bluuebunny/crossref_metadata_2025 huggingface dataset:
Ship Is Essential for Both Endotoxin- and CpG-Induced Tolerance and for Their Cross-Tolerance.
Authors: Sly Laura M. , Omeis Stephanie L. , Rauh Michael J. , Krystal Gerald
Published: October 2019

An initial exposure to lipopolysaccharide (LPS, also known as endotoxin), which activates macrophages via Toll-like receptor 4 (TLR4), induces a transient state of hypo-responsiveness to a subsequent challenge with LPS. The mechanism underlying this phenomenon, termed endotoxin tolerance, remains poorly understood although the activation/upregulation of a number of negative regulators has been implicated. Related to this, we recently demonstrated that the SH2-containing inositol-5′-phosphatase, SHIP, which is a key negative regulator of the PI3K pathway in hematopoietic cells, is essential for endotoxin tolerance. Specifically, we found that SHIP−/− bone marrow derived macrophages (BMmΦs) do not display endotoxin tolerance. Moreover, an initial LPS treatment of wild-type BMmΦs increases the level of SHIP protein 10 fold and this increase is critical for the hypo-responsiveness to a subsequent LPS stimulation. Interestingly, this increase in SHIP protein is mediated by the LPS-induced production of autocrine-acting TGFβ and neutralizing antibodies to TGFβ, as well as antisense oligonucleotides to SHIP, block LPS-induced endotoxin tolerance. In vivo studies with SHIP+/+ and −/− mice confirm these in vitro findings and show a correlation between the duration of endotoxin tolerance and elevated SHIP levels (Sly et al., Immunity, in Press). We have now followed up on these studies by exploring the role of SHIP in the stimulation of other Toll-like receptors. Specifically, we asked whether SHIP plays a role in repressing CpG-induced pro-inflammatory cytokine production, which is mediated via the activation of TLR9. Our results, using SHIP+/+ and −/− BMmΦs, suggest that SHIP negatively regulates this TLR as well and is required for CpG-induced tolerance. Moreover, we found that CpG treated SHIP−/− BMmΦs show higher pro-inflammatory cytokine production compared to their SHIP+/+ counterparts when challenged with LPS. This indicates that SHIP plays a critical role in dampening down inflammatory responses in the innate immune system.
PPAR-gamma Agonists Inhibit Toll-Like Receptor Mediated Activation of Dendritic Cells Via the MAP Kinase and NF-kB Pathways.
Authors: Mirakaj Valdete , Appel Silke , Grünebach Frank , Weck Markus M. , Reichardt Volker L. , Brossart Peter
Published: October 2019

Dendritic cells (DC) play an important role in initiating and maintaining of primary immune responses. However, little is known about the termination of once induced immunological responses. Cyclopentenone prostaglandins (15d-PGJ2, PGD2) are produced during the late phase of inflammation due to upregulation of COX2 and can lead to the resolution of an inflammation by activation of PPAR-gamma dependent and independent pathways. In this study we analyzed the effects of 15d-PGJ2 and the synthetic PPAR-gamma ligand troglitazone (TGZ) on the immunogenicity of monocyte derived DC upon stimulation with toll-like receptor (TLR) ligands. Activation of PPAR-gamma resulted in a reduced activation of DC via the TLR ligands 2, 3, 4 and 7 characterized by downregulation of CD83, adhesion and costimulatory molecules. Moreover, these cells secreted lower levels of cytokines and chemokines involved in T lymphocyte activation and recruitment including IL-12 and RANTES. In contrast to these results, MCP-1 production was increased due to the treatment with PPAR-gamma agonists. To determine the mechanisms by which PPAR-gamma regulates DC function we performed Western blot analyses and found that the inhibitory effects on TLR induced DC activation were mediated via inhibition of the NF-kB and MAP kinase pathways. Incubation of DC during the differentiation from monocytes with 15d-PGJ2 or TGZ resulted in downregulation of TLR ligands induced phosphorylation of ERK1 as well as reduced expression of nuclear localized members of the NF-kB family. No effect on the expression of MyD88, an adaptor molecule involved in the signal transduction of TLR, was observed. Our results demonstrate that inhibition of the MAP kinase and NF-kB pathways is critically involved in the regulation of TLR and PPAR-gamma mediated signaling in DC an represent a novel negative feed-back mechanism involved in the resolution of immunological responses.
M-CSF Drives Human Cord Blood Monocyte Differentiation along an Alternative Pathway into Suppressive CXCR4highIL-10highIL-12absentTNF-adefective Dendritic Cells.
Authors: Li Geling , Broxmeyer Hal E.
Published: October 2019

M-CSF Drives Human Cord Blood Monocyte Differentiation along an Alternative Pathway into Suppressive CXCR4highIL-10highIL-12absentTNF-αdefective Dendritic cells

Geling Li and Hal E. Broxmeyer. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA; Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA; Walther Cancer Institute, Indianapolis, IN, USA

