bluuebunny/crossref_metadata_2025 huggingface dataset:
Curcumin Downregulates NF-kB and Related Genes in Patients with Multiple Myeloma: Results of a Phase I/II Study.
Authors: Vadhan-Raj Saroj , Weber Donna M. , Wang Michael , Giralt Sergio A. , Thomas Sheeba K. , Alexanian Raymond , Zhou Xiao , Patel Priya , Bueso-Ramos Carlos E. , Newman Robert A. , Aggarwal Bharat B.
Published: October 2019

Although the role of NF-kB and STAT3 pathways in proliferation/metastasis of various tumor cells is well established, no agent has been described which could downregulate the activation of these transcription factors in cancer patients (pts). Curcumin has been shown to potently suppress the activation of these transcription factors in cultured cells. Based on these observations, we initiated a clinical trial of curcumin alone (administered orally at 2, 4, 6, 8, or 12 grams/day in 2 divided doses) or in combination with Bioperine (10 mg in 2 divided doses) for 12 weeks in multiple myeloma (MM) pts. The objectives of this study were to evaluate the clinical safety and biologic effects in MM pts who had asymptomatic, relapsed/refractory, or plateau phase disease. Blood was collected for PK/PD and PBMCs were examined (baseline and during treatment) for evaluating the effect of treatment on expression of NF-kB (p65), COX-2 and phospho-STAT3 as surrogate biomarkers. NF-kB activation status was also measured by electrophoretic mobility shift assay. At least 6 pts are enrolled at each dose level; 3 on the curcumin arm and 3 on the curcumin + bioperine arm. Pts with at least stable disease were allowed to continue treatment up to one year. Treatment with curcumin and bioperine has been well tolerated, with no significant adverse events. At the 12 grams dose level, 2 of the 5 pts had difficulty swallowing the large number of capsules. Of the 29 evaluable pts treated so far, no objective responses have been seen. Twelve pts continued treatment for more than 12 weeks and 5 (1 patient at 4 grams, 2 pts at 6 grams, and 2 pts at 8 grams dose levels) completed one year of treatment with stable disease. With few exceptions, little if any free drug was found in the plasma. Total curcumin levels (mostly conjugated drug) were dependent on both dose and the duration of administration. PBMCs from 28 MM pts examined showed constitutively active NF-kB (mean ± STD, 74.2% ± 14.0 positive cells), COX2 (66% ± 15.4), and STAT3 (52.8% ± 19.2). Oral administration of curcumin significantly downregulated the constitutive activation of NF-kB (at 3 months a median reduction of 77%, p<0.0001) and STAT3 (69%, p<0.001), and suppressed COX2 (66%, p<0.0001) expression in most of the pts at each of the monthly time points.

Conclusions: This is the first report to indicate that curcumin, a highly safe agent, is bioavailable and can downregulate NF-kB, STAT3 and COX2 in MM pts. These findings suggest a potential therapeutic role for curcumin that can be further investigated either alone or as a modulator of chemo-resistance in combination with other active agents.
Fucosylation of Cord Blood CD34+ Cells Improves the Rate of Engraftment without Compromising Long-Term Engraftment.
Authors: Robinson Simon N. , Yang Hong , Decker William K. , Xing Dongxia , Steiner David , Ng Jingjing , Thomas Michael W. , Champlin Richard E. , Simmons Paul J. , Brouard Nathalie , Kidd Shannon , Cooper Laurence , Shpall Elizabeth J. , Zweidler-McKay Patrick A.
Published: October 2019

INTRODUCTION: Delayed engraftment following cord blood (CB) transplantation remains a major challenge. While cell dose is clearly limiting, CB-derived hematopoietic stem cells (HSC) also appear to have a deficit in homing and engraftment. CB HSC have low levels of surface fucosylation. Increasing the level of surface fucosylation might improve interactions with selectins expressed by the microvasculature of the hematopoietic microenvironment, potentially improving homing and engraftment.

METHODS: Human CB CD34+ were fucosylated using a recombinant fucosyltransferase (FTVI, Engraftin™, America Stem Cell, Inc., Carlsbad, CA) prior to injection into sublethally-irradiated NOD-SCID IL-2Rγnull mice. Levels of fucosylation were determined by flow cytometry using an anti-sialyl Lewis X antibody (a fucosylated selectin ligand). Each mouse received ∼4.5x104 CD34+ cells. The rate of engraftment was measured by the appearance of human CD45+ cells in the peripheral blood (PB). Following engraftment, multi-lineage reconstitution was measured (lymphoid, myeloid, platelet and erythroid). At termination (11 weeks post-transplant), levels of multi-lineage human engraftment were measured in PB, bone marrow (BM) and spleen.

RESULTS: 1% of CD34+ cells in non-fucosylated samples were heavily fucosylated as compared to >90% in the FTVI-treated sample, confirming that significant de novo fucosylation of CD34+ cells had occurred.(Fig 1) Human engraftment was detected in mice receiving CD34+ cells treated with FTVI ∼2 weeks after transplant, and by ∼3 weeks had reached >1%. In mice receiving non-FTVI-treated CD34+ cells, 1% human engraftment was only realized >4 weeks after transplant. By this time human engraftment in the mice receiving FTVI-treated cells was 5-fold higher.(Fig 2) At 11 weeks post transplant, human engraftment was present in both groups in PB (10±1% vs. 4±1%), BM (33±1% vs. 43±1%) and spleen (42±1% vs. 31±2%). Further, in both groups, evidence of a similar pattern of multi-lineage engraftment was observed in PB, BM and spleen.

CONCLUSION: These data suggest that increasing the levels of fucosylation of CB CD34+ cells using FTVI, dramatically improved the rate of engraftment, provided multi-lineage reconstitution and did not adversely effect long-term engraftment. This study supports further clinical development of fucosylation as a method of improving CB transplantation.

