bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of the bZip Protein Bach2 in Umbilical Cord Blood (UCB) T Lymphocytes.
Authors: Huang Y. , Fanning L. , Masri O. , Kaminski B. , Kadereit S. , Laughlin M. J. 
Published: October 2019

Abstract
Graft versus host disease (GVHD) occurring after allogeneic stem cell transplantation is mediated by donor T cell alloreactivity against major and minor histocompatibility antigens presented by recipient antigen presenting cells (APC). Clinical reports identify that umbilical cord blood (UCB) elicits reduced incidence and severity of GVHD due to reduced cytokine production. Nuclear factor of activated T cells (NFAT1) plays a pivotal role in the transcription of cytokine genes and other immune responses. NFAT1 regulates the expression of cytokine genes through direct interaction with AP-1 (Fos/Jun, basic-lucine zipper bZip proteins). NFAT1 and AP-1 cooperatively bind to a 15-bp DNA that contains both NFAT1 and AP-1 sites. We used Affymetrix oligonucleotide microarrays to compare gene expression of primary purified CD4+ UCB T-cells to adult peripheral blood CD4+ T-cells (AB) at baseline, 6, and 16 hours after primary stimulation with anti-CD3/anti-CD28. NFAT-regulated genes exhibited lower expression in UCB CD4+ T-cells including: GM-CSF, IFN- g, TNF-a, IL-3, IL-4, IL-5, IL-13, IL- 2R (CD 25). In addition, AP-1 (Fos/Jun) did not show a significant change. However, Bach2, an 841- amino-acid-residue containing bZip protein, did show a significant increase in UCB CD4+ T-cells. Previous reports identify from the crystal structure of the quaternary complex of NFAT1/AP-1/DNA (Chen et al., Nature 1998) that NFAT1 is in contact with the lucine zipper region of AP-1 while the basic region of AP-1 binds to the DNA site at TGTTTCA. The amino-acid residues that make contact with DNA in the bZip regions of Jun and Fos are highly conserved in the Jun and Fos families. These amino acid residues are also 100% conserved in the human and mouse Bach2. The high similarity of DNA-contacting amino-acid residues between Bach2 and AP-1 strongly suggests that Bach2 and AP-1 may bind to the same DNA site. We hypothesize that Bach2 may act as a transcriptional repressor of cytokine genes due to the fact that increased Bach2 in UCB may compete with AP-1 for same DNA binding site, which in turn may partly block the interaction of NFAT1 with AP-1 and the expression of cytokine genes in UCB. We are currently pursuing this hypothesis. Ongoing studies are to confirm whether the protein levels of Bach2 and AP-1 are different in UCB compared to with AB CD4+ T-cells. This includes using gel-shift assays in vitro to test Bach2 binding to the DNA site of AP-1 as well as to test potential competitive binding of Bach2 and AP-1 to the same DNA site.
Identification of Immunodominant Alloreactive T-Cell Epitopes on the Jka Red Blood Cell Protein Inducing Either Th1 or Th2 Cytokine Expression: Role of DRB1*01 Molecules in Jka Peptide Presentation.
Authors: Ansart-Pirenne Helene , Zeliszewski Dominique , Lee Ketty , Martin-Blanc Stephanie , Rouger Philippe , Noizat-Pirenne France 
Published: October 2019

Abstract
Background: although antibodies implicated in red blood cell (RBC) blood group antigen alloimmunization are studied for many years, little is known about helper T cell responses that drive their production. The aim of this work was to determine T cell antigenic determinants involved in T cell responses against RBC antigens, and the subsequent patterns of stimulatory cytokine production.
Study Design: the Kidd blood group antigen (Jka), was selected as a model. We investigated cytokine expression by helper T cells after stimulation by Jka peptides. Peripheral mononuclear cells were isolated from Jka primed donors producing anti-Jka antibodies and from non anti-Jka alloimmunized donors as negative controls. Four 16-mer peptides mimicking the Jka sequence, 266–293, including the Asp280 polymorphism, and overlapping each over by 12 amino acids were tested to map alloreactive T cell epitopes. A sensitive method, the real time RT-PCR, was chosen to quantify Th1- and Th2-type cytokines (respectively IL-2 and IL-4) after Jka peptide stimulations. DRB1* and DQB1* low resolution typing, and DRB1* DNA subtyping were performed.
