bluuebunny/crossref_metadata_2025 huggingface dataset:
CD133-Expressing Cells with High Aldehyde Dehydrogenase Activity Represent a Primitive Human Hematopoietic Stem Cell Population with Enhanced Repopulating Potential.
Authors: Hess David A. , Wirthlin Louisa , Craft Timothy P. , Meyerrose Todd E. , Nolta Jan A.
Published: April 2020

Human hematopoietic stem cells (HSC) are commonly purified by the phenotypic expression of cell surface markers such as CD34. We have recently characterized a novel strategy to purify reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin−) followed by selection of cells with high aldehyde dehydrogenase (ALDH) enzyme activity. Lin− cells with high ALDH activity (ALDHhiLin−) represented approximately 0.1% of total UCB mononuclear cells and demonstrated enriched expression of the primitive HSC markers CD34 (91.0±2.9%) and CD133 (70.9±4.0%). Most notably, clonogenic progenitor function and in vivo reconstituting ability in immune deficient mice were exclusive to the ALDHhiLin− population. Here, we have further purified the ALDHhiLin− population based on the expression of CD133, or prominen, a non-restricted surface molecule expressed on primitive progenitor cells of hematopoietic, endothelial, and neural epithelial lineages. ALDHhiCD133− and ALDHhiCD133+ cells, sorted to >95% purity, represented 14.7±2.1% and 23.2±4.3% of the total human UCB Lin− population respectively (n=6). Both ALDHhiCD133−Lin− and ALDHhiCD133+Lin− cells demonstrated clonogenic progenitor function in vitro. However, total colony production was significantly enhanced (p<0.05) in ALDHhiCD133−Lin− cells (1 CFU in 3.5 cells, n=5) when directly compared to ALDHhiCD133+Lin− cells (1 CFU in 10 cells, n=6). Human hematopoietic repopulation was consistently observed in the bone marrow, spleen, and peripheral blood of NOD/SCID (n=23) and NOD/SCID B2M null (n=27) mice transplanted with as few as 103 ALDHhiCD133+Lin− cells, whereas transplantation of up to 2x105 ALDHhiCD133−Lin− cells produced no detectable human engraftment. BM repopulation at limiting dilution demonstrated increased NOD/SCID repopulating ability elicited by ALDHhiCD133+Lin− cells when directly compared to CD133+Lin− cells not selected for ALDH activity. Repopulating ALDHhiCD133+Lin− cells differentiated into cells expressing markers for mature myeloid (CD33, CD14) and B-lymphoid (CD19, CD20) cells. ALDHhiCD133+Lin− cells also supported the maintenance of primitive cell phenotypes up to 8 weeks post-transplantation (2.4±0.7% CD34+CD38−, 5.5±0.6% CD34+CD133+, n=6) and the repopulating function of these cells are currently being confirmed by secondary transplantation. We are also investigating the ability of ALDHhiCD133+Lin− cells to mediate tissue repair in non-hematopoietic organs. Fractionation of human HSC based on combined expression of CD133 and high ALDH activity provides a rigorous selection of purified hematopoietic stem and progenitor cells that maintain primitive characteristics after transplantation, and may be considered a potential alternative to CD34+ cell isolation.
In Vitro Activity of a Novel Fully Human Anti-CD40 Antibody CHIR-12.12 in Chronic Lymphocytic Leukemia: Blockade of CD40 Activation and Induction of ADCC.
Authors: Tong Xia , Georgakis Georgios V. , Li Long , Susan O’Brien , Anas Younes , Mohammad Luqman
Published: April 2020

B-cell chronic lymphocytic leukemia (CLL) is characterized by in vivo accumulation of long-lived CD5+ B cells. However when cultured in vitro CLL cells die quickly by apoptosis. Protection from apoptosis in vivo is believed to result from supply of survival signals provided by cells in the microenvironment. We and others have previously reported that CLL cells express CD40 receptor, and that CD40 stimulation of CLL cells may rescue CLL cells from spontaneous and drug-induced apoptosis in vitro. These observations suggested that blocking CD40-CD40L pathway might deprive CLL cells from survival signals and induce apoptosis. To test this hypothesis, we have generated a fully human anti-CD40 blocking monoclonal antibody in XenoMousemice (Abgenix, Inc.). The antibody CHIR-12.12 was first evaluated for its effect on normal human lymphocytes. Lymphocytes from all 10 healthy blood donors did not proliferate in response to CHIR-12.12 at any concentration tested (0.0001 mg/ml to 10 mg/ml range). In contrast, activating CD40 on normal B-lymphocytes by CD40L induced their proliferation in vitro. Importantly, CHIR-12.12 inhibited CD40L- induced proliferation in a dose dependent manner with an average IC50 of 51 ± 26 pM (n=10 blood donors). The antagonistic activity of CHIR-12.12 was then tested in primary CLL samples from 9 patients. CHIR-12.12 alone did not induce CLL cell proliferation. In contrast, primary CLL cells incubated with CD40L, either resisted spontaneous cell death or proliferated. This effect was reversed by co-incubation with CHIR-12.12 antibody, restoring CLL cell death (n=9). CHIR-12.12 was then examined for its ability to lyse CLL cell line EHEB by antibody dependent cell mediated cytotoxicity (ADCC). Freshly isolated human NK cells from normal volunteer blood donors were used as effector cells. CHIR-12.12 showed lysis activity in a dose dependent manner and produced maximum lysis levels at 0.1 mg/ml. When compared with rituximab, CHIR-12.12 mediated greater maximum specific lysis (27.2 % Vs 16.2 %, p= 0.007). The greater ADCC by CHIR-12.12 was not due to higher density of CD40 molecules on CLL cell line compared to CD20 molecules. The CLL target cells expressed 509053 ±13560 CD20 molecules compared to 48416 ± 584 CD40 molecules. Collectively, these preclinical data suggest that CHIR-12.12 monoclonal antibody may have a therapeutic role in patients with CLL.
HSV Amplicon Transduction Activates an Innate Immune Response in CLL B Cells;Implications for Immune Therapy and Vaccine Development.
Authors: Daubeuf Sandrine , Rosenblatt Joseph D. , Bowers William J. , Urbistondo Natasha T. , Federoff Howard J. , Tolba Khaled A.
Published: April 2020