Figure 1 Figure 1. Figure 2 Figure 2.
A Small Animal In Vivo Imaging Model of Expanded vs. Unexpanded UCB Stem Cells Reveals Differential Patterns of Engraftment.
Authors: Steiner David , Robinson Simon N. , Decker William K. , Marini Frank C. , Shpall Elizabeth J. , Bollard Catherine M. , Champlin Richard E. , Xing Dongxia , Yang Hong , Cooper Laurence J.N. , Gelovani Juri G. , Najjar Amer M. , Di Stasi Antonio , Quintarelli Concetta , Zweidler-McKay Patrick A. , Dotti Gianpietro
Published: October 2019

INTRODUCTION: Use of HLA-mismatched umbilical cord blood (UCB) allows transplant of patients who lack related HLA identical or matched unrelated donors. A major disadvantage of UCB is the limited number of stem cells available within the graft, a contributor to delayed engraftment, especially in adults. Two strategies have been adopted clinically in an attempt to overcome this barrier. 1. ex vivo expansion of some or all of critical graft subfractions; 2. transplantation of two different UCB grafts. These two strategies are not mutually exclusive and are also sometimes used in combination. Little is known about the basic biology that underlies the differential migration and homing of the graft components. To study the biology of complex UCB transplants, we have developed a small animal in vivo imaging model.

METHODS: CD34-selected UCB cells were transduced overnight with a lentiviral construct consisting of GFP or a firefly luciferase/GFP fusion. After 24 hours, SCF, FLT3, G-CSF, and thrombopoietin were added and the cells were cultured for 3 to 8 days. All cultured cells were then injected into non-lethally irradiated, immunocompetent mice (NOD/SCID/IL-2Rγ − /−). Transduced cells were tracked weekly by bioluminescent imaging.

RESULTS: On culture day 3, >99% of cells remained CD34+ and no expansion was observed. After expansion in culture between days 3 and 8, 19% +/− 18.5% of transduced cells were GFP+CD45+ (n= 8). Cells cultured for three days (non-expanded) (3-11x105 CD34+ cells/3-8.6x104 GFP+) were able to create a detectable engraftment signal by post-transplant day 8. The engraftment signal was delayed until day 10 when an 8-fold excess of the expanded cultured cells (4.2x106 total consisting of 1.21x106 CD34+ cells/2.3x105 GFP+ and 6.51x105 CD34-GFP+ cells) were injected. Long term engraftment (>30 days) was unaffected by expansion in culture. However, expanded UCB exhibited a differential pattern of homing and engraftment in comparison to unexpanded UCB. Mice receiving unexpanded UCB exhibited a strong engraftment signal from the area of the calvarium in addition to signals from other marrow spaces. The calvarium signal was only weakly and intermittently observed among mice who received the expanded UCB graft. Pathologic evaluation of these areas is in progress.

CONCLUSIONS: 1. Expanded cells were capable of supporting engraftment, though at a slower rate than their unexpanded counterparts. This delay might ultimately be addressed through the optimization of the expansion technique. 2. The physiologic significance of the differential engraftment pattern(s) is unclear and will require further study.

Figure Figure
Inhibition of Multiple Myeloma Tumor Growth and FGFR3 by the Aurora-Angiogenesis Inhibitor ENMD-981693.
Authors: Hembrough Todd A. , Chen Xiaoru , Burke Patricia A. , Swartz Glenn M. , Bengali Kathleen , Brokx Richard , LaVallee Theresa M. , Bray Mark R.
Published: October 2019

ENMD-981693 is a novel, orally-active antimitotic and antiangiogenic molecule that was discovered through a screening effort directed towards Aurora kinases. In addition to activity against Aurora A (AurA), ENMD-981693 is a potent inhibitor of several tyrosine kinases which drive the neo-vascularization of growing tumors, namely JAK2, VEGFR2 (KDR), PDGFRα and the FGF receptor family members FGFR1, R2, and R3. Multiple myeloma (MM) is a disease in which AurA and angiogenesis have been shown to play a role in disease progression. In addition, up to 15% of MM patients have a t(4;14) translocation, which leads to ectopic overexpression of the FGFR3, and is associated with a poor clinical outcome. In preclinical models, inhibitors of FGFR3 have exhibited antitumor activity towards t(4;14) bearing MM tumors. In in vitro proliferation assays, ENMD-981693 demonstrated an IC50 of ∼2 μM towards several MM cell lines, including H929, U266 and RPMI-8226. In immunoprecipitation studies, ENMD-981693 inhibits the kinase activity of both FGFR3 (IC50 ∼0.8 μM) and JAK2 (IC50 ∼0.5 μM) in the MM cell lines U266 and H929. These activities are in accord with the inhibitory activity of ENMD-981693 on the purified kinase domains of FGFR3 (IC50=0.5 μM) and JAK2 (IC50=0.12 μM). ENMD-981693 also inhibits activation of JAK2 activity in the HEL cell line which expresses the polycythemia vera mutated form of JAK2 (V617F). Mice implanted with H929 tumor xenografts were treated for 4 days with 75 – 225 mg/kg/d of the tartrate salt of ENMD-981693 (ENMD-2076). The amount of phosphorylated (and thus activated) FGFR3 immunoprecipitated from lysates of H929 tumors showed a significant decrease following drug treatment with no change in total FGFR3. In complementary immunoprecipitation studies, a similar decrease in JAK2 activity was seen in these H929 tumors treated with ENMD-981693 tartrate. Daily, oral treatment of RPMI-8226 xenografts with ENMD-981693 tartrate (225 mg/kg) resulted in a significant (67%) reduction in tumor burden. Follow up studies will assess the activity of ENMD-981693 tartrate on MM tumor xenografts which harbor the t(4;14) translocation, and whose growth is largely driven by FGFR3. Taken together, ENMD-981693 has been identified as a single agent that targets multiple critical mediators of MM growth, and angiogenesis. This suggests that ENMD-981693 has the potential for clinical activity in MM, particularly in patients with the t(4;14) translocation and in polycythemia vera patients with mutated JAK2. An IND filing is planned for ENMD-981693 in 2007.
A Phase II Trial of the Oral mTOR Inhibitor Everolimus (RAD001) in Relapsed Aggressive Non-Hodgkin Lymphoma (NHL).
Authors: Reeder Craig B. , Gornet Michel K. , Habermann Thomas M. , Ansell Stephen M. , Micallef Ivana N. , Porrata Luis F. , Johnston Patrick B. , Maurer Matthew J. , LaPlant Betsy , Kabat Brian , Inwards David J. , Colgan Joseph P. , Call Timothy , Markovic Svetomir , Zent Clive S. , Zeldenrust Steven R. , Tun Han W. , Witzig Thomas E.
Published: October 2019