Macrophage colony-stimulating factor (M-CSF) in cooperation with TGF-β1 induces CD34+ hematopoietic progenitor cells to differentiate into Langerhans cells, a subset of immature dendritic cells (DCs) specifically localized in the epidermis. M-CSF levels are dramatically elevated in immunosuppressive conditions and M-CSF deficient mice are osteopetrotic. Therefore, we hypothesized that M-CSF might be involved in the generation of a novel subtype of suppressive DCs which may be located primary in bone marrow where large amounts of SDF-1 are constitutively produced. We also speculated that M-CSF-induced DCs might express the only known ligand of SDF-1, CXCR4 and migrate towards SDF-1. Highly purified umbilical cord blood monocytes (> 98% purity) cultured in the presence of M-CSF, IL-4 and TGF-β1 differentiated into a highly homologous population of cells with typical dendrites and acquired a langerhan cell (LC) phenotype by up-regulated expression of CD1a and E-cadherin. We termed these cells M-LC to distinguish them from regular LCs cultured in the presence of GM-CSF, IL-4 and TGF-β1. In comparison with LC, M-LCs produced a much higher level of IL-10, no IL-12 (detection limit, 4 pg/ml) and a minimal level of TNF-α (21.6 ± 37.5 pg/ml) upon LPS stimulation. LPS-activated M-LCs induced lower allogeneic mixed lymphocyte reaction (MLR) than LPS-activated LCs. Furthermore, exposure to LPS-activated M-LCs rendered a state of low-responsiveness in cord blood naïve CD4+ T cells to further re-stimulation with alloantigens in secondary MLR. M-LCs expressed much higher surface levels of CXCR4 than LCs regardless of their maturation status. M-LCs started to migrate towards SDF-1 at a lower concentration (≥10 fold lower than that required for LCs) and displayed enhanced responsiveness to SDF-1-induced chemotaxis. Our results demonstrate that M-CSF drives monocyte differentiation along an alternative pathway into suppressive CXCR4highIL-10highIL-12absentTNF-αdefective DCs. These novel DCs may be of use to suppress unwanted immune responses in vivo.
The Distinctive Effects of Reactive Oxygen Species (ROS) and Redox Status on the Maturation of Human Monocyte-Derived Dendritic Cells.
Authors: Yamada Hiroko , Arai Toshiyuki , Endo Nobuyuki , Sasada Masataka , Uchiyama Takashi
Published: October 2019

The role of reactive oxygen species (ROS) and redox status on the maturation of dendritic cells (DC) was examined using alpha-phenyl-tert-butylnitrone (PBN) and glutathione reduced form ethyl ester (GSH-OEt). PBN is widely used as a spin trapping agent in electron paramagnetic resonance (EPR) studies. Since PBN reacts with free radical species and stabilizes them, PBN serves as an antioxidant. GSH not only serves as a major antioxidant but also plays a central role in maintaining intracellular redox balance, and GSH-OEt increased the intracellular GSH level. When human monocyte-derived DC were stimulated with LPS, up-regulation of the expression of the surface molecules (HLA-DR, CD40, CD80, CD86 and CD83), production of cytokines (TNF-α and IL-12p70) and allostimulatory capacity were observed. The LPS-induced cytokine production was suppressed by both PBN and GSH-OEt, while the up-regulation of the expression of surface molecules and the allostimulatory capacity were suppressed by only GSH-OEt but not by PBN. The mean values of TNF-α of LPS, LPS+PBN and LPS+GSH-OEt were 1220, 783 and 238 pg/ml, respectively (n=12). Those of IL-12p70 were 632, 358 and 5 pg/ml, respectively (n=12). The EPR study revealed that ROS was generated by LPS and that the ROS generation was attenuated by both PBN and GSH-OEt. Flow cytometric analysis revealed that intracellular GSH content was decreased by LPS and that this reduction was attenuated by GSH-OEt not by PBN. That is, PBN quenched the generated ROS but did not affect the redox status, while GSH-OEt not only quenched the generated ROS but also affect the redox status in the LPS-stimulated DC. The allostimulatory capacity of DC correlates with the expression of surface molecules and it does not depend on IL-12 production. Therefore, our findings suggest that ROS and redox status have distinctive effects on the maturation of DC. The ROS is involved in the cytokine production, while the redox status is involved in the up-regulation of surface molecules and allostimulatory capacity in the LPS-stimulated DC. LPS induces the activation of intracellular signaling cascade via Toll-like receptor 4. The signaling cascade is consist of two distinct pathways, MyD88-dependent and independent pathways. In DC, the MyD88-dependent pathway is involved in the cytokine production, while the MyD88-independent pathway is involved in the up-regulation of CD80 and CD86. The former may be ROS-sensitive, the latter may be redox status-sensitive. Differential regulation of the ROS and the redox status in DC may be possible to adequately modulate inflammatory and immune responses in the disease, such as graft-versus-host disease.

Figure Figure
A Role for Notch Signaling in the Commitment of Human Blood Monocytes to Langerhans Cells.
Authors: Shibasaki Tetsunori , Katayama Naoyuki , Ohishi Kohshi , Masuya Masahiro , Shiku Hiroshi
Published: October 2019

