diagnostic tests for acute GVHD. We first identified 7 potential plasma biomarkers (IL-2R-α, CRP, IL-8, TIMP-1, TNFR1, HGF, CA-19.9) of acute GVHD using a combination of proteomic approaches and antibody microarrays. We next conducted a retrospective analysis using plasma samples from 424 patients at the University of Michigan under IRB approval. We obtained samples at the first clinical signs of acute GVHD prior to treatment and at equivalent time points in patients without GVHD (Table 1). The median duration of follow-up was 420 days with a minimum follow-up of 180 days. Patients with veno-occlusive disease, idiopathic pneumonia syndrome, or septic shock were not included. We measured plasma levels of the 7 proteins by sequential ELISA. Logistic regression models with and without leave-one-out-cross-validation (LOOCV) tested the correlation of the laboratory values with the diagnosis of acute GVHD using area under the receiver-operating-characteristic (ROC) curves (AUC). The training set consisted of 282 randomly selected patients; the validation set included the remaining 142 patients. The final optimal fingerprint of four proteins excluded CRP because of its association with non-specific inflammation and included IL-2R-α, TNFR1, IL-8 and HGF, with AUCs of 0.91 and 0.89 in the training set (without and with LOOCV, respectively) and 0.86 in the validation set. The fingerprint had a strong association with grade of GVHD (p<0.001) and target organ (p=0.002) at onset; interestingly, HGF had the strongest association. Using a predicted probability of acute GVHD of at least 50%, the fingerprint had a 72% sensitivity and 89% specificity. When we categorized the predicted risk of acute GVHD into low (0.00–0.59), moderate (0.60–094) and high (0.95–1.00), the plasma fingerprint predicted long-term survival (Figure 1, p<0.001). We conclude that this plasma protein fingerprint has good sensitivity, high specificity, strong association with initial grade and target organ of acute GVHD, and effectively stratifies patients into three risk groups for GVHD that correlate with long term survival.

Figure Figure Table 1: Patients characteristics GVHD- (N=242) GVHD+ (N=182) Age-yr Median (range) 45 (1–69) 49 (1–71) Donor type (%) MRD: 169 (70%) MRD: 105 (58%) URD: 73 (30%) URD: 77 (42%) Conditioning regimen Intensity (%) Full: 182 (75%) Full: 114 (63%) Reduced: 60 (25%) Reduced: 68 (37%) Day after BMT of samples : median (range) 30 (7–104) 29 (5–119) Grade at GVHD Onset (%) Grade 0 Grade 1 Grade 2 Grade 3–4 242 (57%) 48 (12%) 100 (24%) 34 (7%) Organ Target at GVHD Onset (%) n/a Skin Gut Liver Combined 119 (65%) 38 (21%) 7 (4%) 18 (10%)
Susceptibility of Endocrine, Cardiac, and Macrophage Cell Lines to Iron-Mediated Oxidative Damage and the Cytoprotective Effect of the Orally Active Chelator Deferasirox (Exjade®, ICL670).
Authors: Glickstein Hava , Nick Hanspeter , Cabantchik Zvi I.
Published: April 2020

Systemic iron overload (primary or secondary) affects hepatic and extrahepatic functions by damaging endocrine and cardiac tissue. In vitro studies with pancreatic Min6 and pituitary Att20 cells and with cardiac H9c2 cells (all highly active in endocytotic activity) indicated that their exposure to labile iron, acutely or chronically, lead to major intracellular iron accumulation in organelles (endosomes, mitochondria, cytosol) and increased reactive oxygen species (ROS) formation when redox challenged. Among the functions affected by metal-evoked ROS are permselectivity (calcein leakage), mitochondrial ΔΨ (JC1 test), electron transport activity (Alamar Blue) and cell viability (calcein-propidium iodide). The administration at therapeutically achievable doses of deferasirox (30–100 μM) or deferoxamine (DFO) [10 μM] largely (>70%) prevented labile iron from rising in cells if present in the iron-loading medium; however, only deferasirox reduced iron-evoked cell damage and increased cell viability if incubated with cells prior to or post iron-loading (acute or chronic). Thus, deferasirox has both preventive and corrective potential against iron-evoked damage in iron-loaded endocrine and cardiac cells. A gradual reduction of serum concentration from 10% (normally used in culture conditions) to 1% or less (used for experimental testing of drugs) revealed a commensurate increased susceptibility of endocrine and cardiac cells to deferasirox in the higher concentration range of 50–100 μM (48–72 cell viability test). This indicates that the plasma-binding property of deferasirox has a cytoprotective effect.
Deficient Th-1 Responses from TNFα-Matured Dendritic Cells.
Authors: Decker William K. , Li Sufang , Xing Dongxia , Robinson Simon N. , Yang Hong , Steiner David , Bollard Catherine M. , Komanduri Krishna V. , Shpall Elizabeth J.
Published: April 2020

INTRODUCTION: The ultimate success of dendritic cell (DC) vaccination for the active immunotherapy of neoplasia involves a wide variety of variables including the method of DC generation, loading, and maturation; the DC dose and route of administration. A maturation cocktail comprising IL-1b, TNF-a, IL-6, and PGE2 (ITIP) is commonly employed clinically, as is the use of the single inflammatory cytokine TNF-a. Here we compared these two methods with respect to DC maturation, phenotype, and function.

