bluuebunny/crossref_metadata_2025_split huggingface dataset:
Outcome of Hematopoietic Stem Cell Transplantation for Pediatric Acute Lymphoblastic Leukemia in Third Complete Remission (CR3): A Vital Role for Graft-Versus-Host-Disease/Graft-Versus-Leukemia Effect in Survival.
Authors: Gassas Adam , Ishaqi Kashif , Doyle John 
Published: October 2019

Abstract
Children with acute lymphoblastic leukemia (ALL) who suffer 2 relapses could be salvaged by hematopoietic stem cell transplantation (HSCT) when a suitable stem cell source is available provided they respond to the pre HSCT chemotherapy and at least enter morphological remission. However, these patients are at very high risk for post HSCT relapse and also at a high risk for transplant related mortality (TRM). Our objective, herein, was to review the outcome of children (0–18years) with ALL who received allogeneic HSCT in third complete remission (CR3) at our institution. Between January 1994 – August 2005, twenty-two consecutive children in CR3 received HSCT in the Hospital for Sick Children, Toronto, Canada. Conditioning regimens included single dose of VP16 (60mg/kg infused over 4 hours) and fractionated total body irradiation (TBI; 1200cGy) in six fractions over 3 days (VP16/TBI) in 10 patients (1994–1998) and cyclophosphamide 50mg/kg infused over 1 hour daily for 4 days followed by the same dose of fractionated TBI (CY/TBI) in 12 patients (1999–2005). Graft-versus-host disease (GVHD) prophylaxis included cyclosporine A and a short course of methotrexate for the majority of patients, and all patients were in complete morphological remission prior to HSCT. Median age was 8.4 years (range 3–15.4). Donor source was as follows: matched sibling donor (MSD), 8; matched unrelated donor (MUD) 6; one antigen mismatch related donor (MMRD) 4; one antigen mismatched unrelated donor (MMUD) 3; and one patient received 1 antigen mismatched cord progenitor stem cells. White cell engraftment was successful in all patients at a median of 18 days (range 9–29). Ten patients died of TRM, seven relapsed, one died from other causes and four patients are long term survivors at a median follow up of 3.7 years (range 1–10.2). All patients who did not develop clinical acute or chronic GVHD relapsed and died. Event free survival was (EFS 19% ± 4%). Three out of the 4 survivors received MMUD and all 4 survivors had moderate to severe acute GVHD and three had chronic GVHD, limited in two and extensive in one.
Conclusion: Children with ALL in CR3 receiving HSCT are extremely high risk for relapse and transplant related mortality. These children have already relapsed twice and demonstrated chemotherapy resistance and GVL/GVHD plays a key role in leukemia eradication. Although, TRM is high in such patients and GVHD could potentially increase TRM, there are no survivors without GVHD and exploring means of inducing GVHD by reduction of immunosuppressive medications or other means of immunotherapy should seriously be considered in these patients.
Gleevec as Therapy for CML Relapsed after Allogeneic Stem Cell Transplantation: High Rate of BCR-ABL PCR Negativity and Donor Graft Reconstitution.
Authors: Funke Vaneuza A.M. , Nabhan Samir K. , Oliveira Michel M. , Ruiz Jefferson , Setubal Daniela C. , Azambuja Ana Paula , Lima Denise H. , Kojo Terezinha K. , Bitencourt Henrique , Hamerschlak Nelson , Clementino Nelma , Neves Heliz Regina A. , Pasquini Ricardo 
Published: October 2019

Abstract
INTRODUCTION: CML relapsed after HSCT has been treated with donor lymphocyte infusions (DLI), with responses achieving 60–70%, and even better results in earlier relapses. However, complications as chronic GVHD and myelossupression can be a problem, especially on those who receive larger DLI doses. The present study has the objective of evaluate quality of response and chimerism in patients who received Gleevec as therapy for relapsed CML after HSCT.
PATIENTS AND METHODS: We report 32 CML patients who received Gleevec as therapy for relapse after HSCT. Age: 13 – 56 (Median: 38 y); sex M/F: 17/15; Allogeneic HSCT: 31 pts, syngeneic: 1 pt. Time from HSCT to relapse: 0–122 m (M 16m). Relapse was hematologic in 29 and cytogenetic in 3 patients. Phase at hematologic relapse: CP-14 pts, AP-11 pts and BC-4 pts; clonal evolution: 7 patients. Previous VNTR was available in 16 pts: 10–35% donor in 11, 0% donor in 3 and 95% donor in 2 pts. Previous DLI: 14 pts. Dose: 600mg QD in 15 pts, 400 mg QD - 17 pts.