We previously demonstrated that Notch ligand Delta-1 in concert with GM-CSF and TGF-β1 promotes the differentiation of human blood monocytes into Langerhans cells that are characterized by the expression of CD1a, Langerhans-associated granules Langerin, cutaneous lymphocyte-associated antigen (CLA), CC chemokine receptor 6 (CCR6), and E-cadherin. These data extended the functional scope of Notch ligand Delta-1 in human adult hematopoiesis. HES-1 is known to be the target gene by Notch signaling. We examined the effect of Delta-1 on the expression of HES-1 mRNA in CD14+ blood monocytes in the presence of GM-CSF and TGF-β1, using real-time RT-PCR. When CD14+ blood monocytes were cultured with Delta-1, GM-CSF, and TGF-β1, the expression level of HES-1 mRNA increased approximately 10-fold at 24 hours of incubation, compared with the expression level in freshly isolated CD14+ monocytes. However, the expression level of HES-1 mRNA declined at 48 hours of incubation. This finding suggests that Delta-1 may operate at the early stage of the differentiation pathway from CD14+ monocytes to Langerhans cells. To explore this issue more precisely, we cultured CD14+ monocytes in the presence of Delta-1, GM-CSF, and TGF-β1 for 2 days, and subsequently replated the cells into the cultures without Delta-1. At day 7 of culture, cultured cells were harvested and characterized by phenotypic analysis. This initial 2-day exposure of CD14+ monocytes to Delta-1 gave rise to Langerhans cells, similar to the observation obtained with the supplementation of Delta-1 throughout 7-day culture. In turn, when Delta-1 was added at day 2 of culture, Langerhans cells were not induced, but instead the resulting cells exhibited the features of macrophages. Our results indicate that in response to Delta-1, human blood monocytes appear to initiate the differentiation program toward Langerhans cells, while they are incapable of differentiating into Langerhans cells after their commitment to macrophages.
Thalidomide (T) in Combination with Fludarabine (F) as Initial Therapy for Patients (pts) with Treatment Naïve Chronic Lymphocytic Leukemia (CLL): Results of a Phase I Trial.
Authors: Chanan-Khan Asher Alban , Miller Kena , Koryzna Alexandra , Takeshita Kenichi , McCarthy Philip , Mohr Alice , Donohue Kathleen , Bernstein Zale P. , Alam Arif , Czuczman Myron S.
Published: October 2019

CLL is an incurable disease. Standard treatment with F results in an ORR of 63% (CR 20%). Improved RR are noted when F is combined with biologic agents such as rituximab. All pts eventually relapse with limited salvage options. TNF-a is an important cytokine in the pathogenesis of CLL. T is an immunomodulatory drug with anti- TNF-a, anti-VEGF and immunostimulatory activity. We have completed a phase I study combining T with F as an immunochemotherapeutic approach to enhance anti-CLL activity of F. Treatment-naïve pts requiring therapy for CLL were eligible for this study. T was started at D1 and continued for 6 months stepwise in 3 cohorts of T (100,200,300 mg). Standard dose F (25mg/m2 x 5 D every 4 wks) was given for 4–6 cycles starting on D7. Antitumor activity of T alone was assessed at D7 prior to the first F dose. Low-dose coumadin (1 or 2 mg po qd) was used for prophylaxis against venous thromboembolism (VTE). Thirteen pts (9M, 4F; median age 65,range 38–74 yrs) have been enrolled on 3 dose levels (cohort #1 n =6, #2 n=3, #3 n=4). All pts are available for toxicity analysis. Pts were considered evaluable for response if they completed the intended 6 months of T. 3 pts were removed from study for toxicity (2VTE, 1 in the 1st week of therapy prior to F infusion, 2nd during the 4th cycle, 1 Hep C reactivation presumably secondary to F), without evidence of disease progression. 9 pts completed 6 months of therapy and are available for response (5CR and 4PR, ORR of 100%) median follow-up of 12+ (range 6–18+) months. Response to T alone, assessed on D7 of cycle 1, was noted at all dose levels, and no dose-limiting toxicity was noted. Flare reaction (tender swelling of lymph nodes) was noted in 5/13 pts (38%) and was the most common side effect. Rash, fatigue and constipation were noted in 30%, 23% and 15% of the pts, respectively. Of the 60 cycles given on this study, 9 (15%) and 3 (5%) episodes of Grade III and IV non-hematologic toxicities were noted, respectively. In this phase I study, the combination of T with F was well tolerated with improved ORR over F. T alone appears to be active in CLL with clinical activity noted as early as D 7 at all dose levels studied. The combination does not appear to increase the incidence of VTE compared to T alone. Ongoing phase II portion of this study will further establish the potential role of FT in CLL.
Pentostatin and Cyclophosphamide with or without Rituximab Has Significant Activity in Patients with Previously Treated Chronic Lymphocytic Leukemia and Other Low Grade Lymphoid Neoplasms.
Authors: Lamanna Nicole , Kalaycio Matt , Maslak Peter , Jurcic Joseph , Scheinberg David A. , Gencarelli Alison N. , Horgan Denise , Weiss Mark A.
Published: October 2019

Combination therapy with purine analogs, alkylators, and/or monoclonal antibodies represents a promising new approach in the treatment of patients with chronic lymphocytic leukemia (CLL) and other low grade B cell neoplasms. Most regimens have utilized fludarabine as the purine analog, but the severe myelosuppression and immunosuppression of these combinations requires careful attention to dosing and schedule to minimize these complications. Of the purine analogs in CLL, pentostatin appears to be least myelosuppressive. We have previously reported our experience with pentostatin and cyclophosphamide (PC regimen) in a cohort of heavily pretreated patients with CLL (JCO21:1278, 2003). We have since added rituximab to this active combination (PCR regimen) and have treated a second cohort of patients with CLL and other low grade B cell neoplasms. We now report on our cumulative experience of 69 patients (23 received PC and 46 received PCR) treated with pentostatin combination therapy.