Since gene therapy vectors are designed to shuttle therapeutic genes into various tissues, efficiency transduction is often viewed as the most critical measure of a vector’s clinical usefulness. However, other variables such as activation of the host’s innate immunity may play an equally important role shaping the clinical outcome. We have studied the application of helper-free HSV (hf-HSV) amplicon vectors to deliver costimulatory ligands into chronic lymphocytic leukemia (CLL) B cells for the purpose of enhancing their antigen presenting capacity. In this work, we examine the innate immune response of CLL B cells to transduction with helper-virus free HSV (hf-HSV) amplicon vectors. We screened culture supernatant of CLL B cells transduced with helper-free HSV amplicon for secretion of IL-6, TNF-α and interferon β by ELISA and observed significant induction of these cytokines upon hf-HSV transduction as compared to non-transduced and CLL cells transduced with helper virus-containing (H+-HSV) HSV amplicons. We also compared the effects of hf-HSV and H+-HSV amplicon vector transduction on surface expression of NKG2D ligand. CLL B cells transduced with hf-HSV but not H+-HSV amplicons upregulated the costimulatory NKG2D ligands MICA and MICB. To investigate mechanisms of HSV activation of innate immunity, we screened a panel of human TLR receptors for recognition of HSV amplicons using 293T cells transfected with individual human TLR receptors and assaying NF-κB driven luciferase activity as a read-out. We found that HSV amplicon activates TLR3 (receptor for double stranded RNA), TLR8 (receptor for single stranded RNA) and TLR9 (receptor for unmethylated CpG motifs). CLL B cells express predominantly TLR9 and to a lesser extent TLR8. TLR3, on the other hand is not expressed on resting B cells and is upregulated upon exposure to interferon β. In 293T cells lacking TLR receptors, viable hf-HSV but not UV-inactivated amplicons up-regulated surface expression of both MICA and MICB and activated interferon-β promoter driven luciferase, indicating the presence of a TLR-independent HSV activated innate response. To establish the presence of TLR-dependent NKG2D ligand induction, CLL B cells (expressing TLR7, 8 and 9) were incubated with their specific ligands (Loxaribine and CpG ODN motifs) for 24 hours and MICA and MICB expression confirmed by flow-cytometry. In summary, our data shows that hf-HSV amplicon vectors activate multiple aspects of innate immunity involving both TLR dependent and independent pathways, which translates into expression of cytokines and costimulatory ligands by transduced cells. The presence of helper virus on the other hand completely abolishes this response. In the setting of a vaccine development, the evoked innate response may prime the adaptive T cell response leading to improved clinical outcome.
Triterpenoid Methyl-CDDO Is a Potent Inducer of Apoptosis in CD34+ AML Progenitor Cells Via Activation of SAPK Pathways and Inhibition of MAPK Cascades.
Authors: Konopleva Marina , Ruvolo Peter , Contractor Rooha , Kurinna Svitlana , Shi Yue-Xi , McQueen Teresa , Milella Michele , Andreeff Michael
Published: April 2020

Outcome results in AML are a continued challenge for the development of novel therapeutic strategies. C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid, CDDO-Me, a novel triterpenoid, induces apoptosis in myeloid and lymphoid leukemic cell lines and in primary AML samples in sub-micromolar concentrations. We reported previously that CDDO-Me inhibits the activation of ERK1/2, blocks Bcl-2 phosphorylation, and promoted apoptosis in AML-derived U937 cells (Blood 2002, 99(1):326–35). Here, we examined the effects of CDDO-Me on CD34+ AML progenitor cells in vitro. Exposure to 1μM CDDO-Me induced apoptosis in all but one AML sample (46±4% annexin(+) CD34+ cells, n=20). To assess the anti-leukemia activity of CDDO-Me in vivo, scid mice intravenously injected with U937 cells were treated with liposomally-delivered CDDO-Me (20mg/kg/day IV every other day, starting at day 7, for a total of 9 injections). While CDDO-Me was not toxic to the mice, pathological and flow cytometry analysis revealed reduced (55–86%) leukemia burden in the bone marrow, liver, and spleen of mice. Since we had shown that CDDO-Me inhibits phosphorylation of pERK in U937 cells, a further goal of this study was to assess the role of ERK in CDDO-Me-induced cell death in primary AML samples. ERK was expressed and phosphorylated in all (n=15) patients’ samples studied. CDDO-Me inhibited ERK phosphorylation in 9 of 15 patient samples and promoted apoptosis. However, CDDO-Me still induced apoptosis in 5/6 samples that displayed no significant changes in pERK levels in response to the drug. This finding suggests that ERK is not the sole target of the compound. The stress-activated protein kinases JNK and p38 are related to ERK but in general activate pathways that are opposed to ERK. By Western blot analysis, CDDO-Me induced early (30 min) phosphorylation of JNK and p38, which preceded induction of cell death. Pre-treatment of U937 cells with JNK and p38 inhibitors SP600125 and SB203580 partially abrogated induction of apoptosis, while MEK inhibitor PD-98059 significantly enhanced cytotoxicity. CDDO-Me induced p38 phosphorylation in 5 of 6 primary AML samples tested. Collectively, these finding indicate that CDDO-Me markedly shifts signaling toward the JNK and p38 MAPK stress-related pathways and away from the cytoprotective MAPK cascade. In summary, the triterpenoid CDDO-Me is a potent inducer of apoptosis in primary AML including CD34+ AML progenitor cells. Induction of apoptosis is in part mediated via inhibition of ERK signaling combined with JNK/p38 activation. These studies in primary AML samples and the anti-leukemia activity of the compound in vivo suggest potential utility of CDDO-Me for the treatment of AML patients.
Generation of Human Anti-Leukemia CTL Vaccine Utilizing Dendritic Cell Hybrids.
Authors: Naparstek Elizabeth , Eshel Rinat , Vainas Oded , Ben-Yosef Rami , Shpringer Meirav
Published: April 2020