METHODS: CD14+ monocytes were isolated from normal donor G-CSF-mobilized peripheral blood progenitor cells using the mini-MACS system (Miltenyi Biotec), and cultured for 6 days in AIM-V medium containing GM-CSF and IL-4 (Cellgenix). Immature DCs were loaded with both acute myelogenous leukemia (AML) tumor lysate and mRNA as previously described1 and then matured with the ITIP cocktail, TNF-α alone, or a combination of TNF-α and CD40 agonism (Becton-Dickinson). Mature DCs were analyzed phenotypically and then cultured with autologous T-cells.

RESULTS: Data generated from three different normal donors indicated that DCs matured with TNF-α were deficient in important costimulatory surface molecules CD80, CD83, and CD40 as well as in IL-12 production (p=0.001 in representative experiment). Analysis of T-cell functionality by IFN-γ ELISpot also demonstrated greatly decreased Th-1 functionality among lymphocytes stimulated by TNFα-matured DCs in comparison to ITIP-matured DCs (p=0.004 in representative experiment). Additionally, lymphocytes stimulated by TNFα-matured DCs were not able to proliferate significantly whereas ITIP-matured DCs mediated robust lymphocyte expansions consisting of up to 70% CD8+ cells. The addition of a CD40 agonist antibody to TNF-α during the DC maturation process could partially, but not completely overcome these observed functional deficiencies.

CONCLUSIONS: The data suggest that DCs loaded with primary tumor antigens and then matured with TNF-a might be deficient in the induction of type 1 T-helper responses. The absence of critical costimulatory markers and IL-12 secretion suggest that DCs matured with TNF-α might be suboptimal for the priming of naïve CD8 responses. These findings might help to improve the clinical efficacy of future DC vaccination studies.
Use of Interleukin 6, High Sensitivity CRP, TNFα and Procalcitonin as Early Markers of Sepsis in Febrile Neutropenic Children.
Authors: Kharya Gaurav , Yadav Satya Prakash , Srivastava Lalit M. , Sachdeva Anupam
Published: April 2020

Objective: Children with cancer often develop febrile illnesses after cytotoxic chemotherapy. It is of paramount importance to determine which children have serious bacterial infections in this vulnerable period. We studied the ability of interleukin 6 (IL-6), high sensitivity C-Reactive Protein (hsCRP), tumor necrosis factor alpha (TNFα) and procalcitonin (PCT) to act as a sensitive diagnostic marker for bacterial sepsis in febrile neutropenic children and their utility as a predictive marker for serious infections.

Design: Prospective cohort study.

Setting: Sir Ganga Ram Hospital.

Patients: Sixty-two episodes in children (aged 5 months to 17 yrs) with a known malignancy/aplastic anemia who presented with fever and neutropenia.

Interventions: Serial blood samples were obtained (admission, and 48 hrs), and concentrations of PCT, IL-6, TNF α and hsCRP were determined. Demographic and laboratory data from the patients were collected from the medical records.

Measurements and Main Results: Out of 62 episodes of febrile neutropenia, fifteen (24%) of the children had proven sepsis (13 positive blood cultures, 1 invasive aspergillosis and 1 local abscess with fever). Plasma levels of IL-6, hsCRP, TNFα and PCT were increased at all time points in children with sepsis compared with those without sepsis. In culture positive patients the value of TNF-alpha was higher but statistically not significant. No major differences were found in hsCRP concentrations. IL-6 at 0 and 48 hrs were reliable markers for sepsis (area under the curve 0.73 and 0.77, respectively). Using cutoff values of IL-6 > 137.5 pg/mL at 0 hrs and >69 pg/mL at 48 hrs of admission produced a screening test for sepsis with 67% sensitivity, 75% specificity at 0 hrs and 75% sensitivity and 78% specificity at 48 hrs respectively. Also using the cutoff values of 1.73ng/ml for procalcitonin at 0 hrs was a good screening test with 73%sensitivity and 70% specificity. When IL-6 cutoff was brought down to 20pg/ml at 0 hr then sensitivity increased to 100%. 42/62 children with febrile neutropenia had IL6 >20 which included all 15 proven sepsis cases and all 6 deaths. Rest of 20 patients all had negative cultures and no deaths. PCT value of >1 ng/ml was seen in 39/62 and included 12 of 15 cases of proven sepsis. PCT was < 1ng/ml in a case of invasive aspergillosis and another case of candida line infection and a case of local abscess with fever.

