bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Inosine Monophosphate Dehydrogenase Inhibitor VX-944 Overcomes Conventional Drug-Resistance in Multiple Mueloma Cells in the Bone Marrow Microenvironment.
Authors: Ishitsuka Kenji , Hideshima Teru , Hamasaki Makoto , Noopur Raje , Shaji Kumar , Yasui Hiroshi , Roccaro Aldo M. , Chauhan Dharminder , Podar Klaus , Tai Yu-Zu , Jain Jugnu , Anderson Kenneth C.
Published: April 2020

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA) is a small molecule, selective, uncompetitive novel inhibitor directed against human IMPDH enzyme. IMPDH inhibitors have been demonstrated to induce growth arrest, and extensively investigated as immunosuppressants. Here we show that VX-944 inhibits growth of human multiple myeloma (MM) cell lines, including those resistant to conventional agents, via induction of apoptosis and S phase arrest in vitro. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs), do not protect against VX-944-induced MM cell growth inhibition. We next delineated the molecular mechanism of VX-944-induced MM cell death in the MM.1S human MM cell line. VX-944 induced apoptosis in MM.1S cells, confirmed by PARP cleavage as well as flow cytometric detection of the mitochondrial membrane protein 7A6 and TdT-mediated dUTP nick-end labelling (TUNEL) positive cells, without significant cleavage of caspases 3, 8 and 9. While the pan-caspase inhibitor z-VAD-fmk did not inhibit the VX-944-induced apoptosis and cell death suggesting that VX-944 triggers apoptosis in MM1.S cells primarily via caspase-independent pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin, melphalan and bortezomib, all of which activate caspases in MM cells and induce apoptosis, even in the presence of BMSCs. Taken together, our data demonstrate non-caspase-dependent apoptotic pathway triggered by VX-944 thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional and/or novel agents which trigger caspase activation. Our ongoing studies are delineating the mechanisms whereby VX-944 induces MM cell apoptosis.
Safety and Efficacy Results from a Phase I Trial of Single-Agent Lumiliximab (Anti-CD23 Antibody) for Chronic Lymphocytic Leukemia.
Authors: Byrd John C. , O’Brien Susan , Flinn Ian , Kipps Thomas J. , Weiss Mark A. , Rai Kanti , Lin Thomas S. , Reid Jennifer , Wynne Dee , Leigh Bryan R.
Published: April 2020

The CD23 antigen is expressed at high density on the cell surface of certain B-cell malignancies, including chronic lymphocytic leukemia (CLL). Lumiliximab (L-mab), a macaque-human chimeric anti-CD23 monoclonal antibody, has been reported to have antitumor activity against CLL in preclinical studies. In this Phase I multicenter study, the safety, efficacy, and pharmacokinetics of single-agent L-mab were evaluated in 46 patients with relapsed or refractory CLL. Therapy consisted of intravenous L-mab given as 6 regimens: (1) 125 mg/m2/wk for 4 weeks; (2) 250 mg/m2/wk for 4 weeks; (3) 375 mg/m2/wk for 4 weeks; (4) 500 mg/m2/wk for 4 weeks; (5) 500 mg/m2 for 3 doses during Week 1, then 500 mg/m2/wk during Weeks 2 to 4; and (6) 500 mg/m2 three times a week for 4 weeks. Patient characteristics were as follows: median age of 62 years (range 47 to 80 years), 93% Caucasian, 72% male, 54% fludarabine-refractory, 48% Rai stage III/IV, and 78% WHO Performance Status 1. At study entry, patients had progressive CLL after 1 to 13 prior treatment regimens (median = 4 prior regimens). Antibody infusions, administered over 2 hours in an outpatient setting, were well tolerated. Study-related adverse events (probable, possible, or unknown relationship to study treatment) were reported in 40 of 46 patients (87%). The majority of events were Grade 1 or 2; the most common were headache, constipation, nausea, and cough. Grade 3 or 4 study-related adverse events were reported in 7 of 46 patients (15%) and included neutropenia and dyspnea. Evidence of clinical activity consisted of reductions in absolute lymphocyte counts (ALC) and lymphadenopathy. Decreases in ALC were observed in 42 of 46 (91%) patients, and decreases ≥ 50% were observed in 11 of 40 (28%) patients enrolled at 375 mg/m2/week or higher. Of 37 patients evaluated for change in lymphadenopathy, reductions were observed in 22 (59%). Flow cytometry revealed that L-mab saturated CD23 sites on CLL cells at doses above 375 mg/m2/week without down regulating CD23 expression. These results suggest that single-agent L-mab can be administered safely with evidence of clinical activity in patients with heavily pretreated CLL. Ongoing clinical studies are assessing the potential of L-mab in combination with rituximab and fludarabine-based chemotherapy.
Autologous CMV-Specific T Cells as Effector Cells in Immunotherapy of B Cell Chronic Lymphocytic Leukemia.
Authors: Kater Arnon P. , Remmerswaal Esther E.B.M , Nolte Martijn A. , Eldering Eric , van Lier Rene A.W , van Oers Marinus H.J
Published: April 2020