The therapeutic options for patients with hematological malignancies that relapse after autologous or allogeneic hematopoietic stem cell transplantation (SCT) are limited and their overall prognosis is poor. Therefore new therapeutic approaches such as cellular-based immunotherapies are being investigated. The efficacy of cytotoxic T lymphocytes (CTLs) or dendritic cell (DC) vaccines was already established in the treatment of post transplant CMV infections and in several solid tumors. This strategy however is less effective in hematological malignancies, where the tumor specific antigens are unknown and the cellular immunotherapy is based on the induction of graft-versus-leukemia (GVL) response by allogeneic, non-antigen specific, donor derived T lymphocytes (DLI). One approach to enhance immune recognition and targeting of the leukemic cells, and consequently augment their specific killing by CTLs, is through the use of dendritic cells. DCs are potent antigen presenting cells that can be manipulated to express tumor antigens by various strategies including fusion with tumor cells. Such hybrid cells expressing tumor antigens, presented in the context of MHC class I and II and co-stimulatory molecules of the DC partner, were shown to be effective in several malignancies.

Our aim was to generate human CTLs capable to mount a specific and potent in-vitro anti-leukemia activity, utilizing dendritic-leukemia cell hybrids. We have generated such hybrids by co-culture of immature human DCs with allogeneic (K562) or autologous leukemia cells in the presence of polyethylene glycol (PEG). After maturation, these dendritic-leukemia cell hybrids were utilized to generate specific CTLs, using autologous or allogeneic peripheral blood lymphocytes under IL-2 deprivation. Purified CD8+ cells were then further expanded and tested for their cytotoxicity against the target cells using Alamar Blue cytotoxicity assay.

Both allogeneic and autologous dendritic-leukemia cell hybrids were shown to stimulate a significant CTL responses compared to controls. Dendritic-K562 cell hybrids stimulated a CTL response of 40% lysis of the target K562 cells, while CTLs generated by co-culture with non-fused K562 cells had no detectable cytotoxic activity against their targets. Similarly, CTLs stimulated by dendritic-autologous leukemia cell hybrids showed 15% lysis of the leukemia target cells, compared to 1.5 and 1% lysis observed by CTLs generated by co-culture with autologous tumor or DCs alone. Our results show that in-vitro generation of hybrids of human dendritic and un-manipulated primary leukemia cells is feasible, and that such hybrid cells can elicit the generation of specific autologous or allogeneic anti-leukemia CTLs. Such CTLs may be superior to the currently used non-antigen-specific donor lymphocytes, and may be useful also in the autologous setting.
Selective Induction of Apoptosis in Acute Myelogenous Leukemia Stem Cells by the Novel Agent Parthenolide.
Authors: Guzman Monica L. , Karnischky Lilliana , Li Xiaojie , Neering Sarah J. , Rossi Randal M. , Jordan Craig T.
Published: April 2020

Acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs), which are thought to perpetuate both de novo and relapsed disease. Because LSCs are often refractory to standard chemotherapy agents, it is crucial to investigate the ability of current and novel drugs to target this rare population of cells. Previously, we demonstrated that combined treatment with proteasome inhibitors and anthracyclines induced apoptosis in LSCs while sparing the normal hematopoietic stem cells (HSCs). Using this approach, death of leukemic cells was characterized by NF-kB inhibition and p53 activation. In the present study, we have extended this work by investigating parthenolide (PTL), a sesquiterpene lactone found in the herbal drug feverfew and that has been reported to inhibit NF-kB activation. Primary AML cells treated with increasing concentrations of PTL (1-10 microM) showed a rapid (18h) and dose dependent decrease in cell viability. The PTL concentration and its effect on induction of apoptosis directly correlated with decreased NF-kB binding activity and increased phosphorylation of p53. Importantly, apoptosis induction was also evident in the primitive LSC population, defined by the immunophenotypic profile CD34+, CD38− and CD123+. In contrast, PTL had little to no effect in normal HSCs obtained from either cord blood (CB) or bone marrow (BM). Interestingly, apoptosis induction upon PTL treatment was completely abolished upon treatment with the anti-oxidant, N-acetyl-cysteine (NAC), suggesting that PTL effects involve reactive oxygen species (ROS) induction. This was further corroborated using the fluorescent dyes DCFH-DA and mBBr to measure ROS and thiol content, respectively. When PTL effects were compared to the common chemotherapeutic drug cytosine arabinoside (Ara-C), we observed that PTL selectively targeted leukemic cells whereas Ara-C did not. Functional analyses using in vitro colony assays and the NOD/SCID mouse xenotransplant model were then employed to address whether the drugs used in this study affected stem cell potential. PTL treatment did not affect engraftment of normal cells while leukemic cell engraftment was dramatically impaired. Similarly, methylcellulose cultures showed a severe decrease on colony formation in PTL treated AML cells, while normal colonies were not impaired. Together, these data demonstrates that PTL is able to selectively target LSCs while sparing normal hematopoietic cells. The mechanism of leukemia-specific cell death appears to involve inhibition of NF-kB, activation of p53 and induction of oxidative stress.
Combination Chemoimmunotherapy Using Alemtuzumab, Fludarabine, Cyclophosphamide, and Doxorubicin (Campath-FCD) Is an Effective First-Line Regimen in Peripheral T-Cell Lymphomas.
Authors: Weidmann Eckhart , Hess Georg , Krause Stefan W. , Hebart Holger , Dreyling Martin , Subklewe Marion , Falge Christine , Chow Kai U. , Jaeger Elke , Mitrou Parisis S.
Published: April 2020