RESULTS: Therapy duration: 7–1795 d (M 365 d). 27 (84%) pts reached complete hematologic response (CHR). Time to CHR: 7–60 d (M 28 d). Cytogenetic response was available for 25 pts, being complete in 16 (48%), partial in 2 and absent in 7 pts. Competitive Q-PCR was available in 21 patients, of whom 10 had major molecular responses (3-log reduction at the BCR-ABL/ABL ratio). In 6 (18%) patients, BCR-ABL was negative by nested PCR. Four (13%) pts relapsed after a initial response (1 BC, 3 AP). Nine from 10 pts with sequential VNTR, improved chimerism to more than 95% donor cells after Gleevec therapy. One patient had autologous recovery of Ph negative cells. Hematologic toxicity grade II–IV was observed in 21 pts (63%). Eleven pts developed grade IV neutropenia with a duration of 0–8m (M 2m). Five pts developed non-hematologic toxicity grade III–IV. Dose was held in 4 pts, increased in 6 and reduced in 11 pts. Only two pts developed skin and liver chronic GVHD, and two patients who had c-GVHD before therapy with imatinib did not flare. Estimated 5y OS is 67%.
CONCLUSIONS:Gleevec can be used safely as therapy for BMT relapse, with durable cytogenetic and molecular responses in chronic phase;Complete chimerism is often reestablished by Gleevec therapy after allogeneic BMT;Increased incidence of GVHD was not observed in this group of patients.
Poor Prognosis for Patients after Myelo- and Non-Myeloablative Conditioning Therapy Followed by Allogeneic Peripheral Blood Stem Cell Transplantation Admitted to Intensive Care Unit.
Authors: Neumann Frank , Lobitz Oliver , Fenk Roland , Kondakci Mustafa , Graef Thorsten , Bruns Ingmar , Kelm Malte , Huenerlituerkoglu Ali-Nuri , Haas Rainer , Kobbe Guido 
Published: October 2019

Abstract
Background and Aims: The role of intensive care unit (ICU) support for patients following allogeneic peripheral blood stem cell transplantation (PBSCT) is controversial. In an era of constrained resources, we assessed prognostic factors predictive for survival in patients after myeloablative (MAC) and non-myeloablative allogeneic (non-MAC) PBSCT over a period of seven years.
Patients: Between January 1999 and February 2006 three-hundred and one patients with various hematological malignancies underwent allogeneic stem cell tranplantation in our institution (MAC 196, non-MAC 105). Of these, sixty-four patients (21,3%) with a median age of 47 years (range 18–64 years; female 24, male 40; MAC 49, non-MAC 15) were admitted to the ICU during the first two years following PBSCT (median 55 days, range 1–631 days). We looked for variables defining the SOFA (Sequential Organ Failure Assessment) and the SAPS (Simplified Acute Physiology Score) score on the day of ICU admission and five days later to discriminate patients with poor and good prognosis with regard to survival. We also looked for variables such as age of patients, diagnosis, disease status, donor type, time between transplantation and ICU admission, reason for ICU admission and occurrence of veno-occlusive disease and GVHD.
Results: Mechanical ventilation was required by all patients admitted to the ICU. Median survival following referral to ICU was 22 days (range 0 – 959 days). The main reason for death was sepsis (56%). Among 49 patients who had received MAC 19 (39%) survived the ICU stay with a median survival time of 11 months (range 0–29 months). In the group of patients who had received non-MAC 3 out of 15 patients (20%) could be discharged from the ICU with a median survival time of 5 months (range 4–12 months). Looking at the twenty-two ICU survivors there were seven patients who survived the following year resulting in an overall survival of 11% one year after ICU admission. Only the SOFA score (p = 0.002) on the day of ICU admission was of prognostic relevance for survival.
Conclusion: ICU admission and respiratory failure are associated with poor prognosis after allogeneic stem cell transplantation The probability of survival is independent from the type of conditioning therapy. The SOFA score is a predictor for short term survival but fails to identify long term survivors.
“Designer” Cells for Cellular Immunotherapy: Zinc Finger Nuclease (ZFN) Stimulated Targeted Recombination for the Simultaneous Disruption of the Endogenous Glucocorticoid Receptor and Site-Specific Addition of the IL13-Zetakine.