Background: The phosphatidylinositol 3-kinase/mammalian target of rapamycin (mTOR) signal transduction pathway integrates signals from multiple receptor tyrosine kinases to control cell proliferation and survival. Everolimus (RAD001, Novartis Pharmaceuticals) is an oral investigational antineoplastic agent that targets mTOR.

Objectives: To learn the anti-tumor activity and toxicity of single-agent RAD001 in pts with relapsed/refractory aggressive NHL.

Patients and Methods: Patients were eligible if they had measurable disease, a platelet count >75,000, an absolute neutrophil count >1,000, and a creatinine and bilirubin <2x laboratory upper limit of normal. Patients received RAD001 10 mg PO daily and were evaluated monthly. Dose reductions to 5 mg daily and 5 mg every other day were allowed. Response was assessed after 2 cycles and periodically thereafter. Patients could remain on drug until progression or toxicity.

Results: Thirty-seven pts were treated - 20 (54%) with relapsed diffuse large cell (DLC), 14 (38%) with relapsed mantle cell (MCL), 2 high grade, and 1 follicular grade III. The median age was 72 years (range, 45–92). Patients had received a median of 4 prior therapies (range, 1–15). The overall response rate was 32% (12/37; 95% CI:20–49%) with 1 complete response and 11 partial responses. The ORR was 35% (7/20) in the DLC group and 29% (4/14) in the MCL patients. Patients have received a median of 2 cycles (range, 1–16+) of therapy. The median time to progression for all 37 patients is 3.1 months (95%CI; 2.0 – 4.7). The median duration of response for the 12 responders is 5.5 months (95% CI; 2.3 – 9.2) and 5 pts remained progression free at 6 months. Three patients are currently maintaining a response with a median time of 10.5 months (range, 2.9–15.6+months). RAD001 was well tolerated. The incidence of grade 3 anemia, neutropenia, and thrombocytopenia in this heavily pretreated pt population was 11%, 16%, and 30%. Two pts developed grade 4 neutropenia. Grade 2 hypercholesterolemia occurred in 11%; grade 2 hyperglycemia in 16%; grade 3 hyperglycemia in 11% and 1 pt had grade 4 hypertriglyceridemia.

Summary: RAD001 has single-agent activity in relapsed aggressive NHL and provides proof-of-concept that targeting the mTOR pathway in NHL is clinically relevant.
Retinoblastoma Protein and the Leukemia-Associated PLZF Transcription Factor Interact To Repress Target Gene Promoters.
Authors: Petrie Kevin R. , Guidez Fabien , Zhu Jun , Owen Gareth , Chew Yat Peng , Waxman Samuel , Licht Jonathan D. , Mittnacht Sibylle , Arthur Zelent
Published: October 2019

Translocations of the retinoic acid receptor alpha (RARA) locus with the PLZF or PML genes lead to expression of oncogenic PLZF-RARα or PML-RARα fusion proteins, respectively. These fusion oncoproteins constitutively repress RARα target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARα have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (POZ) and DNA binding (zinc-finger) domains. Additionally, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction between PML or E2F1 with Rb, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. The interaction between PLZF and Rb is further underlined by chromatin immunoprecipitation analysis of PLZF binding to genomic DNA, which shows that PLZF associates with genes controlling cell proliferation known to be regulated by Rb and E2F (for example cdc6). Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to have roles in stem cell biology and, taken together, these data provide a plausible scenario in which interactions between PLZF and Rb function in stem cell commitment or maintenance and self-renewal. The oncogenic PLZF-RARα fusion also interacts with Rb, suggesting that deregulation of Rb function may be a factor in the molecular pathogenesis of PLZF-RARα associated acute promyelocytic leukemia.
The Human Severe Congenital Neutropenia-Associated Gfi1N382S Mutant Critically Requires Deregulated Expression of CSF1 To Block Granulopoiesis.
Authors: Velu Chinavenmeni S. , Baktula Avinash M. , Bourdeau Tristan , Horman Shane R. , Grimes H. Leighton
Published: October 2019

Severe Congenital Neutropenia (SCN) is characterized as a deficiency of mature neutrophils, which leads to recurrent bacterial and fungal infections. Mutations in the Ela2 gene predominate in SCN, but Ela2 mutation in mice does not recapitulate SCN. The Growth factor independent-1 (Gfi1) transcriptional repressor regulates Ela2. The N382S mutation in Gfi1 is associated with human SCN. Unlike Ela2, genetic deletion of Gfi1 results in murine neutropenia. Here we show that the human-SCN-associated Gfi1N382S mutant blocks murine granulopoiesis in a Csf1 dependent manner. Gfi1 functions as a rate-limiting granulopoietic molecular switch, and the DNA-binding-defective Gfi1N382S mutant acts as a dominant negative to block granulopoiesis by selectively derepressing a subset of Gfi1 target genes. One gene repressed by Gfi1 and derepressed by Gfi1N382S is the macrophage colony stimulating factor (Csf1). Chromatin immunoprecipitation and electrophoretic mobility shift analyses demonstrate direct binding of Gfi1 to the Csf1 promoter. Importantly, both antibody absorption of Csf1 and genetic deletion of Csf1 prevent the Gfi1N382S-mediated block to granulopoiesis. These data represent the first identified Gfi1 target gene to be critically required for SCN-like phenotypes engendered by Gfi1N382S, and suggest new potential treatment avenues for Gfi1-mutant SCN patients.
Menin Regulates the Function of Lymphoid Progenitors and Hematopoietic Stem Cells.
Authors: Maillard Ivan , Chen Ya-Xiong , Tubbs Anthony T. , Shestova Olga , Pear Warren S. , Hua Xianxin
Published: October 2019