Peripheral (mature) T-cell lymphomas (PTCL) are represented by distinct lymphoma entities, many of which with a rather unfavourable clinical outcome as compared to B-cell lymphomas. Standard treatment regimens have not been established, except in primary cutaneous T-cell lymphomas (PCTCL). With regard to the unfavourable prognosis we designed a treatment protocol using chemoimmunotherapy in peripheral T-cell lymphomas except PCTCL and ALK-positive large cell anaplastic T-cell lymphomas, consisting of alemtuzumab 3, 10, 30, 30 mg, days 1–4, fludarabine (Flu) 25 mg/m2 days 2–4, cyclophosphamide (CP) 600 mg/m2 day 3, and doxorubicin (Dox) 50 mg/m2 day 4. Included were patients with primary diagnosis or with first or second relapse. Rationales for this regimen were proven efficacy of CP and Dox in T-cell lymphomas, high efficacy of Flu and CP (FC) in other lymphomas and the possible synergism of antibodies with cytotoxic drugs. So far, 23 patients have been included, 18 are evaluable for response and toxicity. Of the latter, 10 patients were diagnosed with PTCL-NOS, 5 with AILD, one with enteropathy-associated T-cell lymphoma, one with NK-cell lymphoma, and one with T-PLL. 9 patients were enrolled with primary diagnosis and 9 patients in relapse. The median age was 60 years (range 21–77); the median non-age adjusted IPI 2.5 (0–4), 2 in patients with primary diagnosis and 3 in patients with relapse. The overall response rate was 61% (11/18). In patients with primary diagnosis the CR rate was 78% (7/9), one patient achieved no change, and one patient died from treatment associated complications before response could be evaluated. All responding patients are in ongoing CR at 2+, 2+, 6+, 11+, 15+,16+, and 17+ months. In the group of relapsed patients one CR and 3 PR (44%) were observed. The main toxicity was leukocytopenia (81% grade III and IV of all evaluable treatment cycles), other grade III and IV toxicities included anemia (18%), thrombocytopenia (39%), infections (18%) nausea/emesis (9%), and allergic reactions (4%). Ten (56%) patients reactivated CMV without CMV-related disease and one patient with suspected CMV-peumonia. In conclusion, this is the first study demonstrating that alemtuzumab can be integrated into a triple-agent chemotherapy regimen. The combination is effective in the first-line treatment of peripheral T-cell lymphomas, however, regarding the general outcome a longer follow-up period of a larger patient population is required.
The Role of IL-12 and IL-23 in the Generation and Maintenance of Memory CD4+ T Cells.
Authors: Cleary Aileen M. , Lewis David B.
Published: April 2020

Human memory CD4+ T cells can be distinguished from antigenically-naïve CD4+ T cells based on their CD45RAlowCD45R0high and CD45RAhighCD45R0low surface phenotypes, respectively. Memory CD4+ T cells from adult peripheral blood can be further divided based on surface expression of the CCR7 chemokine receptor. Th1 memory CD4+ T cells that are CCR7high (putative central memory cells or Tcm) are expected to be preferentially targeted to peripheral lymph nodes where the ligands for CCR7 are expressed in high amounts. These cells have been reported to lack expression of the CCR3 and CCR5 chemokine receptors, which facilitate entry into inflamed tissues, and produce little or no interferon (IFN)-γ after stimulation via the αβ-TCR/CD3 complex. CD45RAlowCD45R0highCCR7low CD4+ T cells account for virtually all IFN-g production by human CD4 T cells after ab-TCR/CD3 stimulation using monoclonal antibodies, and for this reason were termed effector memory cells (Tem). These findings, as well as the observation of shorter telomere lengths for memory CD4+ T cells that are CCR7low compared to those that CCR7high suggest that the Tcm population may be an intermediate between naïve CD4+ T cells and Tem. It has recently been proposed that the level of signal strength and γc containing cytokines play a role in memory T cell generation. However, little is known whether IL-12 or IL-23 are necessary and for this differentiation and/or maintenance. Our laboratory has previously described a patient with IL-12Rβ1 deficiency, which ablates both IL-12 and IL-23 signaling. This patient had a deficiency in Tem number and function, unexpectedly suggesting that IL-12 and/or IL-23 may play a key role in this process. We therefore hypothesized that signaling through IL-12Rβ1 plays a key role in the latter stages of generation and/or maintenance of human memory CD4+ T cells. Preliminary data thus far show CCR7 expression to be slightly decreased on activated Tcm in response to incubation with IL-2 or IL-12 alone, and to a greater extent with IL-2 and IL-12 incubated together. In addition, spontaneous apoptosis of both Tcm and Tem is decreased upon incubation with IL-12. Taken together, these data suggest that IL-12 may play a role in both generation of Tem and maintenance of both Tcm and Tem.
Phenotype, Function, and Regulation of Migrating Hematopoietic Stem Cells.
Authors: Wagers Amy J. , Prohaska Susan S. , Passegue Emmanuelle , Price Jessica , Weissman Irving L.
Published: April 2020