Authors: Reik Andreas , Holmes Michael C. , Urnov Fyodor D. , Zhou Yuanyue , Mendel Matthew , Liu Pei-Qi , Moehle Erica , Rebar Edward J. , Miller Jeffrey C. , Ando Dale , Gregory Philip D. , Jensen Michael 
Published: October 2019

Abstract
Immunotherapy utilizing genetically-modified cytolytic T-lymphocytes (CTL) provides a promising therapeutic approach for treating a variety of diseases, including cancer.
We have demonstrated that CTLs encoding a chimeric T-cell receptor (IL13-zetakine), consisting of an extracellular IL-13 domain and a cytoplasmic CD3 domain, can be re-directed to target malignant glioma both in vitro and in animal models. This chimera re-targets the antigen-specific effector functions of modified CTLs to recognize glioblastomas due to the high expression of IL13R in these tumors. However, practical application of this approach is limited by the fact that patients undergoing surgical resection of the tumor often require treatment with glucocorticoids to control the resulting inflammation. Such treatment blocks the activity of the re-directed CTL clones and thus inhibits their therapeutic action.
To overcome this limitation and render these tumor-specific CTLs resistant to glucocorticoids we have chosen to employ engineered ZFNs to specifically disrupt the endogenous glucocorticoid receptor (GR) gene. Heterodimeric ZFNs, consisting of the cleavage domain of the restriction enzyme FokI linked to engineered zinc finger DNA-binding domains, can be designed to specifically cleave a predetermined site in the genome. We have shown that these ZFN-induced double strand breaks can promote homologous recombination with high efficiency. In the present study we have investigated the use of ZFNs to simultaneously effect functional inactivation of human GR via specifically targeting the integration of the IL13-zetakine expression cassette into the GR locus itself. We can show that GR-specific ZFNs cleave their intended target sequences with high specificity and efficiency - resulting in the disruption of GR and the creation of glucocorticoid resistant cells. Moreover, we can demonstrate that these ZFNs coupled with an appropriate IL13-zetakine containing donor-DNA molecule can stimulate the integration of this chimeric T-cell receptor directly into the GR locus. Thus, this procedure results in the simultaneous knockout of GR and addition of the IL13-zetakine in a genetically defined manner.
These data support the notion that ZFN-modified cells can be engineered to express chimeric antigen receptors from a predetermined genomic locus and may provide a general approach to generating effective cellular immunotherapy strategies.
Tumor-Activated Human NK Cells Specificall Ylyse Autologous and Allogeneic NK-Resistant Tumor Targets.
Authors: Lowdell Mark W. , Bakhsh Ismail , North Janet , Marden Chloe , Addison Elena , Mackinnon Stephen 
Published: October 2019

Abstract
Pre-incubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias and solid tumors, even when HLA-matched; allogeneic or autologous. The primed NK cells remained non-responsive to HLA-C matched and mismatched normal peripheral blood or bone marrow mononuclear cells from multiple donors and did not affect in vitro colony forming unit assays. NK cells isolated from normal healthy donors and co-incubated with irradiated CTV-1 tumor cells synthesized CD69 within 60 min with maximal expression achieved within 6 hr. The tumor-activated NK cells (T-ANKs) lysed NK resistant RAJI and Daudi cells in a 4-hour assay (Fig 1). The degree of lysis was equivalent to that of matched lymphokine activated killer cells (LAK) after non-specific activation with IL-2. CTV-1 cells are HLA-C type 2 homozygous and express HLA-Bw4 alleles. They thus can ligate KIR2DL1 (CD158a) and KIR3DL1 (CD158e1) on NK cells. Co-cultures of HLA-matched or mis-matched NK with CTV-1 showed there was no significant difference in the generation of T-ANK activity in the presence or absence of KIR ligation. T-ANK cells from allogeneic donors were capable of lysis of primary AML cells of all FAB types, the relatively NK-resistant breast cancer cell line, MCF-7, and primary tumor cells isolated from resected tissue from patients with breast cancer and ascities from patients with ovarian cancer. Autologous T-ANK cells were generated from five AML patients in clinical remission; all of whom had low level or undetectable NK activity to their presentation disease. T-ANK cells from all five donors consistently induced significantly greater lysis of autologous AML blasts than the matched NK cells (p<0.05) (Fig 2). To investigate the tumor-restriction of allogeneic T-ANK cells we isolated NK cells from normal donors and either activated them with CTV-1 cells overnight or maintained them in media. These T-ANK cells were then compared with NK cells from the same donor with respect to lysis of normal autologous and allogeneic PBMC. Neither NK nor T-ANK cells lysed autologous PBMC nor did they lyse PBMC from HLA-C mismatched normal donors. To determine the likelihood of bone marrow suppression by T-ANK cells we established hematopoietic colony forming assays with bone marrow from 5 normal donors and added T-ANK from HLA-C mismatched donors at increasing ratios. CFU-GM, BFU-E and CFU-GEMM were not affected by co-incubation with HLA-mismatched T-ANK even at effector:target ratios of 20:1. We believe that this demonstrates a two stage process for NK activation; priming and triggering, the ligands for which are distinct. Tumor priming of NK primes cells capable of lysing a broad spectrum of NK-resistant tumors which may irrespective of KIR ligation which may represent an “off-the-shelf” immunotherapy product.