Introduction. B cell Chronic Lymphocytic Leukemia (B-CLL) is prototypic for human cancers that escape immune surveillance. These cells not only lack immunogenic tumor-specific epitopes but also express low levels of surface molecules that are necessary for optimal interaction with naive T cells. Different strategies have been tried to generate B-CLL specific T cells that can be used for adoptive immunotherapy. Yet it is uncertain whether these T cell specific responses are strong enough to eliminate the neoplastic clone. Previously, we have demonstrated that CMV seropositive patients with B-CLL have significantly increased relative and absolute numbers of CD8+ T cells exhibiting a CD45RA+CD27− cytotoxic CMV specific phenotype. In the present study we investigated the possibility to direct these in vivo generated CMV specific effector cells to the poorly immunogenic B-CLL cells.

Results. A considerable fraction (6%; range 1.0 to 16.4) of the circulating CD8+ T cell pool of the four B-CLL patients studied consisted of CMV-specific T cells, as visualized with either tetrameric HLA-A2.1/NLVPMVATV complexes or HLA-B7.2/TPRVTGGGAM complexes. Activation of these cells by CMV-peptide in the presence of IL-2 at day 0, and restimulation at day 8 with either CMV peptide loaded EBV transformed cell-lines or autologous peptide-loaded B-CLL cells, resulted in a near 100-fold absolute expansion of tetramer positive CD8+ T cells (on average 1.9 x 106 at day 0, and 2.2 x 108 at week 3). These in vitro restimulated CMV-specific T cells were non-cytotoxic for non-peptide loaded B-CLL cells, but showed a very efficient killing of CMV peptide loaded B-CLL cells, with a 50% specific lysis at an effector:target ratio as low as 4:1. Thus, despite their general anti-apoptotic profile, B-CLL cells are excellent targets and undergo apoptosis after CTL attack. This CTL specific killing was found to be completely dependent on the granzyme and perforin pathway. Finally we showed that the CMV specific effector cells in B-CLL patients do not need restimulation to effectuate a cytotoxic response towards CMV peptide loaded B-CLL cells.

Conclusion. Directing virus-specific T cells to B-CLL tumors may overcome the inadequate immunostimulatory capacity of these cells and could be exploited for T-cell mediated immunotherapy.
Novel Anti-CD37 Small Modular Immunopharmaceutical (SMIP) Induces B-Cell-Specific, Caspase-Independent Apoptosis in Human CLL Cells.
Authors: Zhao Xiaobin B. , Biswas Sabyasachi , Mone Andrew , Ledbetter Jeffrey , Ledbetter Martha Hayden , Grosmaire Laura , Espling Erik , Cheney Carolyn , Young Donn , Lee Robert J. , Muthusamy Natarajan , Byrd John C.
Published: April 2020