Results: lymphocytes from non anti-Jka alloimmunized donors produced low level of IL-2 or IL-4 (<10copies), while lymphocytes from all anti-Jka alloimmunized donors responded in a much higher level to at least one of the four Jka peptides. A clear Th1/Th2 dichotomy in the cytokine response was observed in this population. Indeed, among the 10 anti-Jka alloimmunized donors tested, 4 produced IL-2 alone and 6 produced IL-4 alone. Sequences among two peptides (Jka1and Jka2) were together able to elicit a response (IL-2 or IL-4) in 8 among 10 of these anti-Jka alloimmunized donors and represent a pool of immunodominant peptides. This Th1/Th2 dichotomy was not due to delays in kinetics of IL-2 and IL-4 productions. It was neither related to particular donor DRB1* or DQB1* molecules. The DRB1*01 phenotype frequency was increased (82%) in the anti-Jka alloimmunized donors as compared to the expected frequency (18%) in the caucasian population. This observation raised the question as to whether these molecules are associated to genetic susceptibility to anti-Jka alloimmunization.
Conclusion: These data indicate that Th1 and Th2 subsets are associated to the specific memory humoral immune response against Jka RBCs. The cytokine pattern (IL-2 or IL-4) is characteristic of the donor, whatever the peptide tested. DRB1*01 seems to be implicated in the peptide presentation to the T lymphocyte.
A detailed understanding of peptides and cytokines involved in T cell responses to Jka protein and the HLA class II molecules implicated in the peptide presentation constitutes a first step toward evaluation of peptide immunotherapy to prevent or treat anti-RBC alloimmunization.
Update of the Hyper-CVAD and Imatinib Mesylate Regimen in Philadelphia (Ph) Positive Acute Lymphocytic Leukemia (ALL).
Authors: Thomas Deborah A. , Faderl Stefan , Cortes Jorge , O’Brien Susan , Giles Francis , Garcia Guillermo , Kornblau Steven , Andreeff Michael , Beran Miloslav , Verstovsek Srdan , Pierce Sherry , Letvak Laurie , Salvado August , Champlin Richard , Kantarjian Hagop 
Published: October 2019

Abstract
The intensive combination chemotherapy program hyper-CVAD in newly diagnosed Ph+ ALL yields complete remission (CR) rates of 90%, but remissions are brief with median CR duration of 16 months [Kantarjian et al, JCO 18:547, 2000]. The activity of the tyrosine kinase inhibitor imatinib given as a single agent in relapsed or refractory Ph+ ALL or chronic myelogenous leukemia in lymphoid blast phase was 20% [Druker et al, NEJM 344:1084, 2001]. A phase II trial of imatinib and hyper-CVAD was conducted in newly diagnosed Ph+ ALL. Imatinib was given 400 mg days 1–14 of each course of therapy (fractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone alternating with high dose methotrexate and ara-C). Preliminary results were published in the first 20 patients (pts) treated, with CR rates of 100% in the de novo group, and improvement of disease-free survival (DFS) compared with hyper-CVAD alone [Thomas et al, Blood 103:4396, 2004]. To date, 32 pts with Ph+ ALL have been treated from April 2001 to February 2004. Twenty-six patients had active disease, either untreated (n=21) or refractory (n=5) to one induction course without imatinib. Six pts were in CR at study entry after one induction course without imatinib mesylate. Median age was 48 years (range, 17–75); 59% were male. Five had CNS disease (16%). Twenty-five of 26 pts (96%) with active disease at study entry achieved CR (1 failed to meet platelet criteria for CR). Median days to response was 21 days. Two of 26 pts (8%) required 2 courses to achieve CR. Allogeneic stem cell transplant (SCT) was performed in 13 pts in CR within a median of 3 months from start of therapy (range, 1–12). After a median follow-up of 2 years (range, 4–36 months), 1 primary refractory pt relapsed at 12 mos (bcr-abl/abl RT-PCR ratio <.05 at 9 mos), 1 pt relapsed day 149 after matched related SCT despite negative nested PCR for bcr-abl, and 2 pts changed therapy after 5 mos for persistent marrow Ph+ metaphases without overt leukemia relapse. Five pts died in CR, 3 older pts related to comorbid conditions (2 were negative for bcr-abl by nested PCR) and 2 related to complications of allogeneic SCT. Molecular response rate (negative bone marrow RT-PCR for bcr-abl confirmed by nested PCR) was approximately 50% in 19 pts who did not undergo allogeneic SCT. Outcome appears better with the hyper-CVAD and imatinib regimen with 2-year DFS rates of 87% (all pts) compared with 28% for hyper-CVAD alone or 12% for VAD (p<.001). Unexpected toxicities related to the addition of imatinib mesylate were not observed. The hyper-CVAD and imatinib regimen with or without allogeneic SCT continues to appear promising with additional accrual and longer follow-up.