Fig 1 Fig 1. Fig 2 Fig 2.
Playing the Game Together: Coexpression of a Single Chain T Cell Receptor and a T Cell Receptor Constant-Alpha Domain Triggers Tumor Reactivity.
Authors: Thomas Simone , Voss Ralf H. , Intan Ratna , Engel Renate , Kuball Juergen , Huber Christoph , Theobald Matthias 
Published: October 2019

Abstract
Grafting T cells by tumor-antigen specific T cell receptors (TCR) could trigger the initiation of effector function and redirect T cell cytotoxicity towards tumors. We utilized various HLA-A2.1 transgenic mice to bypass human MDM2- and p53-specific self-tolerance. In contrast to the use of HuCD8×A2Kb transgenic mice to generate an MDM2-specific CD8-dependent TCR, we generated a high-affinity, CD8-independent p53-specific TCR in single human A2.1 transgenic mice. The efficiency of double chain (dc) TCR modified T cells could be affected by the incorrect TCR α/β chain pairing between endogenous and transgenic TCR constructs to form hybrid TCR potentially leading to autoimmunity. To address this concern, chimeric A2.1-restricted peptide-specific murine single chain (sc) TCRs were constructed (Vα-Li-VβCβ) and retrovirally transduced into human T cells. Despite detectable surface expression, these chimeric receptors were not able to convey any MDM2- or p53-specific cytolytic activity. Therefore we developed a truncated TCR-alpha domain (Cα) comprising solely the TCRα signal peptide, the ecto-domain, the transmembrane region as well as the cytoplasmic tail and cotransduced these construct with the scTCRs. We anticipated that Cα would stabilize scTCR expression by interacting with the single chain beta chain. Indeed, this approach not only led to increased expression levels of the chimeric scTCRs, but also induced specific lysis of A2.1 positive MDM2 or p53 peptide-pulsed target cells as well as solid tumor cell lines. Recognition of malignant targets by p53 specific scTCR transduced CD4 and CD8-positive T cells was equivalent to that observed with double-chain p53 TCR gene modified effector cells. To test whether this concept is applicable to human TCRs as well, we constructed a human gp100-specific scTCR and a human Cα domain. In contrast to the gp100-specific double chain TCR, only a marginal expression pattern was observed for the human scTCR / Cα constructs. Introduction of an additional disulfide bond within the constant domains in order to stabilize TCR surface expression showed no effect. Since murine TCR are expressed on human T cells to a much higher extent, the human constant β-domain of the scTCR was replaced by murine Cβ. Comparable to the murine scTCR concept, the chimerized scTCR coexpressed with murine Cα demonstrated high cell surface expression and triggered cytotoxicity of malignant A2.1/gp100-positive targets.
In summary, our results lay a commonly applicable conceptual basis for the construction of therapeutic scTCR to prevent recombination of natural and transgenic dcTCR alpha and beta chains.
Effect of Cytosolic Hereditary Spherocytosis Mutations of Human Erythrocyte Anion Exchanger 1 on Its Interaction with Cytoskeletal Protein 4.2.
Authors: Bustos Susan P. , Reithmeier Reinhart A.F. 