Tru 16.4 is a single chain protein with a modified CD37 binding Fv domain linked to a modified human IgG1 hinge, CH2 and CH3 domains. It is a member of a novel composition class called small modular immunopharmaceuticals (SMIP) construct. CD37 is a stable marker for B-cell chronic lymphocytic leukemia (BCLL) and non-Hodgkins lymphoma (NHL). Although radiolabeled murine anti-CD37 antibody MB-1 has been evaluated in NHL patients and shown to be able to target tumors, efficacy was not established. There has been no evaluation of an unconjugated anti-CD37 antibody in human clinical trials. Here we report that Tru 16.4, binds to CD37 on primary CLL cells surface, and induces caspase-independent apoptosis that is further enhanced by a secondary crosslinking with goat anti-human antibody. The percentage of dead cell population over control based upon Annexin V/PI staining of 12 BCLL patient samples (28.3%, SD 15.4%) is significantly greater than that of isotype control, trastuzumab (3.4%, SD 7.6%). The expression of CD37 on T cells is low, and the exposure of Tru 16.4 under the same conditions does not demonstrate significant binding or induce noticeable apoptosis on normal T cells at 24 hours (0.47%, SD 0.99%). This provides a selective advantage for BCLL antibody therapy. Results from a comparison study utilizing 6 additional patient samples demonstrated that Tru 16.4 induced apoptosis (31.8%, SD 12.2%) is significantly higher than that of the anti-CD20 antibody, rituximab (21.8%, SD 5.25%) and similar to that observed with the anti-CD52 antibody, alemtuzumab (36.6%, SD 19.5%). The cell death induced by Tru 16.4 is dependent upon dose and duration of treatment, indicating 5μg/mL as a saturation concentration and 24 hours as the optimal time for maximal in vitro apoptosis. The degree of CLL cell death was found to be directly proportionate to cell surface membrane expression of CD37, based on nonparametric Spearman rank correlation analysis of the mean fluoresecnce intensity (MFI) after PE-labelled anti-CD37 antibody immunostaining and percentages of Annexin V/PI positive cells, with a correlation factor rs = 0.80 ( P < 0.01). Ongoing studies suggest Tru 16.4 induces intracellular signaling following CD37 antigen ligation and current efforts are focused on identifying the specific death pathways mediated by this treatment. Overall, these findings indicate that Tru 16.4 mediates B-cell specific apoptosis through a novel caspase independent mechanism and supports effort for further clinical investigation in CLL.
Modification of B-CLL Cells Via Infection with a Replication-Defective MVA Virus Encoding Three Costimulatory Molecules: A Potential Approach to Tumor Cell Immunotherapy of B-CLL.
Authors: Palena Claudia , McIntosh Loni , Schlom Jeffrey , Foon Kenneth , Panicali Dennis , Tsang Kwong Y.
Published: April 2020

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a lymphoproliferative disorder characterized by the accumulation of slowly dividing, apoptosis-defective, clonal CD5+ B cells that typically involve the bone marrow, lymph nodes, spleen and blood of affected individuals. New therapeutic approaches are necessary to be investigated for the treatment of B-CLL. Several characteristics of the neoplastic B cells make B-CLL a good candidate for immunotherapy; evidence exists that B-CLL cells can be recognized by autologous anti-tumor T cells in an MHC-restricted manner. B-CLL cells, however, are themselves ineffective antigen-presenting cells mainly because of their inadequate costimulatory capacity. We have investigated the ability of in vitro manipulated B-CLL cells that express a triad of costimulatory molecules, designated TRICOM, to stimulate effective anti-tumor T-cell responses and their potential as cancer vaccines. Delivery of transgenes to the B-CLL cells could be accomplished through the use of a modified vaccinia virus strain Ankara designated MVA. This is a highly attenuated, non-replicative virus that has been demonstrated to be safe and well tolerated in both animals and humans. Here we report the use of a recombinant MVA vector to deliver the simultaneous expression of three human costimulatory molecules (B7-1, ICAM-1, LFA-3) on the surface of B-CLL cells. Infection was conducted using various multiplicity-of-infection (MOI) of MVA-TRICOM and the efficiency of the vector to enhance the expression of the encoded transgenes was evaluated by FACS analysis. The infection of B-CLL cells with MVA-TRICOM markedly increased the expression of B7-1, ICAM-1 and LFA-3 on the cell surface. The ability of the MVA-TRICOM-infected B-CLL cells to activate specific T cells was subsequently analyzed in proliferation assays, cytotoxic, and cytokine production assays. Proliferation of both autologous and allogeneic T cells was observed when MVA-TRICOM-infected B-CLL cells were used as stimulators in mixed lymphocyte reactions. Autologous CTLs were then generated in vitro using MVA-TRICOM-infected B-CLL cells as APC; these CTLs were capable of lysing uninfected autologous as well as allogeneic B-CLL cells. From our results we conclude that MVA-TRICOM-infected B-CLL cells can be used to induce B-CLL-specific CTLs that, in turn, can mediate the lysis of unmodified B-CLL cells. Our findings have implications for a potential use of MVA-TRICOM-infected B-CLL cells as tumor vaccines in the immunotherapy of B-CLL.
Constitutive Activation of Akt and mTOR by Flt3 Internal Tandem Duplications Mediates Myeloid Leukemogenesis and Can Be Inhibited by Rapamycin.
Authors: Brandts Christian H. , Sargin Bulent , Rode Miriam , Biermann Christoph , Lindtner Beate , Schwable Joachim , Buerger Horst , Muller-Tidow Carsten , Choudhary Chunaram , McMahon Martin , Berdel Wolfgang E. , Serve Hubert L.
Published: April 2020

Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, a tyrosine kinase receptor. The signalling pathways and downstream targets are only partly understood. We demonstrated that the presence of Flt3-ITD constitutively activated Akt (PKB), a key serine-threonine kinase within the PI3-kinase pathway. The constitutive activation of Akt induced phosphorylation and transcriptional inhibition of the transcription factor Foxo3a. Also, forced Foxo3a activity reversed Flt3-ITD-mediated growth properties. Using a novel Akt inhibitor, we showed that Flt3-ITD-mediated Foxo3a phosphorylation was dependent on Akt. Conditional Akt activation targeted to the cell membrane conferred cytokine-independent survival, proliferation and transformation of myeloid progenitor cells. Induction of Akt activity in vivo promoted the development of a myeloproliferative disease in a syngeneic mouse model. Importantly, phosphorylation of Akt was frequently found in primary myeloid blasts, supporting that this signalling pathway may be important in the development of AML. Furthermore, activation of Akt constitutively activated the downstream kinase mammalian target of rapamycin (mTOR). mTOR has several important biological functions, notably in nutrient sensing and protein translation. Treatment with rapamycin, a specific inhibitor of mTOR, inhibited mTOR kinase activity at 1nM in all examined cell systems. Rapamycin caused cell cycle arrest, apoptosis and completely inhibited transformation in FLT3-ITD-positive cells. These effects were most pronounced in the low nanomolar range, which are therapeutic concentrations of rapamycin-treated patients. The biological advantage in Akt-expressing cells was fully reversed by treatment with rapamycin. In all examined primary blasts from AML patients, mTOR was inhibited by rapamycin. In short term proliferation assays, twelve out of 18 (67%) examined showed marked sensitivity with an IC50 below 10nM rapamycin, while one third were found to be more resistant.

Taken together, increased survival, proliferation and leukemic transformation by Flt3-ITD is mediated by Akt and mTOR, and can be inhibited by rapamycin. We conclude that rapamycin may be an attractive substance for the treatment of patients with AML.
HIV-1 Protease Inhibitor Ritonavir Potentiates the Effect of 1,25-Dihydroxyvitamin D3 to Induce Growth Arrest and Differentiation of Human Myeloid Leukemia Cells Via Inhibition of CYP24.
Authors: Ikezoe Takayuki , Yang Yang , Bandobashi Kentaro , Sekiguchi Nobuo , Sakai Shuichi , Koeffler H. Phillip , Taguchi Hirokuni
Published: April 2020

Ritonavir (RTV) is a potent inhibitor of cytochrome p450 (CYPs) enzymes. This study explores the effects of RTV on CYP24 which converts 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its inactive form 1,24,25,(OH)3. Real-time RT-PCR showed that exposure of HL-60 cells to 1,25(OH)2D3 (10−7 M, 1h) induced expression of CYP24 by 80-fold, and pre-incubation of these cells with RTV (2X10−5 M, 3h) decreased 1,25(OH)2D3-stumulated expression of CYP24 transcripts by 30 %, resulting in increased levels of 1,25(OH)2D3 in culture media (50.7 vs 82.4 nmol/L). Importantly, pre-incubation of HL-60 cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) also increased intracellular levels of 1,25(OH)2D3 (1.20 vs 2.46 nmol/L) and potentiated the ability of 1,25(OH)2D3 to induce growth arrest of HL-60 cells. Clonogenic assay showed that either RTV (5X10−6 M) or 1,25(OH)2D3 (10−8 M) alone inhibited colonal growth of HL-60 cells by either a mean 30 ± 12 (±SD) % and 40 ± 12 %, respectively; when both were combined, colony formation was inhibited by a mean 80 ± 13 % (p<0.01). Measurement of CD14 expression, NBT reduction, and non-specific esterase staining showed that RTV augmented the prodifferentiative effects of 1,25(OH)2D3 in HL-60 cell. For example, 1,25(OH)2D3 (10−8 M, 2 days) induced approximately 48 % of HL-60 cells to become CD14 antigen positive. RTV (2X10−5 M, 48h) alone did not stimulate expression of CD14 antigen; when 1,25(OH)2D3 was combined with RTV, approximately 65 % of cells expressed CD14. Furthermore, androgen-independent DU145 human prostate cancer cells, that are resistant to growth inhibition by 1,25(OH)2D3, expressed 80-fold higher levels of CYP24 transcripts compared to HL-60 cells, and intracellular levels of 1,25(OH)2D3 were not detectable (<0.3 nmol/L) after exposure of these cells to 1,25(OH)2D3 (10−7 M, 24 h). Of note, pre-incubation of these cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) increased intracellular levels of 1,25(OH)2D3 to 0.94 nmol/L and sensitized DU145 cells to growth inhibition mediated by 1,25(OH)2D3 as measured by MTT assay. Taken together, inhibition of CYP24 might open a new paradigm for therapy using vitamin D compounds.
Leukemia Proto-Oncoprotein MLL Forms a SET1 like Complex with Menin to Regulate Hox Gene Expression.
Authors: Yokoyama Akihiko , Wang Zhong , Wysocka Joanna , Sanyal Mrinmoy , Aufiero Deborah J. , Kitabayashi Issay , Herr Winship , Cleary Michael L.
Published: April 2020