The PC regimen consisted of pentostatin 4mg/m2 and cyclophosphamide 600mg/m2, given every 3 weeks for a total of 6 treatments. In the 2nd cohort of CLL patients, rituximab 375mg/m2 was added to this regimen starting with cycle 2. Supportive measures in both studies included hydration with each treatment (and monitoring of renal function) and prophylactic administration of filgrastim, sulfamethoxazole/trimethoprim, acyclovir, and antiemetics. In the first cohort of patients treated with the PC regimen, the median number of prior treatments was 3 (range 1–5) with the median age being 64 (32–77). There were responses achieved in 74% of patients with 17% complete responses seen in this heavily pretreated group. Similar (or perhaps slightly better) results were obtained with patients treated with the PCR regimen. For this cohort the median age was 62 (30–80) and the median number of prior regimens was 2 (1–7). Of the 32 patients with CLL 28 are evaluable for response: 79% responded and this includes 29% who achieved a complete response. Ten of the 14 patients with other low grade B cell diseases (SLL 8 patients, Waldenstrom’s macroglobulinemia 2 patients, follicular lymphoma 4 patients) are evaluable for response. The overall response rate for these patients was 50% (all PRs). These regimens were generally well tolerated with grade 3/4 toxicity consisting primarily of myelosuppression and its complications. We conclude that PC and PCR are highly active, well tolerated regimens even in heavily pretreated patients. We plan to conduct a multicenter study of PCR as initial therapy in patients with CLL.
Runx1 Deficiency Predisposes Mice to T-Cell Lymphoblastic Lymphoma.
Authors: Kundu Mondira , Compton Sheila , Garrett Lisa , Stacy Terryl , Starost Mathew , Eckhaus Michael , Speck Nancy A. , Liu Pu Paul
Published: October 2019

Chromosomal rearrangements affecting RUNX1 and CBFB are common in acute leukemias. These mutations result in the expression of fusion proteins that act in a dominant negative manner to suppress the normal function of the CBF β/RUNX1 complex. In addition, loss-of-function mutations in RUNX1 have been identified in sporadic cases of acute myeloid leukemia (AML) and in association with familial platelet disorder with propensity to develop AML (FPD/AML). In order to examine the hypothesis that decreased gene dosage of RUNX1 may be a critical event in the development of leukemia, we treated chimeric mice generated from Runx1lacZ/lacZ embryonic stem (ES) cells that have homozygous disruption of the RUNX1 gene, as well as Runx1+/lacZ mice with N-ethyl-N-nitrosurea (ENU). The heterozygous Runx1+/lacZ mice did not show increased incidence of any malignancy. On the other hand, we observed an increased incidence of precursor T-lymphoblastic lymphoma in Runx1lacZ/lacZ compared to wild-type chimeras, and confirmed that the tumors were of ES cell origin. It was determined by PCR that Runx1lacZ/lacZ ES cells contributed to the T cell progenitor population in the chimeras prior to leukemia development, which may explain the tissue-specificity of the malignancy we observed. Our results suggest that deficiency of Runx1 can indeed predispose mice to hematopoietic malignancies.
Gender-Dependent Up-Regulation of the VWF-Cleaving metalloprotease ADAMTS-13 in Mice with Obesity and Hypercholesterolemia.
Authors: Liu Anne , Wu Huaizhu , Tao Zhenyin , Choi Hiuwan , Bergeron Angela , Ballantyne Christie M. , Dong Jing-Fei
Published: October 2019

The development of atherothrombosis is associated with hypercholesterolemia and systemic inflammation. Recent studies have shown that adipose tissue may directly promote inflammation. Since one of the hallmark events of atherosclerosis is the deposition of von Willebrand factor (VWF) on vascular endothelial cells, the release and proteolysis of ultra-large (UL) and hyperreactive VWF may play an important role in the development of thrombotic diseases. We therefore investigated how the synthesis of ADAMTS-13, the newly characterized metalloprotease that cleaves the hyperreactive ULVWF to smaller and less active form, may be regulated in mice with obesity and hypercholesterolemia. Wild-type C57BL/6 and Apo E −/− C57Bl/6 mice (18 in each group with equal gender distribution) were fed with regular Chow or western-type high fat diet for 6 months and then analyzed for ADAMTS-13 mRNA in the liver (by quantitative PCR), the primary site of ADAMTS-13 synthesis, and ADAMTS-13 activity under flow condition. We found that ADAMTS-13 mRNA was more than doubled for wild-type mice on high fat diet than those on regular diet (4.14±0.46 vs. 1.72±0.57 units, respectively, p < 0.01). The increase was 47.9% for Apo E−/− mice on high fat diet compared to mice on regular diet (2.61±0.31 vs. 3.86±0.72 units, respectively, p = 0.05). When the data were stratified for gender difference, the increase was primarily observed in male mice. For male mice, ADAMTS-13 mRNA on high fat diet was more than four and two folds of those on regular diet for wild-type and Apo E−/− mice, respectively. , For female mice, however, the increase was less than 40% and only detected in wild-type mice fed with high fat diet. Consistent with this observation, ADAMTS-13 activity in general was lower in female (61±12%) than males (81±10%, p < 0.05). For both wild-type and Apo E−/− mice, we detected no difference in ADAMTS-13 activity in males, but a significantly lower activity in the female Apo E−/− mice on high fat diet (19.6±10.5% vs. 49.1±9.2% for high fat and regular diets, respectively, p < 0.05). These results demonstrate that the synthesis of ADAMTS-13 is up-regulated in obese mice (wild-type on high fat diet) and mice with hypercholesterolemia (Apo E−/− mice on high fat diet). The increase may serve as a protective measure to encounter the likely increase in ULVWF release due to systemic inflammation and endothelial cell stimulation seen in these mice. Interestingly, male mice showed a much stronger up-regulation than females. The low rate of ADAMTS-13 synthesis may contribute to the lower ADAMTS-13 activity found in female mice, suggesting that the machinery of ULVWF proteolysis in female mice may be less responsive to obesity/hypercholesterolemia and systemic inflammation.
Oral Mucositis Incidence and Severity after Methotrexate and Non-Methotrexate Containing GVHD Prophylaxis Regimens.
Authors: Cutler Corey , Li Shuli , Kim Haesook T. , Laglenne Patricia , Ford Colleen , Ho Vincent , Lee Stephanie J. , Alyea Edwin , Soiffer Robert , Antin Joseph H. , Sonis Stephen
Published: October 2019

Oral mucositis (OM) occurs in up to 75% of recipients of high-dose chemoradiotherapy conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (HSCT). OM affects patient quality of life by causing oropharyngeal pain, and by impairing communication and swallowing. As a result, narcotic analgesia and total parenteral nutrition (TPN) are commonly required in the recovery period after HSCT. Methotrexate (MTX), an antiproliferative agent used as GVHD prophylaxis, impairs mucosal regeneration after conditioning-related injury, and worsens and prolongs OM. We assessed the impact of sirolimus, a novel immunosuppressive agent used in lieu of MTX for GVHD prophylaxis, on outcomes associated with OM.