Published: October 2019

Abstract
Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Human AE1 has two functionally independent domains: its 52 kDa C-terminal membrane domain catalyzes the exchange of chloride for bicarbonate across the membrane while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the membrane to the cytoskeleton, giving the red cell its stability and flexibility. Several proteins bind to cdb3 including cytoskeletal protein 4.2, ankyrin, glycolytic enzymes and deoxyhemoglobin. Three mutations in cdb3 (E40K, G130R and P327R) are associated with the hemolytic anemia hereditary spherocytosis (HS) and decreased levels of erythrocyte protein 4.2 while maintaining a normal amount of AE1 at the red cell membrane. Wild-type and mutant cdb3 proteins were expressed in E. coli and purified and it was shown through a variety of biophysical methods that these three HS mutations do not cause major structural changes in this domain. Each of these mutations introduces a positive charge at the surface of the protein that is predominantly negatively charged, which may have an effect on protein interactions while maintaining its native folded structure. Full-length wild-type AE1 or HS mutants were co-expressed with protein 4.2 in HEK-293 cells in order to study their interaction in a mammalian cell line. All three HS mutant proteins were expressed at similar levels to wild-type in these cells and were shown to be present at the membrane using immunofluorescence and confocal microscopy. These proteins were also co-expressed in LLCPK-1 cells for the purpose of co-localization studies using immunofluorescence. A series of GST fusion proteins of protein 4.2 domains were designed based on a homology model of protein 4.2 and regions of the protein known to interact with AE1. These fusion proteins were expressed in E. coli and purified on glutathione-Sepharose resin and were used to study their interaction with purified wild-type and HS mutant cdb3 proteins in vitro. Blot overlay analysis showed that a protein 4.2 GST fusion protein containing a putative β-hairpin region (Asp145 to Glu203) binds specifically to wild-type cdb3 while GST does not. It is hypothesized that since these three HS mutations do not cause major structural changes in cdb3, the decreased level of protein 4.2 in the red cells of these patients is a result of impaired binding that occurs due to these mutation sites.
Gfi1b Association to Human Hematological Abnormalities.
Authors: Villegas Ana , Gonzalez Fernando A. , Anguita Eduardo 
Published: October 2019

Abstract
Lineage specific transcription factors play essential roles in regulation of hematopoietic development. Transcription factor abnormalities have been frequently described in acute leukemia, mostly through cytogenetic changes. Nevertheless, point mutations can be easily missed. Recently, mutations in the erythroid and megakaryocyte specific transcription factor GATA1 have been discovered in patients with dyserythropoietic anemia and acute megakaryoblastic leukemia (AML-M7) with Down syndrome. Besides GATA-1, located on the X-chromosome, point mutations have been described in biallelic genes. This is the case of AML1 (RUNX1). PU1 and C/EBPalpha also represent examples of transcription factors in which point mutations are found in leukemia.
A new zinc finger transcription factor involved in erythropoiesis is Gfi1b. Gfi1b was recently identified by sequence homology with oncogene Gfi1. Gfi1b knockout has demonstrated that this gene is essential for development of both erythroid and megakaryocytic lineages, and in its absence no enucleated erythrocytes are produced.
Several Gfi1b DNA and protein targets (GATA1, Gfi1, AML1, p21WAF1, IL-6 Socs1 and Socs2) have been described that might be involved in malignancy. These findings indicate that Gfi1b is at the centre of hematopoiesis and may be a good candidate gene to be involved in hematological abnormalities.
We have searched for Gfi1b point mutations in 122 patients with acute leukemia of all FAB types at diagnosis or relapse and 9 cases of congenital dyserythropoietic anemia. We have amplified Gfi1b promoter, coding and non-coding exons (Nucleic Acids Res2004;32:3935–46, MN 004188) by high fidelity PCR and screen for point mutations through dHPLC (Wave, Transgenomic) followed by sequencing of the cases with abnormal pattern. SNIPs in the promoter and exons were further confirmed in at least another PCR, cloned in pGEM-T easy vector system (Promega) and sequenced. Alleles with promoter SNIPs were cloned in pGL3-Enhancer vector (Promega), and transiently cotransfected with pEGFP-C2 (Clontech) to K562 cells. Luciferase activity was determined with Dual-Luciferase Reporter Assay (Promega).
We found two promoter SNIPs in sequences conserved from chicken to human, one of them affecting a GATA-1 site, reducing promoter in vitro activity by 60 and 50% respectively. We also discovered a congenital exonic SNIP causing a mammalian conserved serine change to leucine. We excluded these to be frequent polymorphisms by dHPLC analysis of 96 blood donors. Although we cannot at present establish a clear relation between the former SNIPS and leukemia, we will discuss the presence of other milder hematological abnormalities. So far this is the first report of Gfi1b mutations that can be related to human pathology.