MLL (Mixed Lineage Leukemia) is a proto-oncogene that is mutated in a variety of human leukemias. Oncogenic MLL fusion genes created by chromosomal translocations cause acute myeloid or lymphoid leukemias often in infants. MLL, a homolog of Drosophila trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression. But its regulatory mechanism is poorly understood. Our previous studies showed that MLL undergoes a variety of post-translational modifications to assume its mature form. Most uniquely, MLL is proteolytically processed after translation into two major polypeptides, MLL-N and MLL-C, which possess opposite transcriptional properties and associate with each other through their intra-molecular interaction domains. In order to understand the function of MLL and its regulatory mechanism, we performed the biochemical purification of MLL. MLL-C was found to associate with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including ASH2L (highly conserved protein of unknown function whose Drosophila homolog genetically interacts with trithorax), WDR5 and RBBP5 (two WD repeat proteins implicated in histone binding) to form an MLL-C histone methyltransferase (HMT) sub-complex centered on its SET domain. Two other members of the novel MLL complex identified here are Host Cell Factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL-N (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties. Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1 MDa MLL complex. Abrogation of menin expression phenocopies loss of MLL, and reveals a critical role for menin in the maintenance of Hox gene expression. Oncogenic mutant forms of MLL retain an ability to interact with menin, but not other identified complex components. These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis.
Dysregulation of Actin Polymerization in Hematopoietic Cells Transformed by Bcr-Abl.
Authors: Dai Zonghan , Li Yingzhu , Clough Nancy , McNiece Ian
Published: April 2020

Actin polymerization is a fundamental cellular process that controls cytoskeletal functions such as adhesion, migration, and homing. This dynamic cellular process is spatiotemporally regulated in normal cells but its dysregulation is often observed in tumor cells and is believed to contribute to tumor metastasis. Hematopoietic cells isolated from Bcr-Abl positive leukemia patients exhibit multiple abnormalities in cytoskeletal function. The mechanism associated with these abnormalities is not completely understood. Previously, we showed that the expression of Bcr-Abl in hematopoietic cells induces tyrosine phosphorylation of Ableson interactor (Abi) 1 and ubiquitin dependent degradation of Abi 2. The dysregulation of Abi 1 and Abi 2 requires the SH3 domain and C-terminal proline-rich sequences of Bcr-Abl. Deletion of these sequences in Bcr-Abl not only abrogates Abi-1 tyrosine phosphorylation and Abi-2 degradation but also impairs leukemogenic activity of Bcr-Abl. Notably, the mutant Bcr-Abl with deletion of these sequences failed to induce splenomegaly in a bone marrow transplant mouse model. Because Abi proteins have been shown to function as a key regulator of actin polymerization, we examined the F-actin organization in Ba/F3 cells transformed by wild type Bcr-Abl (p185wt) and a mutant Bcr-Abl defective in signaling to Abi (p185ΔSH3ΔC). Actin polymerization is increased in Ba/F3 cells transformed by either p185wt or p185ΔSH3ΔC, as compared to control Ba/F3 cells. However, the pattern of F-actin organization in these cells appears different. The expression of p185wt induces a localized abnormal F-actin structure which is not observed in control Ba/F3 cells and the Ba/F3 cells transformed by p185ΔSH3ΔC. We have characterized this abnormal F-actin structure and show that it co-localizes with Bcr-Abl. Investigation is now underway to determine whether the Abi/WAVE pathway is involved in Bcr-Abl-induced abnormal F-actin structure. These studies may provide insight into the mechanism by which Bcr-Abl induces cytoskeletal abnormalities in CML cells.
Venous Thromboembolism (VTE) Risk Factors among Nursing Home Residents: A Population-Based Case-Control Study.
Authors: Petterson Tanya M. , Leibson Cynthia L. , Heit John A. , Bailey Kent R. , Melton L. Joseph
Published: April 2020

Confinement to a nursing home (NH) is an independent risk factor for VTE and accounts for 13% of all VTE occurring in the community, but the question of which NH residents are at risk is largely unanswered.

Objective: To determine VTE risk factors among NH residents.

Methods: Using the Minnesota State Case Mix Review Program Public Research Files, we performed a case-control study of all Olmsted County, MN NH patients with an ICD-9 code-identified diagnosis of VTE from 1988–1994. From the same source, we identified one age-, sex-matched control and used conditional logistic regression to estimate odds ratios for an association of VTE with 46 characteristics assessed in the Minnesota Case Mix data.