Methods: Two cohorts of patients were prospectively analyzed for OM severity, and chart reviews were performed to assess correlative outcomes. All patients underwent HLA-matched sibling PBSC transplantation after Cy-TBI conditioning. GVHD prophylaxis consisted of sirolimus/tacrolimus (ST) in the study group and tacrolimus/methotrexate (TM) in the control group. OM was assessed 3x/week using a prospectively validated 6-point assessment tool, by members of the Oral Medicine Service. At each assessment, the evaluators systematically evaluated for the presence of erythema and ulceration of the oral mucosa at nine pre-defined locations within the oral cavity. The use of narcotics and TPN was recorded from the time of transplantation to hospital discharge.

Results: 30 patients received ST and 24 patients received TM as GVHD prophylaxis after HLA-matched PBSCT between 10/2000 and 5/2003. The two groups were balanced for demographic variables, including age, sex, and disease status at transplant. OM severity was reduced in the ST group. Mild, moderate and severe OM was noted in 37, 57 and 7% of the ST group and 8, 42 and 50% of the TM group (p=0.0002). As a result, TPN use was reduced in the ST group (17 vs. 43% of hospital days, p=0.02), and a higher proportion of subjects required no TPN in the ST group (47 vs. 21%, p=0.08). The total number of hospital days where narcotics were required for pain control was lower in the ST group in comparison with the TM group (14 vs. 16 days median, p = NS). The time from transplant to first hospital discharge was shorter in the ST group compared with the TM group (18 vs. 22 days median, p= 0.07)

Conclusions: The use of ST for GVHD prophylaxis is associated with less severe oropharyngeal mucositis than TM, despite equivalent conditioning regimen intensity. As a result, TPN use was reduced, narcotic use was reduced and the duration of hospitalization was shortened. The use of less toxic GVHD regimens without MTX may have significant impact on patient quality of life, patient outcomes and economic outcomes associated with allogeneic stem cell transplantation.
An Externally Active Endogenous Platelet Galactosyl Trasferase (Gal-T).
Authors: Hoffmeister Karin M. , Wandall Hans H. , Sankaranarayanan Vasanthi , Clausen Henrik , Stossel Thomas P. , Hartwig John H.
Published: October 2019

Over 30 years ago, Jamison and Barber proposed that externally disposed glycosyl transferase (GT) activity mediates platelet adhesion and other functions. Subsequent work ruled out ecto-GT activity in nucleated cells and established the Golgi as the site of such enzymes, although no further studies examined platelets. We recently reported a platelet-associated galactosyl transferase (β4Gal-T) catalyzing galactose coupling in a β1,4 linkage to exposed N-acetylglucosamine (GlcNAc) residues on N-linked glycans of the GPIbα subunit of the von Willebrand factor receptor complex. This activity, also present in plasma, mediates galactose transfer to GPIbα following addition of UDP-galactose (UDP-Gal) alone. Since galactosylation of GPIbα prevents clearance of chilled platelets by liver macrophages after transfusion, sufficiently active endogenous endogenous β4Gal-T could facilitate platelet cold storage after the mere addition of substrate UDP-Gal (Science301: 1531–1534, 2003). We now give evidence that human platelets contain a highly active (0.05 nmol/min) plasma membrane-associated ecto-β4Gal-T with properties of an isoform designated as type 1 (β4Gal-T1). A specific β4Gal-T1 antibody immunoprecipitates β4Gal-T1 activity from platelet lysates and the inhibition profile activity by α-lactalbumin and benzyl-β-D-GlcNAc are characteristic of β4Gal-T1. β4Gal-T1 has a transmembrane domain, and platelet β4Gal-T, although releasable by non-ionic detergent, remains cell- and membrane associated despite extensive washing. The platelet enzyme incorporates UDP-[C14]Gal into a single polypeptide with the apparent Mr of GPIbα, identifying this protein as its unique endogenous target. Nevertheless, washed platelets are as active as recombinant β4Gal-T1 in galactosylating exogenous substrates, benzyl-β-D-GlcNAc and ovalbumin. In conclusion, we have revived the concept of an ecto-glycosyl transferase on blood platelets and demonstrated that platelet β4Gal-T is probably sufficiently active to accommodate reliable enzymatic conversion of platelets by simple addition of UDP-Gal.
Characterization of Regulatory Elements in the 5′-Upstream Region of the Human Gαq Gene in a Human Megakaryocytic Cell Line.
Authors: Gauthami Jalagadugula S. , Dhanasekharan Danny N. , Rao A. Koneti
Published: October 2019