Hematopoiesis in adult animals is maintained by a small population of clonogenic, multipotent hematopoietic stem cells (HSC), which maintain throughout life the capacity to self-renew and to differentiate to give rise to progeny cells that ultimately generate all lineages of mature blood cells. In adult mice and humans, the majority of HSC are found in the bone marrow (BM); however, HSC are also constitutively present at low levels in the circulation. The frequency of HSC in the blood can be significantly increased through the use of “mobilizing” agents, including cytotoxic drugs and/or cytokines, which often act both to drive HSC proliferation and to induce HSC migration from the BM into the bloodstream. Yet despite the increasingly common clinical exploitation of HSC in bone marrow and mobilized peripheral blood progenitor cell transplantation, both the evolutionary rationale and the molecular mechanisms that underlie the remarkable migratory capacity of HSC remain largely unknown. Therefore, to begin to elucidate the mechanisms and regulators of these events, we have used parabiotic and transplantation models to characterize normal blood-borne HSC. Our data clearly demonstrate that HSC are constitutively present in the blood of untreated mice and maintain a cell surface phenotype in the blood highly similar to their BM counterparts. Blood-borne HSC in normal mice can engraft both irradiated and non-irradiated BM niches, and subsequently are phenotypically and functionally indistinguishable from endogenous, host-type cells. These data suggest that BM homing of transplanted HSC in irradiated recipients and HSC mobilization in cytokine-treated animals likely makes use of pre-existing pathways that support the constitutive recirculation of these cells in normal animals. Finally, to extend these data and begin to uncover factors likely to play a role in stimulating HSC migration in both normal and mobilized mice, we have employed cDNA microarray technology to compare global gene expression profiles of normal and pre-migratory BM HSC, and have thus identified multiple candidate genes, including cell cycle regulators, signaling molecules, and transcription factors, that may be involved in HSC expansion or in HSC retention in and/or egress from the BM. Taken together, these findings provide significant insight into the dynamic nature and function of HSC, and may ultimately suggest novel and improved strategies for clinical hematopoietic cell transplantation.
Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.
Authors: Narayan A. Daisy , Chase Jessica L. , Ersek Adel , Thomson James A. , Lewis Rachel L. , Kaufman Dan S. , Zanjani Esmail D.
Published: April 2020

We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.
Coagulation and Resuscitation Patterns Following Transfusion with Polymerized Hemoglobin Based Oxygen Carrier (HBOC-201) after Severe Hemorrhage in Swine.
Authors: Arnaud Francoise , Hammett Michael , Philbin Nora , Rice Jennifer , Dong Frank , Flournoy Shannon , Pearce Bruce , McCarron Richard , Freilich Daniel
Published: April 2020

INTRODUCTION: Blood substitutes can alleviate problems related to blood typing and blood availability in civilian and combat trauma casualties. The aim of this study was to assess the effect of transfusions of a blood substitute, HBOC-201 (Biopure Corp, Cambridge, MA) (HBOC), as a resuscitation fluid in severe controlled hemorrhagic shock (HS).

METHODS: Yucatan pigs underwent a 55% EBV controlled hemorrhage by catheter withdrawal. Animals (n=24) were either non-resuscitated (NON) or resuscitated with HBOC or with buffered hydroxyethyl starch (HEX) at an initial rate of 10 ml/kg. Additional infusions were given at 5 ml/kg if hypotension or tachycardia persisted during the 4 hr period following HS. Thereafter, animals received simulated hospital care up to 72 hr with access to blood transfusions for Hb < 7g/dl. In-vivo parameters (vital signs and in vivo bleeding time (BT)), as well as coagulation (PT, PTT, fibrinogen), thromboelastography (TEG) and in vitro bleeding time (PFA-CT) were assayed on blood samples obtained at various times after HS.

RESULTS: Although not statistically significant, HBOC allowed 100% survival compared to 75% with HEX and 25% for NON. It also restored the mean arterial pressure more rapidly compared to HEX. HBOC and HEX groups received similar resuscitation fluid volumes and showed similar hemodilution during the prehospital phase with comparable BT. PFA-CT increased abruptly for both groups remaining elevated at 24 h for HBOC. Other hemostasis parameters were better controlled in the HBOC group compared to HEX: TEG-MA was less suppressed and PT was lower with HBOC. TEG-R was unchanged but indicated delayed hypocoagulation in the HBOC group by 24 hr. At simulated hospital arrival 4 hr after HS, only 10% of animals in the HBOC group required blood transfusions compared to 100% in HEX group. By 72 hr most hematological parameters returned to normal.

CONCLUSIONS: Although HBOC-201 caused less coagulopathy than HEX during fluid resuscitation, it resulted in mild, delayed, and transient coagulopathy by 24 hr. Moreover, HBOC-201 reduced blood transfusion requirements, supporting use of this oxygen-carrying fluid for hemorrhagic shock.