Results: Univariate associations between characteristics and odds of VTE are described below. Significantly increased odds were observed for residents assessed following hospitalization (a nearly 4-fold increase, p=0.002, Table) and residents considered incapable of self-preservation (i.e., unable to cope with potentially harmful situations) (> 2.0 fold increase, p=0.03). Non-significant 2–3 fold increased odds were observed for limitations in certain specific Activities of Daily Living (ADL) (including walking and dressing) and for limitations in more than 3 ADLs. Similar increased odds were observed for residents using a wheelchair, taking more than 3 oral medications, enrolled in professional activity programs, and needing behavior management.

Conclusions: Need for assistance (especially problems with immobility), lack of self preservation, and recent hospitalization may identify a subset of NH residents that are at especially high VTE risk and who may warrant consideration of prophylaxis.

Univariate VTE Risk Factors for Nursing Home Residents

Characteristic Odds Ratio 95% CI P-value Oral medications > 3 3.00 0.97-9.30 0.06 Activities of Daily Living High/Medium (4-8) vs. low (0-3) 2.20 0.76-6.33 0.14 Help dressing 2.75 0.88-8.64 0.08 Help walking 3.00 0.61-14.86 0.18 Wheelchair use 2.50 0.97-6.44 0.58 Needs behavior management 3.00 0.62-14.86 0.18 Help toileting 2.00 0.68-5.85 0.20 Lack of self-preservation 2.33 1.07-5.10 0.03 Transfer from hospital 3.86 1.68-8.86 0.0015 Anti-depressant drugs 0.50 0.19-1.33 0.16 Professional activity program 2.25 0.69-7.3 0.18
Endothelial Cells Derived from Human Vessel Walls Contain a Complete Hierarchy of Endothelial Progenitor Cells.
Authors: Ingram David A. , Mead Laura E. , Woodard Wayne , Fenoglio Amy , Murphy Brian , Harvey Celeste , Moore Daniel B. , Yoder Mervin C.
Published: April 2020

Endothelial progenitor cells (EPCs) can be isolated from adult peripheral and umbilical cord blood. EPCs are thought to originate from bone marrow, circulate in peripheral blood, and migrate to sites of angiogenesis. However, the number of circulating EPCs in peripheral blood is remarkably low, and recent genetic studies show that the contribution of bone marrow derived EPCs to newly formed vascular networks is minimal. Further, while endothelial cells (ECs) derived from vessel walls are widely considered to be differentiated mature ECs, these cells retain extensive proliferative potential and can be passaged for at least 40 population doubling in vitro. Based on these observations, we tested whether EPCs potentially reside in vessel walls utilizing a newly developed single cell deposition assay (Blood, 2004). Analogous to a paradigm established in the hematopoietic cell system, we can utilize this assay to reproducibly identify the following EPCs: (1) high proliferative potential - endothelial colony forming cells (HPP-ECFC), which form macroscopic colonies that form secondary and tertiary colonies upon replating, (2) low proliferative potential - endothelial colony forming cells (LPP-ECFC), which form colonies greater than 50 cells, but do not form secondary colonies upon replating, (3) endothelial cell clusters (EC-clusters) that contain less than 50 cells, and (4) mature terminally differentiated endothelial cells (EC), which do not divide. Utilizing this assay, we compared the clonogenic potential of 1000 single adult human dermal microvascular endothelial cells (HMVECds), human umbilical vein endothelial cells (HUVECs), human umbilical artery endothelial cells (HUAECs), human coronary artery endothelial cells (HCAECs), and human aortic endothelial cells (HAECs) to the potential of adult peripheral and umbilical cord blood derived EPCs. We conducted four independent experiments. Remarkably, we demonstrate that a complete hierarchy of EPCs can be identified in EC populations derived from every vessel wall tested (Table I and n=4). Further, we show that ECs derived from each vessel wall cell population tested contain more proliferative EPCs (LPP-ECFCs and HPP-ECFCs) compared to EPCs derived from adult peripheral blood.