Menin is the product of the Men1 gene, a tumor suppressor gene that is mutated in patients with multiple endocrine neoplasia type I (MEN1). In addition to its effects in endocrine tissues, Menin interacts with the Mixed Lineage Leukemia (Mll) gene product as part of a multiprotein complex with H3K4 methyltransferase activity. Menin is required to mediate malignant transformation induced by Mll gene rearrangements, an activity associated with transcriptional activation of Homeobox (Hox) gene expression, presumably through epigenetic regulation. To explore the normal function of Menin in hematopoiesis, we studied bone marrow (BM) progenitors after inactivation of the Men1 gene in adult mice. Loss of Menin led to a modest reduction in peripheral blood neutrophil, lymphocyte and platelet counts. In the absence of hematopoietic stress, numbers of multilineage and myeloerythroid BM progenitors were preserved, but pro-B cells and downstream B lineage progenitor subsets were significantly decreased. Competitive BM transplantation assays revealed a marked defect in the function of Menin-deficient hematopoietic stem cells (HSCs), including long-term HSCs. Furthermore, Menin-deficient mice had impaired hematopoietic recovery after chemoablation with 5-fluorouracil. However, expression of Hox genes in BM HSCs was not impaired in the absence of Menin. These observations reveal an essential role of Menin in the homeostasis of hematopoietic stem and progenitor cells. Furthermore, they suggest that Menin may regulate normal hematopoiesis through mechanisms that are distinct from its role in Hox gene-dependent malignant transformation.
Foxo3 Modulation of ATM and Oxidative Stress Mediates Distinct Functions in the Regulation of Hematopoietic Stem and Progenitor Cell Fate.
Authors: Yalcin Safak , Luciano Julia P. , Zhang Xin , Vercherat Cecile , Taneja Reshma , Ghaffari Saghi
Published: October 2019

FOXO transcription factors are required for hematopoietic stem cell self renewal. In this study, we demonstrate that Foxo3 plays a specific and essential function in the regulation of both hematopoietic stem and progenitor cell fate. Foxo3 null mice display a myeloproliferative syndrome characterized by splenomegaly, a major expansion of the myeloid compartment in the blood, bone marrow and spleen, cytokine hypersensitivity of progenitors in hematopoietic organs and associated with the repression of the B lymphoid compartment. In addition, loss of Foxo3 leads to significant defects in hematopoietic stem cell numbers and activity. In particular, the numbers of long-term culture initiating cells (LTC-IC) was significantly reduced and the ability to repopulate lethally irradiated mice was severely compromised in Foxo3-defcient mice. This effect was mediated at least partially by enhanced accumulation of reactive oxygen species (ROS) in Foxo3-deficient hematopoietic stem cells as demonstrated

by reduced QRT-PCR expression of several anti-oxidant enzymes leading to accumulation of ROS, (as measured by chloromethyl,dichlorodihydrofluorescein diacetate assay) in Foxo3 null hematopoietic stem cells, and in vitro and in vivo rescue of the phenotype using ROS scavengers.

Furthermore, we demonstrate that while ROS accumulation results in suppression of Foxo3 null hematopoietic stem cell compartment, it enhances the activity of multipotential cells. By measuring RNA versus DNA content, and BrdU uptake, we determined that Foxo3-deficient hematopoietic stem cells exit quiescence (G0) and are impaired in their cycling at the G2/M phase. In particular, we identified ROS activation of p19ARF/p53 pathway and ROS-independent modulation of ataxia telangiectasia mutated (ATM) gene and p16INK4a, as major contributors to the interference with Foxo3-deficient hematopoietic stem cell self renewal and cycling. Loss of ATM has been shown to lead to hematopoietic stem cell deficiency. Importantly, we show that ATM gene expression is significantly suppressed in Foxo3-deficient hematopoietic stem cells suggesting that ATM lies downstream of Foxo3. Retroviral expression of a constitutively active form of Foxo3 in Foxo3-deficient bone marrow mononuclear cells enhances significantly the ATM expression suggesting that Foxo3 regulate expression of ATM gene. These combined findings suggest that Foxo3 functions in a tumor suppressor network to protect hematopoietic stem cells against deleterious effects of oxidative damage, to maintain hematopoietic lineage fate determination and to restrict the activity of long term repopulating hematopoietic stem cells. These findings provide insights into the mechanisms underlying hematopoietic stem cell fate.
Association of γ’ Fibrinogen with Risk Factors and Prevalent Cardiovascular Disease in the Framingham Heart Study.
Authors: Lovely Rehana S. , Massaro Joseph F. , D’Agostino Ralph B. , Benjamin Emelia J. , Kazmierczak Steven C. , O’Donnell Christopher J. , Farrell David H.
Published: October 2019

Background: γ’ fibrinogen is a newly-emerging cardiovascular disease (CVD) biomarker that has shown significant associations with coronary artery disease in small case/control studies. γ’ fibrinogen is an isoform with an altered γ chain, and constitutes ∼10% of total fibrinogen. Both elevated γ’ and total fibrinogen cause fibrinolytic resistance in vitro. However, data are lacking from community based studies on the association of γ’ fibrinogen with CVD risk factors or with clinical CVD.