Development of Lymphohematopoietic Progenitors during Human Embryonic Stem (hES) Cell Differentiation on OP9 Stromal Cells.
Authors: Slukvin Igor I. , Vodyanik Maxim A. , Bork Jack A. , Thomson James A. 
Published: October 2019

Abstract
hES cells provide an unique opportunity to study the earliest stages of hematopoietic commitment which are not easily accessible in the human embryo. To model early hematopoietic development, we cultured H1 and H9 hES cell lines on OP9 stromal cells without the addition of cytokines. On day 2 of co-culture, hES cells up-regulated brachyury expression and began to form mesodermal-like colonies. A transient population of blast colony-forming cells (CFCs) with the potential to differentiate into blood and endothelial cells was detected on days 3–6 of co-culture. CD34+ cells first appeared on day 3–4 of co-culture, which was coincident with induction of the transcription factors GATA-1, GATA-2 and SCL. CD43+ and CD41a+ cells along with CFCs emerged 2 days later within CD34+ population; 3–4 days before the appearance of CD45+ cells. We were able to obtain up to 20% of CD34+ cells from hES/OP9 co-culture and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8–9 days of culture. The hES cell-derived CD34+ cells were highly enriched in CFCs, displayed CD90+CD117+CD164+CD38- phenotype of primitive hematopoietic progenitors, and contained ALDHhigh cells as well cells with verapamil-sensitive ability to efflux rhodamine 123. Isolated CD34+ cells differentiated into lymphoid (NK cells) as well as myeloid (neutrophils and macrophages) lineages when cultured on MS-5 stromal cells in the presence of SCF, Flt3-L, IL7 and IL3. These data indicate that hES cell/OP9 co-culture reproduces the major events that are observed during embryonal hematopoietic development, including the formation of lympho-myeloid progenitors. We employed OP9 system for identification of the phenotype of early hematopoietic progenitors in humans and to directly differentiate hES cells into different blood lineages.
ZAP-70 Expressed in CLL Is an Hsp90 Client Protein and Its Level of Expression Can Be Downregulated by Specific Hsp90 Inhibitors.
Authors: Prada Carlos E. , Loria Olivier , Kamal Adeela , Burrows Francis J. , Kipps Thomas J. , Castro Januario E. 
Published: October 2019

Abstract
Hsp90 is a ubiquitous molecular chaperone protein that is involved in the folding, activation and assembly of many client proteins, including mediators of signal transduction, cell-cycle control and transcriptional regulation. In order to exert its function on client proteins, Hsp90 requires the formation of an active protein complex composed of cochaperone molecules and an active ATP binding site. Proteins identified as Hsp90 client proteins include transmembrane tyrosine kinases [HER-2/neu, epidermal growth factor receptor (EGFR), MET and insulin-like growth factor-1 receptor (IGF-1R)], metastable signaling proteins (Akt, Raf-1 and IKK), mutated signaling proteins (p53, Kit, Flt3 and v-src), chimeric signaling proteins (NPM-ALK, Bcr-Abl), steroid receptors (androgen, estrogen and progesterone receptors), cell-cycle regulators (cdk4, cdk6) and apoptosis related proteins. It has been postulated that malignant progression and cancer prognosis may be associated with the presence of activated Hsp90 which exists in heightened complexes with cochaperone proteins.