Percent of 1,000 Single Cells Plated

Mature EC EC-Cluster LPP-ECFC HPP-ECFC HUVEC 42±6 18±2 29±9 11±5 HAEC 37±3 23±8 21±4 20±6 HMVECd 65±9 21±6 12±4 2±0.6 HCAEC 46±2 18±2 20±2 16±2 HUAEC 41±1 10±1 27±4 21±2 Adult EPC 81±9 9±1 12±8 0.2±0.2 Cord EPC 50±20 7±2 20±10 23±9

Thus, this study provides evidence that a diversity of EPCs exists in human vessels and provides a new conceptual framework for determining both the origin and function of EPCs in maintaining vessel integrity and contributing to new sites of angiogenesis.
Hyperhomocysteinemic Mice Have Increased Susceptibility to Carotid Artery Thrombosis.
Authors: Wilson Katina M. , Dayal Sanjana , Bottiglieri Teodoro , Lentz Steven R.
Published: April 2020

Hyperhomocysteinemia is a risk factor for cardiovascular disease, stroke, and venous thrombosis. In animal models, hyperhomocysteinemia produces endothelial dysfunction due to decreased bioavailability of endothelium-derived nitric oxide (NO). Because NO has antithrombotic properties, we tested the hypothesis that hyperhomocysteinemia accelerates photochemically-induced carotid artery thrombosis induced in mice. Mice heterozygous for a targeted disruption of the cystathionine β-synthase (Cbs) gene (Cbs+/−) and wild type littermates (Cbs+/+) were fed either a control diet or a high methionine/low folate (HM/LF) diet for 8 months. Plasma total homocysteine was elevated by the HM/LF diet compared with the control diet in both Cbs+/+ (11.6±1.2 vs. 3.7±0.5 μmol/L; p<0.05) and Cbs+/− mice (26.4±3.8 vs. 6.2±0.6 μmol/L; p<0.001). The time to stable occlusion after photochemical injury (rose bengal and green laser) of the carotid artery was 50% shorter in mice fed the HM/HF diet (17.5±2.3 and 18.2±4.0 minutes for Cbs+/+ and Cbs+/− mice, respectively) compared with mice fed the control diet (44.0±9.1 and 31.4±4.7 minutes for Cbs+/+ and Cbs+/− mice, respectively; p<0.001). Using the oxidative dye, dihydroethidium (DHE), increased production of superoxide was detected in Cbs+/+ and Cbs+/− mice fed the HF/HM diet compared with the control diet. To determine whether lack of endothelium-derived NO may accelerate carotid artery thrombosis, mice deficient in endothelial nitric oxide synthase (Nos3−/ −) were also studied. Time to occlusion did not differ between Nos3−/ − mice and wild type mice (55.0±10.2 vs. 42.1±7.1 minutes, p=0.31). We conclude that hyperhomcysteinemia increases susceptibility to experimental thrombosis through a mechanism that is independent of endothelium-derived NO, but may involve oxidative stress.
Cerebral Vascular Dysfunction in Methionine Synthase-Deficient Mice.
Authors: Dayal Sanjana , Devlin Angela M. , McCaw Ryan B. , Liu Mei-lan , Arning Erland , Bottiglieri Teodoro , Shane Barry , Faraci Frank M. , Lentz Steven R.
Published: April 2020

Methionine synthase (MS) is a key enzyme involved in remethylation of homocysteine to methionine. We tested the hypothesis that deficiency of MS influences endothelial function. Mice heterozygous for disruption of the Ms gene (Ms+/−), and wild type littermates (Ms+/+), were fed either a control diet or a low folate (LF) diet. Plasma total homocysteine was similar in Ms+/+ and Ms+/− mice fed the control diet (4.5±0.3 and 5.3±0.4 μmol/L, respectively), and was mildly elevated in Ms+/+ or Ms+/− mice fed the LF diet (7.5±0.7 and 9.6±1.2 μmol/L, respectively; p<0.001 vs. control diet). Dilatation of cerebral arterioles (~30 μm diameter) to the endothelium-dependent dilator, acetylcholine (10 μM), was blunted in Ms+/− mice compared with Ms+/+ mice fed the control diet (21±4 vs. 32±4%; p<0.05). Both Ms+/+ and Ms+/− mice exhibited impaired dilatation of cerebral arterioles to acetylcholine when they were fed the LF diet (12±2 and 17±3%, respectively; p<0.01 vs. Ms+/+ mice fed the control diet). Dilatation of cerebral arterioles in response to the endothelium-independent dilator, nitroprusside, was similar in all groups of mice. Relaxation of aortic rings to acetylcholine was not influenced by Ms genotype or diet. Elevated levels of superoxide and hydrogen peroxide were detected by dihydroethidium (DHE) and dichlorodihydrofluorescein (DCF), respectively, in cerebral arterioles of Ms+/− mice fed the control diet and in both Ms+/+ and Ms+/− mice fed the LF diet. These findings demonstrate that defective homocysteine remethylation caused by deficiency of either MS or folate produces endothelial dysfunction and increased oxidative stress in the cerebral microcirculation of mice. Interestingly, impairment of cerebral vascular function was independent of plasma tHcy concentration.
Modulation of Tissue Factor-Factor VIIa Coagulant and Signaling Functions by Membrane Cholesterol and Caveolae.
Authors: Awasthi Vineet , Rao L. Vijaya Mohan , Pendurthi Usha
Published: April 2020