GTP-binding protein Gαq plays a major role in platelet signal transduction. We studied the transcriptional regulation of the human Gαq gene in a megakaryocytic cell line, human erythroleukemia (HEL). 10 nM phorbal myristate acetate (PMA) was used to induce megakaryocytic lineage in HEL cells. Western blot analysis on untreated vs PMA-treated HEL lysates showed enhanced Gαq expression with PMA. Firefly luciferase reporter gene constructs carrying various lengths of the Gαq gene upstream promoter region −1/−2727 bp from ATG codon, were transiently transfected into PMA-treated HEL cells. Gαq promoter driven luciferase expression was measured by Dual Luciferase Reporter Assay system (Promega). These studies indicated that the Gαq gene is regulated by positive and negative elements. Two positive regulatory sites were identified in the proximal region (−138/−238 bp) and distal region (−731/−1116 bp); a negative regulatory site was observed further upstream (−1117/−2727 bp). The proximal region −138/−238 was resolved into a repressor (−207/−238) and a positive (−138/−207) site. Consensus sequences for two well recognized megakaryocytic transcription factors (TFs) PU.1 (−225/−230) and GATA-1 (−208/−211) in the repressor site were deleted; this had no effect on promoter activity. The positive region (−138/−207) was further resolved into repressor (−162/−186) and positive (−187/−207) regions by functional studies. In the repressor region a CCACC (−175/−179) consensus motif for Sp/XKLF family of TF was found and deleted; the repressor activity was lost indicating a functional effect of the CCACC-motif. The positive region contained a Sp1/AP2 consensus sites between −152 and −203. Gel shift and super shift experiments on double stranded DNA oligo 1 (−175/−203) and oligo 2 (−152/−174) using HEL extracts demonstrated nuclear protein binding to these regions. However, antibodies against Sp1 and AP2 or the consensus oligos did not alter the binding. Gel shift mutant analysis was performed on both oligos to define the functional sequences mediating the nuclear protein binding. Mutations made at −190/−194, which has a consensus sequence for EGR1/EGR2 and, at −180/−184 and at −156/−160, abolished the protein binding suggesting that they are preferred sequences for DNA-protein interaction. The distal upstream positive region −731/−1116 also revealed positive and negative elements. Mutation in the EGR-2 consensus sequence (−909/−919) decreased the promoter activity to 50% and mutation in the AP2/EGR-1/Sp1 consensus sequence (−885/−893) abolished the promoter activity. Our results suggest that the sequences −156/−160, −180/−184, and −190/−194 in the proximal region, and −885/−893 and −909/−919 in the distal region have a critical role in the transcriptional regulation of Gαq in megakaryocytic cells.
Differential Agonist Stimulated Cytokine Release from Platelets: A Proteomics Approach.
Authors: Coppinger Judith A. , O Connor Roisin I. , Cagney Gerard , Cox Dermot , Maguire Patricia
Published: October 2019

Dept of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin 2 Coppinger JA, O Connor RI, Cagney G, Cox D & Maguire PB. Recent evidence suggests that certain cytokines secreted by activated platelets can promote the development of atherosclerosis and thrombosis e.g the platelet specific cytokine platelet factor 4 (PF4) facilitates macrophage differentiation during inflammation and has shown to be incorporated into atherosclerotic plaques. In this study, we examined the release of several cytokines and other secretory markers from platelets stimulated by three different agonists in the presence and abscene of the anti-platelet drug aspirin. Using antibody array technology we detected the presence of 22 cytokines in the platelet releasate. High levels of abundant platelet cytokines RANTES, PDGF and EGF were seen throughout the arrays for all releasate fractions. The interleukin family were generally underrepresented with the exception of interleukin 8 and GRO. Several interesting angiogenic factors such oncostatin M (not previously reported in platelets) were present in some of the releasate fractions. Higher levels of cytokines were detected in the Collagen and TRAP stimulated fractions than in the ADP fractions suggesting that platelet secretion is an agonist dependent response. The levels of platelet secretory markers thrombospondin and PF4 identified from one dimensional gels by mass spectrometry were stronger in the Collagen and TRAP releasate fractions in keeping with the microarray results. Interestingly, unique protein profiles were obtained for the three different agonists. As platelet activation is COX-dependent we examined the effects of aspirin on cytokine release. Certain cytokines for example GRO were down regulated in the ADP and Collagen treated fractions but no significant decrease in cytokine levels were detected in the TRAP releasate. Cytokines released during platelet activation play diverse roles in vascular biology such as smooth muscle cell proliferation. These results indicate that that the release of platelet cytokines can be in part COX dependent, which may explain some of the anti inflammatory properties of aspirin.
A Possible Mechanism for Synergy in Transcriptional Activation between AML1 and PU.1: Exclusion of the Co-Repressor CBFA2T3 (ETO-2, MTG16).
Authors: Baraoidan Kristine , Ibanez Vinzon , Mittal Chetna , Yimer Habte , Chakraborty Suman , Sinha Kisle , Ackerman Steven , Nucifora Giuseppina , Saunthararajah Yogenthiran
Published: October 2019

AML1 and PU.1, important regulators of hematopoietic differentiation, interact with each other and are known to synergize in transcriptional activation (Zhang et al, 1996). PU.1 and AML1 also interact with mSin3, a component of a co-repressor complex that can include N-CoR, HDAC and CBFA2T1 (ETO, MTG8) or CBFA2T3 (ETO2, MTG16). CBFA2T3 is highly expressed in hematopoietic cells and is a target of a chromosomal translocation found in acute myeloid leukemia (t(16;21)). In transfected 293T cells, we demonstrate that both AML1 and PU.1 co-immunoprecipitate with the conserved N-terminal TAFH domain of CBFA2T3 but not the C-terminal MYND domain. Although AML1 and PU.1 independently co-immunoprecipitate with CBFA2T3, when all three proteins are over-expressed in 293T cells, AML1 and PU.1 co-immunoprecipitate with each other while excluding CBFA2T3. CBFA2T3 interacts with the non-runt portion of AML1 (AML1 C-terminus) while PU.1 can interact with both the runt domain of AML1 and AML1 C-terminus. Presumably, the interaction between AML1 and PU.1 shields the CBFA2T3 binding sites on both proteins. Since this region includes the binding site for mSin3, other co-repressors may also be excluded from an AML1/PU.1 complex. This may be one basis for the co-operation between AML1 and PU.1 in transcriptional activation.
Molecular Characterization of a PU.1 Transcription Complex Formed on the IL-1β Proximal Promoter.
Authors: Hoang Trang , Grondin Benoit , Lefrancois Martin , St Denis Marianne , Tenen Daniel G. , Waga Kazuo
Published: October 2019