Methods: γ’ fibrinogen levels were measured using an ELISA assay in 3284 men and women in the Framingham Heart Study Offspring Cohort 7th examination cycle (2002–05). Associations of γ’ fibrinogen were examined using linear regression for continuous CVD risk factors and logistic regression for prevalent CVD and other dichotomous measures.

Results: The ELISA CV was 9.3% at mean γ’ fibrinogen levels. Participants (mean age 60 years, 53.4% women) with prevalent CVD (0.297 ± 0.007 mg/ml; mean ± SE) had significantly higher mean concentrations than those without CVD (0.255 ± 0.002 mg/ml). There were significant (all p<0.05) associations between γ’ fibrinogen levels with several cardiovascular risk factors, including age, BMI, systolic blood pressure, diabetes mellitus, cigarette smoking, and total cholesterol, and an inverse association with HDL cholesterol. γ’ fibrinogen was associated with prevalent CVD with an odds ratio of 1.76 (95% CI 1.06–2.92) for the highest compared to the lowest tertile, after adjustment for all of the above risk factors. As expected, since γ’ fibrinogen constitutes a subset of total fibrinogen, γ’ fibrinogen was correlated with total fibrinogen. However, if we included both γ’ and total fibrinogen in further analyses of association with CVD, the odds ratio was 3.08 (95% CI 1.41–6.72) for the combined tertile of both the highest γ’ fibrinogen and highest total fibrinogen, an odds ratio that was substantially higher than that for either marker alone.

Conclusions: γ’ fibrinogen is associated with multiple CVD risk factors and with prevalent CVD, accounting for CVD risk factors, over and above total fibrinogen alone. Prospective studies are warranted to examine the ability of γ’ fibrinogen to predict CVD.
Interleukin-7 Produced by Antigen Presenting Cells Regulates the Homeostatic Peripheral Expansion of Naive CD4 T Cells.
Authors: Guimond Martin , Veenstra Rachelle G. , Grindler David J. , Cui Yongzi , Na Risu , Hennighausen Lothar , Zhang Hua , Merchant Melinda S. , Mackall Crystal
Published: October 2019

Interleukin-7 (IL-7) is the only cytokine demonstrated thus far to directly support the development and maintenance of naive T cells. Interleukin-7 is produced mainly by the stroma of lymphoid organs but also to a lesser extent by antigen presenting cells. Dynamic changes in the relative availability of IL-7 during lymphopenia support T cell homeostatic peripheral expansion (HPE). Whereas HPE is very efficient at reconstituting the pool of mature CD8 T lymphocytes, regeneration of CD4 T lymphocytes via HPE is more problematic and deficits in CD4 T lymphocytes persist for years in humans following lymphodepletion. We demonstrate that in mice, CD4+ T cells undergo substantially diminished HPE compared to CD8+ T cells during lymphopenia. We hypothesized that CD4+ T cells uniquely require both MHC Class II plus IL-7 for survival and expansion during lymphopenia and that the availability of these signals are limited by the availability of IL-7 producing APCs. Consistent with this hypothesis, studies performed in chimeric mice showed that cells derived from APCs, but not stromal cells, provided the endogenous IL-7 that is require for naive CD4+ T cell HPE. Furthermore, whereas systemic exposure to high levels of rhIL-7 did not induce efficient homeostatic peripheral expansion of CD4+ cells, in vivo modulation of Class II expressing cells by flt3 ligand promoted homeostatic expansion of CD4+ T cells. Surprisingly however, we found that APCs respond to elevated systemic IL-7 by diminishing production of IL-7, a loop mediated by IL-7Rα and Stat5. As a result, elevated systemic IL-7 levels present during lymphopenia paradoxically diminish the production of IL-7 by APCs and thereby diminish CD4 HPE. This was directly demonstrated by experiments wherein CD4+ HPE was dramatically augmented in chimeric mice generated such that bone marrow derived cells lacked IL7Rα or Stat5. This work demonstrates a critical role for APC derived IL-7 in CD4+ regeneration and provides a new paradigm for understanding why prolonged CD4+ lymphopenia occurs following T cell depletion in humans.
B Cell Lineage-Specific Deletion of Icsbp/IRF8 Reveals Roles for IRF8 in the Regulation of Marginal Zone and Follicular B Cell Development.
Authors: Wang Hongsheng , Feng Jianxun , Lee Chang Hoon , Morse Herbert
Published: October 2019

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a transcription factor that expresses in T cells, B cells and macrophages and plays a role in myeloid development. Targeted deletion of IRF8 in mice (IRF8−/−) induced progressive increase in the numbers of granulocytes in various lymphoid organs and development of a syndrome similar to human chronic myelogenous leukemia. In addition to defective development of macrophages and dendritic cells, B cell development was also impaired in IRF8−/− mice. This includes decreased numbers of early B cells, expanded marginal zone (MZ) B cells and diminished follicular (OF) B2 cells. Because abnormal myeloid cells could alter microenvironment required for normal B cell development, we have generated IRF8 conditional knockout mice to specifically investigate the function of IRF8 in B lineage cells. Mice were engineered to have exon 2, encoding the DNA binding domain of IRF8, flanked by loxP sites (designated IRF8f/+). These mice were then crossed with the CD19Cre strain in which the expression of Cre-recombinase is controlled by the endogenous CD19 locus. Homozygous mice (designated (IRF8f/f x Cre)F1) underwent germline excision of IRF8 in CD19+ B lineage cells. As a result, there was no detectable mRNA and protein of IRF8 in their splenic B cells. Flow cytometry analysis revealed expanded MZ B cells and reduced OF B2 cells in the spleen of (IRF8f/f x Cre)F1 mice. Interestingly, the expression level of CD23 on OF B cells was significantly decreased in (IRF8f/f x Cre)F1 mice, indicating that IRF8 is required for maintaining a normal OF phenotype. In the peritoneum of (IRF8f/f x Cre)F1 mice, while the numbers of B1a and B2 cells were slightly decreased, the number of B1b cells was slightly increased. Furthermore, BXH2 mice carrying a mutation (C915T) in the Icsbp1 gene exhibited similar expansion of MZ B cells and low expression of CD23 in OF B cells. Taken together, these analyses indicate that IRF8 is required for development of normal MZ and B2 cells.
Transcriptional Regulation of NGAL by the Pro-Inflammatory Cytokines IL-17 and TNFα.
Authors: Karlsen Joachim R. , Borregaard Niels , Cowland Jack B.
Published: October 2019