The zeta-associated protein of 70 kD (ZAP-70), is a protein kinase found in a subset of patients with chronic lymphocytic leukemia (CLL) and its expression correlates with disease progression, poor clinical outcome, decreased overall survival and early requirement for treatment. ZAP-70 promotes phosphorylation of downstream signaling molecules after engagement of the B cell receptor (BCR). Since ZAP-70 is a marker of disease progression in CLL we hypothesized that its abnormal over-expression and function in CLL B cells may depend on activated Hsp90. To test this, we performed immunoprecipitation, western blot and flow cytometry analyses on CLL samples from patients that differ in their level of ZAP-70 expression (ZAP-70 positive and negative based on a cutoff expression of 20% by flow cytometry). We observed that in ZAP-70 positive CLL samples (n=4) the majority of Hsp90 present in the cytoplasm was in a complexed-activated form, whereas in ZAP-70 negative samples (n=4) most Hsp90 was found to be non-complexed. In addition, we found that ZAP-70 co-immunoprecipitated with Hsp90, suggesting that it is an Hsp90 client protein. In addition, the level of ZAP-70 expression was decreased 30–40% in cells treated for 24 hours with Hsp90 specific inhibitors at nanomolar concentrations.
Together, our data show that CLL B cells that express ZAP-70 have larger amounts of activated Hsp90 than those with negligible expression of this tyrosine kinase protein. In addition, we found that ZAP-70 is an Hsp90 client protein and its level of expression could be downregulated by using specific Hsp90 inhibitors. These findings suggest an important role for Hsp90 as a prognostic marker in CLL and provide the rationale to investigate, in clinical trials, the activity of Hsp90 inhibitors and their effect on ZAP-70 expression.
Rituximab-Mediated Inhibition of the Constitutive NIK/IKK/IκB/NF-κB Signaling Pathway in Non-Hodgkin’s Lymphoma (NHL) B-Cell Lines: Role in Chemo-Sensitization.
Authors: Jazirehi Ali R. , Huerta-Yepez Sara , Cheng Genhong , Bonavida Benjamin 
Published: October 2019

Abstract
The chimeric anti-CD20 antibody rituximab (Rituxan, IDEC-C2B8) is widely used in the clinical treatment of patients with non-Hodgkin’s lymphoma (NHL). Rituximab sensitizes NHL B-cell lines to drug-induced apoptosis via selective down-regulation of Bcl-xL expression (Jazirehi et al., Mol. Cancer Therapeutics 2:1183, 2003). We hypothesized that rituximab-mediated down-regulation of Bcl-xL expression may be due, in part, to inhibition of constitutive NF- κB activity that regulates Bcl- xL expression. This hypothesis was tested following treatment with rituximab of CD20+ drug-resistant Ramos (Bcl-2−/ Bcl-xL+) and Daudi (Bcl-2+/ Bcl-xL+) cell lines. Rituximab decreased the phosphorylation of NIK, IKK and κB-Iα, diminished IKK kinase activity and decreased NF- κB DNA-binding activity. These events and down-regulation of Bcl-xL expression occurred with similar kinetics and were observed 3–6 h post rituximab treatment. The role of NF- κB in the regulation of Bcl-xL transcription in both Ramos and Daudi cells was demonstrated by using 1) promoter reporter assays in which deletion of the two tandem NF- κB binding sites in the upstream promoter region significantly reduced the luciferase activity 2) IκB super-repressor expressing cells and 3) by NF- κB specific inhibitors. The underlying mechanism of the inhibition of the NF-κB signaling pathway by rituximab was shown to be due, in part, to upregulation of Raf-1 kinase inhibitor protein (RKIP) expression, thus, interrupting the NF- κB signaling pathway through the physical association between NIK and RKIP, which was concomitant with Bcl-xL down-regulation. The direct role of Bcl-xL in drug-resistance was evaluated by using Bcl-xL over-expressing Ramos cells, which exhibited higher resistance to drugs that was partially reversed by rituximab. These findings reveal a novel mechanism of action of rituximab-mediated signaling by inducing RKIP expression that negatively regulates the constitutive NF- κB pathway resulting in Bcl-xL down-regulation and chemo-sensitization of the NHL B-cells. Furthermore, these findings identify several targets, namely RKIP, Bcl-xL and the components of the NF- κB signaling pathway, for therapeutic intervention in combination with cytotoxic agents to reverse adaptive and acquired resistance of B-NHL.
Preclinical Evaluation of Hematide™, a Novel Erythropoietic Receptor Agonist for the Treatment of Anemia Caused by Kidney Disease.
Authors: Woodburn Kathryn W. , Fan Qing , Leuther Kerstin K. , Holmes Christopher P. , Zhang Jim J. , Velkovska Svetlana , Chen Min-jia , Schatz Peter J. 