The opinions contained herein are the ones of the authors and are not to be construed as official or reflecting the views of the Navy department or the naval service at large.

Figure Figure
Transfusion Related Acute Lung Injury (TRALI): A Tertiary Care Hospital Experience.
Authors: Strupp Annie , Ilstrup Sarah
Published: April 2020

Background: The 500 bed tertiary care hospital provides Level 1 trauma services, solid organ transplant services and an extensive cardiovascular surgery program.

Methods: Transfusion reaction records for January 2000 – June 2004 were reviewed.

Results: 39 suspected transfusion related acute lung injury reactions reported during this time at a rate of 1:1036 transfused blood products (1:4961 for RBC, 1:1031 for Apheresis Platelets, and 1:522 for FFP). There were 26 male patients, average age of 62.2 years (17–87 years) and 13 females, average age of 62.5 years (24–82 years).

All 39 patients developed acute lung injury within 6 hours of transfusion characterized by acute onset of hypoxia, without evidence of volume overload and chest xrays (performed in 34/39) showing evidence of new bilateral pulmonary infiltrates.

Patients received an average of 2.5 blood products (1–8), and 13/39 (33%) received only one product. FFP was 66% (65/98 units) of implicated product transfused, leukoreduced RBC 24% (24/98, all additive solution products), and leukoreduced platelet pheresis 9% (9/98 units). Of the patients that only received one product, 8/13 (62%) were transfused with a RBC.

Many (74%, 29/39) patients experiencing possible TRALI reactions had underlying risk factors (some patients had more than one underlying problem) including: probable sepsis (5), multiple trauma (2), cardiopulmonary bypass (6), underlying pulmonary disease (5), liver transplant (2), cardiac transplant (1), hematologic malignancy (4), TTP (3), recent surgery (5).

The reaction occurred during or immediately following transfusion in 60% (23/39) of patients, and in 77% (30/39) within 30 minutes of completion of transfusion. Of the recipients that reacted immediately 39% (9/23) of the implicated donors had HLA antibodies (Class I and Class II), and of those reacting within 30 minutes 47% (14/30) of donors had HLA antibodies. 7.7% (3/39) reactions were fatal.

Of implicated donors, 59% (58/98) are female, and 41% male (40/98). 10 donors gave a history of transfusion (1 male, 9 females). 74% (42/57 (1 female couldn’t be contacted)) had been pregnant, with the average number of pregnancies 4.0 (range 1–9), with 67% (28/42) of these donors having 3 or more pregnancies.

Donor antibody testing (Granulocyte Agglutination Assay (GA), Granulocyte Immunofluorescence Assay (GIF), and HLA Antibody Screen (HLA)) was performed only on donors that had been pregnant and/or had received a transfusion, therefore only one male donor was tested and was negative. Of the female donors 62% (36/58) were tested, 29% (17/58) did not meet testing criteria and 9% (5/58) were not available for testing. Of those tested, 31% (11/36) were negative. 69% (25/36) had HLA antibodies. 8% (2/25) with HLA antibodies also had antibodies with granulocyte specificity.

Conclusion: The etiology(ies) and prevention of TRALI reactions require additional investigation of both blood donors and recipients, although HLA antibodies appear to be involved.
Time-Sequenced G-CSF (Lenograstim) for the Treatment of Adult Acute Lymphoblastic Leukemia - Seven Year Follow-Up of the Polish Adult Leukemia Group 4–96 Study.
Authors: Giebel Sebastian , Holowiecki Jerzy , Krawczyk-Kulis Malgorzata , Kyrcz-Krzemien Slawomira , Hellmann Andrzej , Dmoszynska Anna , Paluszewska Monika , Robak Tadeusz , Konopka Lech , Seferynska Ilona , Skotnicki Aleksander , Baran Wojciech , Adamczyk-Cioch Maria , Wrzesien-Kus Agata , Jagoda Krystyna
Published: April 2020

The goal of the study was to evaluate long-term outcome of patients treated within 4–96 trial by the Polish Adult Leukemia Group (PALG). Sixty four patients with newly diagnosed acute lymphoblastic leukemia (ALL) (median age 26 years, range 16–58) were randomly assigned to receive chemotherapy alone (n=31) or chemotherapy and glucosylated G-CSF (lenograstim) (n=33). Both groups were well-balanced in terms of age, initial WBC, immunophenotype and bcr/abl positivity. Induction therapy consisted of epirubicin+vincristin (Epi/Vcr) on days 1, 8, 15, 22, prednisone on days 1–28, L-asparaginase 8 doses starting from day 13; consolidation treatment included twice methotrexate+etoposide (Mtx/Vep), twice high dose cytarabine and cyclophosphamide (HDAraC/Ctx), CNS irradiation and intrathecal Mtx. In T-derived ALL, additional HDAraC/Ctx was administered instead of the first Mtx/HDAraC. During induction patients received G-CSF 150 μg/m2 sc. on days 2–6, 9–13, 16–20, 23-until the neutrophil recovery >1.0x109/L, starting 36 hours after Epi/Vcr, finishing 48 hours before the next dose; in consolidation - following each HDAraC/Ctx course on days 5–16. High risk patients having a donor were performed allogeneic hematopoietic cell transplantation in first complete remission, whereas those without a donor were given autologous transplant. At seven years the overall survival rate equalled 42% for G-CSF group and 24% for the controls (p=0.11). There was also a trend to higher probability of leukemia-free survival in advantage of patients receiving the cytokine (38% vs. 20%, p=0.17). The above differences might have resulted from: 1) Higher CR rate in the G-CSF group compared to the controls (94% vs. 87%), 2) Better adherence to the chemotherapy protocol (faster completion of induction-consolidation programme by 19 days, p=0.005, less dose reductions or delays), which in turn might have influenced the risk of relapse.