IL-17 is a proinflammatory cytokine synthesized by a newly discovered subset of helper T-cells called TH17. In our search for targets that are regulated by IL-17, we have found that the gene for the bacteriostatic protein neutrophil gelatinase associated lipocalin (NGAL) is strongly induced in epithelial cells by this cytokine. NGAL is a siderophore-binding protein that binds bacterial catecholate-type ferric siderophores and exerts a bacteriostatic effect under iron limiting conditions. It has been observed that NGAL synthesis is induced in inflamed epithelium of the lungs and colon. Using the type II pneumocyt-derived cell line A549 as a model, we found that NGAL was up-regulated 27-fold at RNA level, and 14-fold at protein level, when co-stimulated for 24 hours with IL-17 (200ng/ml) and TNFα (20ng/ml), whereas no induction was seen with either cytokine alone compared to unstimulated cells. The expression of NGAL in A549 cells, when stimulated with IL-17 and TNFα, was dependent on de novo protein synthesis as demonstrated by inhibition of NGAL mRNA production by cycloheximide. Deletion and substitution analysis of the NGAL promoter and subsequent stimulation of transfected cells with IL-17 and TNFα showed that a functional NF-κB-binding site was essential for promoter activation whereas AP-1 and C/EBP-sites were dispensable. Quantitative RT-PCR showed a 13-fold induction of IkB-ζ mRNA in A549 cells when stimulated with IL-17 and TNFα with peak level at 1½ hours and a slow decline for up to 24 hours. Cells stimulated with IL-17 alone resulted in 8-fold mRNA induction at 1½ hours followed by a decline to a 3-fold induction level of mRNA, which lasted for 24 hours. In contrast, cells stimulated with TNFα showed a 9-fold mRNA induction at 1½ hours but then rapidly returned to back-ground level at 3 hours post-induction. These data, and previous studies of the NGAL promoter (Cowland et al. (2006), 176, 5559), demonstrates that the NGAL promoter requires binding of the transcription factor NF-κB as well as the NF-κB-binding co-factor IkB-ζ for transcriptional activation. Our data indicate that TNFα stimulates transcription of the IkB-ζ gene and NF-κB binding to the NGAL promoter whereas IL-17 stabilizes IkB-ζ mRNA and thus enables translation of this co-factor. Only when acting together the two cytokines induce the formation of an NF-κB:IκB-ζ-complex that can stimulate NGAL transcription. We have found that human b-defensin 2 (hBD2) is regulated in the same manner. The functions of NGAL and hBD2 in the innate immune response are well characterized, and the present data demonstrates that IL-17, which is synthesized only by the newly discovered TH-17 cell population, is involved in transcriptional regulation of these effector molecules. The data furthermore demonstrate that cells of the adaptive immune system is able to activate genes of the innate immune system.
Critical Role for CD81 in B Cell Activation.
Authors: Sanyal Mrinmoy , Fernandez Rosemary , Levy Shoshana
Published: October 2019

CD81 is a component of the CD19/CD21 signaling complex in B cells. CD81 was originally discovered as target of an anti-proliferative antibody in a human B cell lymphoma. However, the exact role of CD81 in B cell function is not known. Here we studied B cells from CD81 knockout mice. We demonstrate that upon BCR induction these B cells

flux higher intracellular free calcium ion; increase the phosphorylation of BCR-related proximal and distal substrates and increase their proliferation. Similarly, polyclonal activation of CD81-deficient B cells with LPS induced increased proliferation and antibody secretion.

Consistent with these intrinsic B cell capabilities, CD81-deficient mice mounted significantly higher immune response upon antigenic stimulation. In addition, bone marrow perisinusoidal B cells (IgM+IgD+) capable of mounting T-independent immune responses against blood-borne pathogens were over represented in CD81-deficient mice. These cells also displayed increased calcium influx kinetics as splenic B cells and produced higher amounts of antibody after polyclonal stimulation. Taken together, these results suggest that CD81 is involved in suppressing B cell activation.
Treatment of Primary CNS Lymphoma with Induction High-Dose Methotrexate, Temozolomide, Rituximab Followed by Consolidation Cytarabine/Etoposide: A Pilot Study with Biomarker Analysis.
Authors: Issa Samar , Shen Arthur , Karch Jon , Kadoch Cigall , Shuman Marc , Damon Lloyd , Rubenstein James
Published: October 2019

Background: There is no consensus on the optimal treatment for patients diagnosed with primary CNS lymphoma (PCNSL). The goals of this study were:

to determine the safety and efficacy of a methotrexate (MTX)-based induction therapy followed by high-dose consolidation chemotherapy and the elimination or deferral of whole brain irradiation, to identify molecular markers in PCNSL which predict sensitivity to chemotherapy and outcome.

Methods: 23 newly diagnosed, CD20-positive, immunocompetent PCNSL patients were treated with combination high-dose intravenous MTX (8 gm/m2), temozolomide (150 mg/m2/day) and intravenous rituximab (375 mg/m2) (MTR). Patients in complete remission (CR) after eight courses of MTX were offered consolidation with high-dose cytarabine (2 g/m2 x 8 doses) and etoposide (40 mg/kg over 96 hours) (AE). Candidate markers of outcome in PCNSL were identified by gene expression profile analysis of an independent, multicenter dataset of PCNSL tumors. Immunohistochemical analysis of one of these markers, death-associated protein-1 (DAP-1), was performed on paraffin sections of tumors from 18 of the patients treated with the MTR regimen.