Published: October 2019

Abstract
Hematide™ is a PEGylated synthetic dimeric peptide. Initial peptide sequences were identified by screening a human erythropoietin receptor (HuEPOr) with Recombinant Peptide Diversity libraries possessing > 1010 different chemical structures. Following extensive chemical analoging and introduction of novel architectural features, a compound containing a dimeric peptide having no detectable sequence identity to natural human EPO was created and this compound is substantially equipotent to natural EPO. The portion of the molecule containing the peptide was linked to polyethylene glycol to yield Hematide, a potent stimulator of erythropoiesis with sustained plasma persistence. Antibodies generated experimentally to Hematide peptide do not cross-react with recombinant human EPO and therefore this peptide-based HuEPOr agonist is unlikely to cause aplastic anemia and loss of potency for either recombinant erythropoietin receptor agonists or for the recipient patient’s endogenous EPO. Pharmacokinetic studies in rats, dogs and monkeys have demonstrated the extended plasma half-life of Hematide. In the rat, elimination half-life is between 21.5 to 30.7 hours, steady state volume of distribution (Vss) is between 42.7 to 65.6 mL/kg and clearance is slow at 1.3–2.3 mL/h/kg. In the dog, elimination half-life was 73.7 hours while clearance and Vss were 0.69 mL/h/kg and 69.6 mL/kg, respectively. Prolonged half-lives and slow clearance times are also evident in the monkey. At a dose of 1.35 mg/kg, a half-life of 58.4 hours was obtained with a clearance rate of 0.96 mL/h/kg. Erythropoietic activity of Hematide has been demonstrated in the normocythemic mouse, rat, dog, and monkey, and in the nephrectomized rat. Pharmacology studies following single dose administration in mice, rats (normocythemic and nephrectomized) and monkeys indicate that the rise in hemoglobin is dose-dependent. Erythropoietic activity was demonstrated in Sprague-Dawley rats when Hematide was given every week for 6 weeks at doses up to 1.35 mg/kg. There was a pronounced erythropoietic response and no anti-Hematide antibodies were detected in any of the dose groups. In conclusion, Hematide is a potent erythropoietin receptor agonist with prolonged half-life and slow clearance times. It is anticipated that similar prolonged clearance will be observed in the clinic, potentially enabling dosing intervals of 3 to 4 weeks that may translate into improved patient convenience and compliance.
Phosphatidylinositol 3-Kinase p85 Subunit-Dependent Interaction with BCR/ABL: Molecular Mechanism and Biological Consequences.
Authors: Ren Shuyue , Bolton Elisabeth , Skorski Tomasz 
Published: October 2019

Abstract
BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Mutagenesis approach combined with pull-down assays indicated that both N-terminal and C-terminal SH2 domains (nSH2 and cSH2, respectively) and SH3 domain of p85 play an important role in the interaction with BCR/ABL. SH2 domains exerted their function through binding to the tyrosine-phosphorylated motifs, and SH3 domain recognized proline-rich regions. Disruption of these functions by introduction of point-mutations in nSH2, cSH2 and SH3 domains abrogated their interaction with BCR/ABL. p85 mutant protein (p85-mut) bearing these mutations was not able to interact with BCR/ABL, while its binding to the p110 catalytic subunit of PI-3k was intact. In addition, binding of Shc and Gab2, but not Crk-L, to p85-mut was abrogated. When expressed in BCR/ABL-transformed hematopoietic cells p85-mut diminished activation of Akt kinase, the downstream effector of PI-3k. This effect was associated with the inhibition of BCR/ABL-dependent growth of hematopoeitic cells line and murine bone marrow cells. Interestingly, addition of IL-3 rescued BCR/ABL-transformed cells from the inhibitory effect of p85-mut. SCID mice injected with BCR/ABL-positive hematopoietic cells expressing p85-mut survived longer in comparison to the animals inoculated with BCR/ABL-transformed counterparts transfected with empty plasmid. In conclusion, we have identified the domains of p85 responsible for the interaction with BCR/ABL. Moreover, we demonstrated that expression of p85-mut, which binds to the p110 catalytic subunit of PI-3k but is not able to interact with BCR/ABL, affected the growth ability of BCR/ABL-positive leukemia cells.