Conclusions: Our data shows the utility of a rapid and sensitive assay for interleukin-6 along with procalcitonin as a sensitive diagnostic marker of bacterial sepsis in febrile neutropenic children. The use of these markers as a clinical tool may allow for early discharge on oral antibiotics or no antibiotics in patients with IL6 less than 20 pg/ml and PCT less than 1 ng/ml.
Identification of Specific TCR Vβ3 and Vβ13 Genes Relate to Diffuse Large B-Cell Lymphoma-Associated Antigen.
Authors: Yin Qingsong , Li Yangqiu , Tan Huo , Chen Shaohua , Yang Lijian , Ye Jingmei , Li Bo
Published: April 2020

Diffuse large B-cell lymphomas (DLBL) are the most common type of non-Hodgkin’s lymphoma (NHL), account for 30% to 40% of NHL. Treatment with conventional combination chemotherapy produces complete remission rates of 50 to 70%. Target therapy such as anti-CD20 monoclonal antibody (rituximab) had shown favourable results. In recent years, specific adoptive immunotherapy with tumor-specific T-cells was considered as a new and promising treatment in malignancy. Thereby, researches are focused mainly on the generation of effective antigen specific T-cells, both autologous and allogeneic specific T cells. The antigen specific T cells could be determined by analysis of the T-cell receptor (TCR) Vα or Vβ repertoire, which display monoclonal or oligoclonal expansion. In order to develop the specific cellular immunotherapy in DLBL, in the present study, we analysis at first the distribution and clonality of TCR Vβ repertoire in peripheral blood T cells by RT-PCR and genescan technique, which was expected to define the DLBL associated TCR Vβ subfamily T cells. 6 cases with DLBL were used in the study. The skew distribution and clonal expansion of TCR Vβ subfamily T cells could be found in all patients with DLBL. Monoclonal expanded T cells were identified in 3 cases, which used the Vβ3 (1 case) and Vβ13 (2 cases) subfamilies, the PCR products from the monoclonal T cells were analyzed by direct sequencing to define CDR3 sequence. The CDR3 sequences from these 3 TCRβ genes were identified, which were not identical in the reported TCR sequences in Genebank. The whole sequences of Vβ3 and Vβ13 found in 3 patients with DLBL were amplified and cloned, which are being registered in NCBI Genebank. These specific TCR Vβ genes will be farther used for research on generation of CTL by TCR gene-modified technique, and so on. In the clinical observation showed that patients who had specific reactive T cell clones had a preferable prognosis, suggesting that these detectable clonal expanded T cells might play a crucial role in tumor eradication.
Effect of Staphylococcal Enterotoxin A on the Distribution and Clonal Expansion of TCR Vβ Subfamilies and the Cytotoxicity of T Cells Stimulated by PML-RARα Peptid.
Authors: Lin Chen , Chen Shaohua , Yang Lijian , Bai Xue , Li Yangqiu
Published: April 2020

Bacterial superantigens (SAgs) are potent T cell activators. Superantigens stimulate some population of T cells, based on Sag binding to specific T cell receptor variable region β chains, a property that distinguishes SAgs from conventional T cell mitogens. The biologic properties of SAgs make them attractive for use in immunotherapy. However, the T cell stimulatory effects of SAgs cannot be confined to specific antigens, its important role might be to enhance the immune effect for tumor antigens. PML-RARα peptide is a specific antigen associated with acute promyelocytic leukemia, which can induce specific proliferation of T cells from healthy human donor in vitro. But the immunogenicity of PML-RARα peptide is weak based on our previous study. In order to investigate the differentiation and proliferation of T cells induced by PML-RARα peptide that combined with Staphyloccucal enterotoxins A(SEA), T cell phenotype, TCR Vβ repertoire usage, clonality and cytotoxicity of T cells were analyzed. Peripheral blood mononuclear cells from healthy donors were cultured with PML-RARα peptide and SEA for 20 days. After induction, the distribution and clonal expansion of TCR Vβ subfamilies of T cells were examined by RT-PCR and GeneScan. The cytotoxicity of T cells induced against NB4 cell lines were examined by Cell Counting Kit-8 (CCK-8). The CD4 and CD8 surface marker on the harvested CD3+ T cells were detected by flow cytometry (FCM). And cytokine levels of IFN-γ, IL-4 and IL-10 in the cultured supernatants were measured by Enzyme-Linked Immunosorbent Assay (ELISA) as well. The results showed that restricted expression of Vβ repertoire (11 Vβ subfamilies) was found, which were Vβ1, Vβ2, Vβ3, Vβ6, Vβ7, Vβ8, Vβ13, Vβ14, Vβ16, Vβ17, Vβ19 subfamilies. Clonal expanded T cells could be identified in some Vβ subfamilies, which were preferentially used in Vβ13 and Vβ14. The specific cytotoxicity of T cells induced by PML-RARα peptide with SEA was higher than that from PML-RARα peptide alone induction group against NB4 cells. The FCM assay showed that the activation of CD8+ T cells was in priority induced by PML-RARα peptide whether with SEA or not, while it came out to be an equal between CD4+ and CD8+ T cells induced only by SEA. And the IFN-γ level of Th1-type cytokine was higher than that of the Th2-type cytokines (IL-4 and IL-8) in both cultured supernatants of T cells induced by PML-RARα peptide with or without SEA. In conclusion, PML-RARα peptide can induce specific T cells with clonal expansions of TCR Vβ subfamilies. SEA could enhance the induction of the specific PML-RARα peptide. The T cells induced by combination of PML-RARα peptide and SEA have higher specific antileukemia effect.