Results: MTR induction followed by AE consolidation was well tolerated with no treatment-related mortality or evidence for neurotoxicity. Thirteen patients (56.5%) attained CR with induction; 8 received consolidation; 5 in CR refused AE. Median progression-free (PFS) and overall survival (OS) has not yet been reached with a median follow-up of 33 months. Karnovsky performance status (KPS) correlated with improved survival (p<0.0281). Expression by lymphoma cells of DAP-1, a regulator of apoptosis, was associated with improved progression-free survival (p<0.03) and overall survival (p<0.038).

Conclusions: Combination MTR followed by AE is well tolerated in PCNSL. PFS appears at least similar to regimens that contain whole brain irradiation. A multi-center study has been initiated to further evaluate this regimen. DAP-1 may be a tumor suppressor whose expression in PCNSL predicts a favorable response to MTX-based therapy.
A Phase 1/2a Study Evaluating the Safety, Pharmacokinetics, and Efficacy of ABT-263 in Subjects with Refractory or Relapsed Lymphoid Malignancies.
Authors: Wilson Wyndham H. , Tulpule Anil , Levine Alexandra M. , Dunleavy Kieron , Krivoshik Andrew P. , Hagey Anne E. , Shovlin Margaret , Gloria Melanie A. , Greco Renee , Xiong Hao , Chiu Yi-Lin , Elmore Steven W. , O’Connor Owen A.
Published: October 2019

Background: ABT-263 is a novel small molecule Bcl-2 family protein inhibitor that binds with high affinity (Ki ≤ 1 nM) to multiple antiapoptotic Bcl-2 family proteins including Bcl-XL, Bcl-2, Bcl-w, and Bcl-B. ABT-263 displays potent mechanism-based cytotoxicity (EC50 ≤ 1 mM) against human tumor cell lines derived from lymphoid malignancies and small cell lung cancer (SCLC). Pre-clinical experiments on primary patient derived chronic lymphocytic leukemia (CLL) samples with ABT-737, a first generation Bcl-2 family protein inhibitor, induced robust, concentration-dependent apoptosis. The anticipated ABT-263 associated toxicities are mechanism based, in particular, Bcl-XL mediated decrease in circulating platelet survival, Bcl-w mediated testicular toxicity, and Bcl-2 mediated effects on lymphocytes. ABT-263 is currently being investigated in 3 Phase 1/2a studies in patients with lymphoid malignancies, SCLC/solid tumors, and CLL.

Methods: In this Phase 1/2a study of ABT-263 in subjects with lymphoid malignancies, the Phase 1 dose escalation portion of the study was designed to determine the maximum tolerated dose (MTD), dose limiting toxicity (DLT), and recommended Phase 2 dose (RPTD). For each 21-day cycle, subjects receive ABT-263 orally QD for 14 consecutive days followed by 7 days off drug. Pharmacokinetic (PK) assessment was performed at study initiation and on Day 14 of drug dosing. The Phase 2a portion of the study will evaluate ABT-263 at the RPTD in up to 40 subjects with follicular and aggressive NHL to obtain additional safety information and a preliminary assessment of efficacy.

Results: Currently 17 subjects have been enrolled in the lymphoma study. Three subjects completed each of the 10, 20, 40, and 80 mg cohorts enrolled thus far. A Grade 3 DLT (URI resulting in hospitalization treated with antibiotics) occurred in the 160 mg cohort, thus the cohort was expanded to 6. Two patients with bulky SLL/CLL in the 40 and 160 mg cohorts had 95% and 64% tumor reductions after cycles 4 and 2, respectively, and continue on treatment. The PK profile of ABT-263 appeared to be linear between the 10 mg and 160 mg dose levels. The average terminal half-life of ABT-263 varied between 14 and 25 hours across all dose levels. ABT-263 reduced the platelet level in a dose-dependent manner. One subject experienced a Grade 3 thrombocytopenia (platelets 45K/uL) on C1D14, 4 hours post-dosing with 80 mg, resolving 8 hours post-dosing to a platelet count of 162 K/uL. The decreased platelet count did not result in any medical sequelae.

Conclusions: ABT-263 is a novel, orally bioavailable and active small molecule Bcl-2 family protein inhibitor that has shown preliminary evidence of activity in lymphoid malignancies.
Biomarkers of Vorinostat Resistance in Cutaneous T-Cell Lymphoma.
Authors: Zhang Chunlei , Li Baoqiang , Talpur Rakhshandra , Yin C. Cameron , Duvic Madeleine
Published: October 2019