We conclude that time sequenced G-CSF administration may improve long-term outcome of adult ALL patients although the study including larger population is required to confirm this hypothesis.
Re-Targeting of an NK Cell Line (NK92) with Specificity for CD19 Efficiently Kills Human B-Precursor Leukemia Cells.
Authors: Romanski Annette , Uherek Christoph , Bug Gesine , Muller Tina , Rossig Claudia , Kampfmann Manuela , Krossok Nicola , Hoelzer Dieter , Seifried Erhard , Wels Winfried , Ottmann Oliver G. , Tonn Torsten
Published: April 2020

The continuously growing Natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origin without affecting normal human cells. It is the only NK cell line that has entered clinical trials to date. Acute lymphoblastic leukemias (ALL) show variable sensitivity towards NK cells including NK-92-lysis. All T-ALL cell lines tested (MOLT-3, MOLT-4 and CEM-T) display moderate sensitivity to NK cytotoxicity with approximately 22±2 – 24±2% killing at an effector to target (E:T) ratio of 10:1, respectively. Comparable results were obtained with primary patient derived T-ALLs (n=4), displaying specific killing rates of 17±5 – 20±2%. In contrast, B lineage ALL cell lines (NALM-6, SEM, REH, JKB, BV173, SupB15, TOM-1, TMD5) and cells derived from patients (n=11) were resistant to NK-92-mediated lysis, with specific killing rates ranging from 2±1% to 13±3% at an E:T ratio of 10:1. Approaches to overcome resistance of B-precursor ALL by re-targeting may shed light on the specific reasonable mechanism conferring resistance and lead to novel therapeutic strategies. Here we have generated genetically modified NK-92 cells expressing a chimeric antigen receptor specific for the pan B cell antigen CD19 which is universally expressed by B lineage leukemia cells. This receptor fragment kindly provided by H. Zola (Child Health Research Institute North Adelaide, Australia) consists of the CD19 specific scFv(CD19) antibody fragment, a flexible hinge region, the CD3 ζ chain and a Myc-tag. Transduced cells were selected with G418, and surface expression of the chimeric scFv(CD19)-ζ construct was verified by FACS analysis using the Myc-tag-specific mAb 9E10. No difference in cytotoxic activity of NK-92 and transduced NK-92-scFv(CD19)-ζ cells towards CD19 negative targets (K562, MOLT-4) was found. In contrast, NK-92-scFv(CD19)-ζ cells specifically and efficiently lysed CD19 expressing B precursor leukemia cells (NALM-6, SEM, B173, SupB15, TOM-1, TMD5) including cells that were completely resistant to cytolytic activity of parental NK-92 cells. Killing of SupB15 and BV173 cells was 10,0±0,8% and 12,0±8,5% with parental NK92 vs. 58,2±14,8% and 52,3±4,1% lysis with NK92 cells expressing the CD19 specific scFv(CD19) antibody fragment. These results demonstrate that efficient retargeting of NK-cell cytotoxicity can be achieved, and might allow the generation of potent cell-based therapeutics. Since the widespread resistance of B precursor ALL blasts to NK cell cytotoxicity is not caused by mechanisms commonly relevant in tumors, this model may allow dissection of possible mechanisms of resistance.
The Acquired CD40L Deficiency in B-CLL Is Reversible and Due To Contact Dependant and Independent Factors.
Authors: Buggins Andrea G.S. , Richards Julie , Patten Piers E.M. , Mufti Ghulam J. , Devereux Stephen
Published: April 2020

Interactions between CD40L (CD154) and its receptor CD40, are of central importance in T-cell mediated B-cell activation, proliferation and isotype switching and the regulation of antigen presentation by dendritic cells. Peripheral blood T lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) have an acquired defect of activation induced CD40L expression that might contribute to the immunodeficiency characteristic of the disease. In view of recent reports that T-cells within bone marrow and lymph node pseudofollicles express CD40L we have re-examined the mechanism of the deficiency in PB cells. Activation of normal resting naïve and memory (CD45RA+ and −) T-cells with PMA and ionomycin increased the number expressing surface CD40L from a mean of 2 and 0% to 81.2 and 66.5% respectively. In line with previous reports, up-regulation of CD40L by CD4+45RA+ and CD4+45RA− T-cells from patients with B-CLL was reduced to a mean of 11.8% and 2.6%. This defect is reversible since removal of T-cells from the malignant clone by CD3 selection increased the number of cells able to up-regulate CD40L to 62.2% and 61.8% for naïve and memory CD4 subsets. To investigate whether this phenomenon is due to cell contact or soluble mediators, washed leukemic cells or supernatant (SN) harvested from the same cell number were incubated with normal donor T-cells for 48 hours then stimulated for 4 hours with PMA and Ionomycin. B-CLL SN reduced CD40L up-regulation by a mean of 51% (range 14–86, n=17, p<0.0001) but co-culture reduced this further to a mean of 17% (range 7–33%, n=8, p<0.0001). Other markers of T-cell activation were similarly affected, for example T-cell IL-2 production was reduced to 40.1% ( 6.2% SEM, p < 0.0001) of the level seen in the absence of B-CLL SN. Although SN and cell contact both prevented CD40L up-regulation, only cell contact caused its down-regulation in pre-activated T-cells (reduced to 94% of normal with SN, [range 93–96%, n=3, p=0.5] and 14% of normal with co-culture [range 4.3–21.7%, n=6, p p<0.0001]). The acquired CD40L deficiency observed in patients with B-CLL is thus reversible and mediated by contact with leukemic cells and soluble mediator(s). B-CLL cells are known to secrete a number of factors that might produce this effect. Studies using blocking monoclonal antibodies and immuno-adsorption excluded the most likely candidates including TGF-beta, soluble CD40 and soluble IL-2R. These findings indicate that regulation of the CD40/CD40L system in B-CLL is more complex than previously reported however the impact of this on disease pathogenesis needs to be determined.
Identification of a Subset of Aplastic Anemia Patients Highly Responsive to Danazol.
Authors: Yamazaki Hirohito , Chuhjo Tatsuya , Sugimori Chiharu , Ishiyama Ken , Nakao Shinji
Published: April 2020