The gene coding for the pro-inflammatory cytokine IL-1β is induced at the transcription level in differentiating macrophages and in stress response. Interestingly, PU.1 and C/EBPβ, two transcription factors implicated in IL-1β gene expression are not induced by stress exposure, while c-Jun is strongly induced. Strikingly, this upregulation of c-Jun is required for IL-1β induction, as cells expressing a c-Jun antisense construct fail to respond to stress exposure. We have mapped the induction of IL-1β gene expression to its proximal promoter and show that it is mediated by the transcriptional synergy between C/EBPβ, c-Jun and PU.1 via specific DNA binding sites for C/EBPβ and PU.1 only. To elucidate how PU.1 and C/EBPβ cooperate with c-Jun at the molecular level, we have optimized a DNA binding assay based on IL-1β promoter fragments immobilized on beads to isolate protein complexes from nuclear extracts, which were subsequently eluted and identified by Western blotting. We show that PU.1 or C/EBPβ alone directly bind this promoter fragment via specific sequences while purified recombinant c-Jun fails to do so. However, the presence of either PU.1 or C/EBPβ on the promoter allows for a recruitment of c-Jun to the DNA template, mediated by direct protein-protein interaction. Interestingly, the leucine zipper domain of c-Jun is essential for its interaction with C/EBPβ while dispensable for PU.1 interaction in vitro whereas its basic domain is required for both interactions. Furthermore, we show that PU.1 and C/EBPβ cooperatively bind the IL-1β promoter, resulting in a synergistic recruitment of c-Jun. Finally, we show that the strength of interaction of c-Jun mutants with PU.1 or C/EBPβ determine the strength of transcription output and c-Jun mutants that fail to associate with either PU.1 or C/EBPβ are transcriptionally inactive. In contrast, c-Jun mutants exhibiting increased homodimerization are more active that the wild type protein. Taken together, our data suggest that c-Jun homodimers can be targeted to the IL-1β promoter in the absence of a specific DNA binding element, and conclude that PU.1 and C/EBPβ are specifically tethered to the IL-1β promoter while c-Jun cooperatively binds these proteins and acts as a transcriptional co-activator. We propose a mechanism based on an initial binding of PU.1 and C/EBPβ to the IL-1β promoter followed by a cooperative recruitment of c-Jun, resulting in transcriptional synergy and IL-1β gene expression in stress response.
Identification of Common Target Genes of the BCR/ABL and FLT3-ITD Tyrosine Kinases by Microarray Analysis.
Authors: Banerji Lolita , Scheijen Blanca , Griffin James D.
Published: October 2019

Human myeloid leukaemias are commonly associated with activating mutations in tyrosine kinases. The BCR-ABL oncogene causes CML and is generated by the Philadelphia (Ph) chromosome translocation t(9;22)(q34;q11). The FLT3 tyrosine kinase receptor is mutated in about 30 % of all cases of AML, most often through a mechanism that involves an internal tandem duplication (ITD) of a small number of amino acid residues in the juxtamembrane domain. Both BCR/ABL and FLT3 (ITD) produce a constitutively active tyrosine kinase that enhances proliferation and viability of myeloid cells, and induce a similar myeloproliferative disorder when transplanted into mice. In an effort to identify any common downstream targets of BCR/ABL and FLT3(ITD) we generated paired Ba/F3 cell lines in which either Bcr/Abl or FLT3-ITD could be induced under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline rapidly induced expression of BCR/ABL or FLT3-ITD proteins, respectively proteins. The gene expression profile of these conditionally expressing BCR/ABL and FLT3-ITD BaF3cells was examined using the U430A gene chip (Affymetrix), which contains more than 34000 well substantiated genes. With a 2.5 fold cut-off, Bcr/Abl altered the expression of 336 genes and FLT3-ITD altered expression of 231 genes at 24 hours after induction. Fifty one genes were regulated by both Bcr/Abl and FLT3-ITD, including Cyclin G2, cyclin D2, CXCR4, osteopontin and FKBP12. Following induction of BCR/ABL and FLT3-ITD, cyclin D2, osteopontin and FKBP12 are strongly upregulated, whereas cyclin G2 and CXCR4 are downregulated. We validated the expression pattern of these genes by real-time PCR analysis. To further validate that these genes are directly regulated by the kinase activity of BCR/ABL and FLT3-ITD, cell lines were treated with specific small molecule kinase inhibitors, resulting in both cases in a significant decrease in cyclin D2, osteopontin, and FKBP12 and an increase in cyclin G2 and CXCR4 expression. The functional role of several of these genes is under investigation. For example, we found that the PI3K/AKT pathway is important for regulating osteopontin gene expression, and that osteopontin is involved in mediating the increase in cell growth caused by both BCR/ABL and FLT3-ITD. The identification of downstream targets, such as cyclins, osteopontin, and others, that are shared in common by several oncogenic tyrosine kinase oncogenes suggest that they could have value as therapeutic targets.
The Protein Subunit of Telomerase Is Downregulated with Normal Myeloid Differentiation, but Reactivated in Leukemic Progenitors.
Authors: Jamieson Catriona , Gilison D. , Allsopp R. , Weissman I. , Artandi S.
Published: October 2019