Profiling gene expression with DNA microarray technology has elucidated novel therapeutic targets and led the approval of a number of targeted therapeutic agents for the treatment of cancer. Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a pan-histone deacetylase (HDAC) inhibitor that has demonstrated an overall response rate of approximately 24–30% in two phase II studies of cutaneous T cell lymphoma (CTCL) patients. There are currently no known specific biomarkers to indicate resistance to vorinostat. To identify genes resistant to vorinostat we compared profiles using the Aligent whole human genome oligo microarrays containing ∼41,000 genes/transcripts in vitro in vorinostat-resistant MJ and -sensitive HH CTCL cell lines treated with 1 μM of vorinostat for 24 hours and compared them to patients’ peripheral blood mononuclear cells (PBMCs) before and during oral therapy. There were 3151 (7.7%) genes/transcripts differentially expressed in vitro in treated resistant MJ cells compared to untreated vehicle control (p < 0.001). We also studied differential gene expression in two clinically resistant Sézary patients’ PBMCs taken at baseline and four weeks after oral vorinostat (400 mg daily or 300 mg bid 3 days/wk). In patients’ PBMCs, 585 (1.4%) and 2744 (6.7%) differentially expressed genes/transcripts (p < 0.001) were identified, respectively. Genes that were up-regulated both in vitro and in vivo included a tumor necrosis factor receptor super-family member 11a (TNFRSF11a or RANK), matrix metallopeptidase 9 (MMP9), suppressor of cytokine signaling 3 (SOCS3), vinculin (VCL) and KIAA1840. Genes that were down-regulated in both included adenylate kinase 3-like 1 (AK3L1), leucine rich repeat and fibronectin type III domain containing 4 (LRFN4), and AL359650. Increased RANK, MMP9 and SOCS3 mRNA expression in MJ compared to HH cells and in three resistant versus three vorinostat responding Sézary patients’ PBMCs was confirmed using quantitative real-time PCR. In conclusion, our results suggest that oligonucleotide microarray analysis may identify biomarkers of resistance to vorinostat which would be helpful to select patients who may not benefit from treatment. These findings provide the rationale for future functional studies and development of more effective use of HDAC inhibitors for CTCL patients.
Receptor-Based Identification of Novel Peptide Ligands as Inhibitors of Blood Stage Malaria.
Authors: Li Xuerong , Lauterbach Sonja B. , Lanzillotti Roberto , Chen Huiqing , Kane Ravi , Coetzer Theresa L. , Chishti Athar H.
Published: October 2019

Malaria parasite Plasmodium falciparum uses multiple receptors on the surface of human red blood cells to attach and invade host cells during blood stage infection. Glycophorins, including glycophorin A, B, and C have been implicated as host receptors and play an important role during Plasmodium falciparum invasion in human erythrocytes, particularly in the sialic acid-dependent parasite strains. To identify the parasite proteins that could bind to human glycophorins, we screened a phage display cDNA library of P. falciparum (FCR3 strain, a sialic acid-dependent strain) using human glycophorins and native intact human erythrocytes as bait. After four rounds of library screening and panning, 35 phage clones were identified that bound to purified glycophorins immobilized on the plastic surface. DNA sequencing of 12 phage clones revealed that they encode the same 7-amino acid sequence, ETTLKSF. Using immobilized intact human erythrocytes, a similar screening strategy led to the isolation of additional 20 phage clones, and the DNA sequencing of 6 such clones again revealed the same 7-amino acid sequence, ETTLKSF. In vitro binding of synthetic ETTLKSF peptide to purified glycophorins and intact erythrocytes was confirmed by ELISA and indirect immunofluorescence assays. Pull-down experiments demonstrated that the ETTLKSF peptide specifically interacts with glycophorin C, but not with glycophorin A and B, on human erythrocytes. The synthetic ETTLKSF peptide blocked merozoite invasion of P. falciparum in human erythrocytes in a dose-dependent manner, whereas the control peptides were without any effect. We have named the ETTLKSF peptide as GBL-1, the Glycophorin Binding Ligand-1. We propose that further characterization of GBL-1, the first peptide ligand that specifically binds to human glycophorin C, could lead to the development of novel anti-malarial agents that prevent P. falciparum invasion in human erythrocytes via the glycophorin C receptor.
Quantitative Bioluminescent Method Appropriate for Bimodality Analysis To Improve and Follow a Syngenic Murine Model of B-Cell Lymphoma Expressing Human CD20.
Authors: Daydé David , Dommange Florence , Lerondel Stéphanie , Golay Josée , Le Pape Alain , Paintaud Gilles , Bardos Pierre , Watier Hervé , Cartron Guillaume
Published: October 2019

The anti-CD20 monoclonal antibody rituximab (C2B8) has shown promising results in the clinical treatment of patients with B-cell Non-Hodgkin’s Lymphoma (B-NHL). However, its therapeutic effect is variable whereas a better knowledge of factors affecting rituximab response could lead to improve its efficacy. It has been suggested that tumour burden could influence rituximab exposure and response in human. The lack of method to assess precisely tumour burden has however prevent to confirm such influence. Study of factors affecting antibody exposure requires bimodality analysis allowing to precise factors related to antibody from those related to tumour. We purposed to develop a quantitative method of tumour burden using bioluminescence imaging (BLI) and simultaneously scintigraphic study of monoclonal antibody biodistribution and exposure (indium-111 labelled ibritumomab, 2B8). Firstly, we developed a murine model of T-lymphoma cells transduced with human CD20 cDNA (EL4-hCD20) and transfected with luciferase plasmid (EL4-hCD20-luc). C57Bl6J mice and C57Bl6J TyrC2-J mice (albino phenotype) were injected with 8.10^3 EL4-hCD20-luc lymphoma cells by i.v. route. All mice died within 30 days with liver, spleen, bone marrow and lymph nodes involvement confirmed by immunohistochemistry and PCR analysis. In order to evaluate tumour growth and dissemination, potassium luciferin (2 or 4 mg) were injected bi-weekly intra peritoneally and tumour burden was analysed using Hamamatsu® CCD imaging system. Background noises and cosmic rays, which disturb the signal of bioluminescence, were eliminated from merging image before bioluminescent activity analysis. The sensitivity of analysis was below than 10^2 El4-hCD20-luc cells and there was proportionality between bioluminescent activity and cells number from 10^3 to 10^9 cells. The use of different binning allowed the follow-up of tumour burden evolution from 9 days after inoculation until the death of mice. We validated the used of black phenotype C57Bl6J compared to C57Bl6J TyrC2-J phenotype and demonstrated that the amount of luciferin could influence tumour growth. Regions of interest were delimited by segmentation method and we developed a reliable quantification method of bioluminescence activity taking into account absorption coefficients of the cells light emitted according to their localization. Such model and quantification method makes possible the follow-up of tumour growth and monoclonal antibody efficacy and exposure studied by bimodality analysis. D.D. is supported by a grant from Région Centre.