Treatment of patients refractory to immunosuppressive therapy is a major problem in the management of aplastic anemia (AA). Some patients who are ineligible for allogeneic stem cell transplantation have been successfully treated with anabolic steroids although little is known about the common characteristics of these patients. In order to characterize high responders to danazol, we retrospectively studied 44 AA (13 male and 31 female) patients who were treated with 300 mg of danazol daily for at least 3 months. All patients had been previously treated with ATG+CsA or CsA alone without appreciable effects before danazol therapy. Peripheral blood of these patients was examined for the presence of PNH-type cells and clonality in granulocytes using sensitive flow cytometry (Blood100: 3897, 2002) and the improved HUMARA assay (Blood102: 1211, 2003). 24 of 44 (55%) patients attained PR or CR according to the response criteria proposed by Camitta. All 3 lineage cells increased in the responders. The rate of response in female patients (21/31, 68%) was significantly higher than that (3/13, 23%) in male patients (P=0.0058). There was no difference in the response rate between older (>50 years) patients (75%) and younger (<50 years) patients (60%) in female patients, indicating that the effect of danazol is not affected by a female sex hormone. Increased PNH-type cells were detectable in 2 (8.3%) of 24 responders and in 6 (40%) of 15 non-responders. The HUMARA assay revealed presence of clonal granulocyte population in 13 (76%) of 17 responders and 2 (33%) of 6 non-responders, respectively. For a female patient without increased PNH type cells who has not responded to immunosuppressive therapy, the rate of response to danazol was 64%. Probability of 3-year survival in responders to danazol was 100% while that in non-responders to the therapy was 90%. Univariate analysis revealed that negativity for increased PNH-type cell and being female were significantly associated with good response to danazol. These findings indicate that danazol is a useful drug in the treatment of a subset of female AA patients characterized by refractoriness to conventional immunosuppressive therapy and absence of increased PNH-type cells. Given the high frequency of clonal hematopoiesis in responders to danazol, this anabolic steroid may stimulate expansion of a small number of stem cells that survived non-immune marrow insult.
Decreased Expression of Stem Cell-Specific Genes in Aplastic Anemia; Possible Regulation of HOXB4 Gene by GATA-2.
Authors: Yokoyama Hisayuki , Harigae Hideo , Takahashi Shinichiro , Okitsu Yoko , Yamamoto Johji , Tohmiya Yasuo , Fujiwara Minami , Ishizawa Kenichi , Kameoka Junichi , Minegishi Naoko , Kaku Mitsuo , Sasaki Takeshi
Published: April 2020

Aplastic anemia (AA) is characterized by reduced hematopoiesis resulting in pancytopenia. It is suggested that a certain immunological attack to hematopoietic stem cells play an important role in developing AA. However, limited information is available for the intrinsic characteristics of stem cells in AA. Previous work in our laboratory showed decreased expression of GATA-2 gene in CD34 positive cells in AA, suggesting that there is an aberrant expression of stem cell-specific genes in stem cells in AA. Recently it is emerged that some genes such as HOXB4 and BMI-1, function for the proliferation and maintenance of stem cells. In this study, we examined expression levels of HOXB4 and BMI-1 in CD34 positive cells by quantitative PCR in 10 patients with AA and 13 with idiopathic thrombocytopenic purpura (ITP). Between these two factors, the expression level of HOXB4 was markedly decreased in AA compared with in ITP, whereas that of BMI-1 was not. Moreover, the expression level of GATA-2 in these populations was significantly correlated to HOXB4 gene expression (Spearman’s rank correlation, r=0.6573 p<0.01) compared to that of BMI-1(r=0.4107, p>0.05). As they functions as a transcription factor, these results raise the possibility that GATA-2 and HOXB4 regulate each other. To explore this possibility, first, we cloned HOXB4 5′flanking region by PCR and performed promoter analysis. Since the previous report showed that the region from −164 to −116 was important for promoter activity of HOXB4 gene, we focused on a GATA element located at −160. When this element was deleted, the reporter activity was decreased to 60% of wild-type in K562 cells. Furthermore, co-transfection of GATA-2 expression vector significantly activates the reporter gene in a dose dependent manner. EMSA revealed that GATA-2 binds specifically to this element. On the other hand, the active region both of exon 1S and 1G promoter of GATA-2 gene, which was identified by the promoter analysis, did not contain the consensus sequence recognized by HOXB4. These findings suggest that in stem cells in AA, the decreased expression of GATA-2 gene lead to the reduced HOXB4 gene expression, which may responsible for the development of the disease.