Telomeres are comprised of TTAGGG nucleotide repeats and a large protein complex that together protect chromosomal ends. Telomeres shorten progressively with cell division in the absence of telomerase, a reverse transcriptase that synthesizes telomere repeats. Critical telomere shortening has potent effects on cancer development, either blocking cancer progression by impairing proliferation and survival or promoting cancer by enhancing chromosomal instability. Although telomerase is upregulated in the vast majority of human cancers, where it is thought to endow cancer cells with unlimited proliferative capacity, the regulation of telomerase during the course of normal lineage development or during leukemogenesis remains unclear. Here, we show that expression of TERT, the protein catalytic subunit of telomerase, is tightly regulated during myeloid development in both human and mouse. Real time PCR analysis of RNA from purified human stem cell and progenitor cell populations showed that TERT mRNA was highest in human HSCs (100%, values normalized to HSC). TERT mRNA remained high in common myeloid progenitors (CMP, 47%), but was dramatically decreased with differentiation to granulocyte-monocyte progenitors (GMP, 3%) or megakaryocyte-erythroid progenitors (MEP, 0.2%). TERT was similarly down regulated during normal myeloid development in mouse bone marrow. In marked contrast, TERT was reactivated in leukemic GMP in a transgenic Faslpr/lprhMRP8BCL2 mouse model of acute myelogenous leukemia. Notably, transplantation experiments demonstrated that leukemic GMP provided the most robust leukemic transplantation potential compared with HSC or blasts suggesting that TERT reactivation may provide an important final step in leukemic pathogenesis by enhancing the replicative life-span of leukemic progenitors. Currently, we are in the process of assessing TERT regulation in human leukemic progenitors isolated from patients with CML in chronic, accelerated and blast phases. Together, these data indicate that TERT is most highly expressed in stem cells and multipotent progenitors. This pattern of TERT expression may reflect a need for efficient telomere maintenance in these stem/progenitor compartments. Furthermore, these data are consistent with our recent data showing novel, telomere length-independent functions for TERT in stem/progenitor cell compartments.
The Expression of Genes 5′ in the Hox-A Cluster Is Co-Ordinated Both in Normal and Leukemic Hematopoiesis.
Authors: Kumar Ashish R. , Kersey John H.
Published: October 2019

Hox genes are known to play critical roles in hematopoiesis and are probably important in the pathogenesis of leukemias. Disruption of the Hoxa9 gene in mice leads to defects in erythroid, lymphoid and myeloid hematopoiesis while over-expression of Hoxa9 leads to leukemia in mice. Transcription of Hoxa9 is partly regulated by the Mixed lineage leukemia gene (Mll) product. Bone marrow cells of mice carrying the leukemic Mll-AF9 fusion gene as a knock-in mutation (henceforth called Mll), display an increase in expression of several Hox genes - Hoxa5, Hoxa6, Hoxa7, Hoxa9 and Hoxa10, when compared to age matched wild type mice (Kumar et. al, 2004, Blood). Since the over-expressed Hox genes all belong to the same Hox-a cluster, we hypothesized that these genes might be co-regulated during normal hematopoiesis. Co-regulation of neighboring genes within the same cluster has been reported by others for the Hox-b and c clusters. To test this hypothesis for the 5′ Hox-a cluster genes, we compared expression levels of Hoxa5, Hox7 and Hoxa10 in Hoxa9 homozygous knockout (Hoxa9−/−) and wild type bone marrow by real-time quantitative RT-PCR using Taqman primers/probe sets (Applied Biosystems, CA). Expression levels of Hoxa5, Hoxa7, and Hoxa10 were all reduced by 65% ± 2% in Hoxa9−/− mice compared to wild type mice. In contrast, levels of Hoxb4 and Meis1 - homeobox genes that are not part of the Hox-a cluster - were identical in Hoxa9−/− and wild type mice. These results show no compensatory increases in expression of other 5′ Hox-a genes in the absence of Hoxa9, but instead demonstrate that disruption of Hoxa9 decreases the expression of neighboring genes in the Hox-a cluster. The hematopoietic defects seen in Hoxa9−/− mice (leucopenia, lymphopenia and blunted granulocytic response to G-CSF) might thus be attributable to the deficiency of multiple Hox-a gene products, rather than of Hoxa9 alone. To further evaluate the extent of this co-regulation, we studied the expression levels of these genes in Mll mice that lacked Hoxa9 (Hoxa9−/−/Mll-AF9+/−, henceforth called Mll/Hox). The Mll/Hox mice develop leukemia at the same rate and time course as the Mll mice, but with a phenotype that is relatively more immature. In Mll/Hox mice, the expression levels of Hoxa5 and Hoxa7 were increased 13 fold and 4 fold respectively, while those of Hoxa10 remained decreased at 35% of wild type. These results indicate that the decreased expression of neighboring Hox-a genes in Hoxa9−/− mice was reversed by Mll-AF9 for Hoxa5 and Hoxa7, but not for Hoxa10, suggesting a second level of co-regulation for Hoxa9 and Hoxa10. Overall, our findings demonstrate a co-regulated relationship between the 5′ Hox-a cluster genes during normal hematopoiesis, and provide evidence that deregulation of a single Hoxa9 gene significantly alters the expression of neighboring Hox-a cluster genes with implications for understanding the pathogenetic mechanisms of leukemia.