Cholesterol in the membrane not only regulates flexibility and mechanical stability of the membrane but also plays a critical role in differentiating and maintaining cell surface microdomains of differing lipid composition, particularly sphingolipid rafts. Cholesterol- and sphingolipid-rich rafts in association with a structural protein, caveolin, form caveolae, flask-shaped invaginations in the plasma membrane. Lipid rafts and caveolae are shown to regulate various cellular functions, including receptor function, endocytosis, intracellular trafficking of receptors and signaling pathways. In the present study, we investigated the role of membrane cholesterol and caveolae in modulating coagulant and signaling functions of tissue factor (TF)-factor VIIa (VIIa) complexes on tumor cells. Breast carcinoma cells, MDA-MB-231, were treated with ß-methyl cyclodextrin (CD), which depletes membrane cholesterol and thereby disrupts caveolae, or with filipin, which disrupts caveolae without depleting the membrane cholesterol. TF-VIIa coagulant function was measured in factor X activation assay and the signaling function was evaluated in IP3 hydrolysis and IL-8 gene induction. As expected, CD (10 mM) treatment of tumor cells depleted cellular cholesterol level by more than 70% whereas filipin (5 μg/ml) treatment did not reduce the cellular cholesterol level. Both the treatments had no effect on the cell viability as measured in trypan blue exclusion method and MTT assay. CD treatment, in a dose-pendent manner (1 to 10 mM CD), impaired both TF-VIIa coagulant function and the signaling function (both IP3 hydrolysis and IL-8 gene expression). In contrast, varying effects were observed with filipin treatment. Filipin treatment (5 μg/ml) increased TF-VIIa coagulant function, reduced the TF-VIIa-induced IP3 hydrolysis and no effect on TF-VIIa-induced IL-8 gene expression. Detergent (Triton X-100) extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF was distributed both in lipid rafts and soluble fractions. CD and filipin treatments slightly reduced TF association with lipid rafts. Overall these data suggest that the membrane cholesterol modulates TF-VIIa proteolytic function and hence the TF-VIIa protease-induced signaling. In contrast, disruption of caveolae uncouples selective signaling pathways activated by TF-VIIa without impairing TF-VIIa protease activity.
Phase II Study of SGN-30 (Anti-CD30 Monoclonal Antibody) in Patients with Refractory or Recurrent Hodgkin’s Disease.
Authors: Leonard John P. , Rosenblatt Joseph D. , Bartlett Nancy L. , Gopal Ajay , Younes Anas , Fisher David , Foss Francine , Forero Andres , Bernstein Steven , Cheson Bruce , Carabasi Matthew H. , Bociek R. Gregory , Hart Bruce , McDonald Michael
Published: April 2020

SGN-30 is a chimeric monoclonal antibody which recognizes the CD30 antigen found on tumor cells from patients with Hodgkin’s disease (HD) and anaplastic large cell lymphoma (ALCL). Preclinical studies with this agent have demonstrated anti-lymphoma effects in both in vitro cell line assays and in vivo murine model systems. The results of a multi-dose phase I study showed minimal toxicity associated with doses from 2 to 12 mg/kg administered as six weekly IV infusions over 120 minutes each. Of the 21 patients with Hodgkin’s Disease accrued to the phase I study, four patients had stable disease (SD). A phase II multi-dose study is currently underway to further evaluate the safety, antitumor activity and pharmacokinetics of six weekly IV infusions of 6 mg/kg of SGN-30 in patients with relapsed or refractory HD or systemic ALCL (sALCL). Fifteen subjects (6M, 9F) with HD have been enrolled, with baseline data as follows: median age 34 (range 20–65), median number of prior therapies 3 (range 1–5), and 11 patients (73%) have disease which progressed after prior high-dose chemotherapy and stem cell transplant. Multiple doses of SGN-30 have been well tolerated in all subjects. Drug-related adverse events have been typically mild and consistent with monoclonal antibody administration. The most common drug-related adverse event has been fatigue. No grade 3/4 events have occurred. Twelve patients are currently evaluable for response, with 6 having had stabilization of their disease. Assessment of duration of disease stabilization and response is continuing. While the acceptable safety profile and frequency of stable disease following therapy in this heavily pretreated patient population are encouraging, a 12 mg/kg/dose regimen is now being tested in subsequent subjects to further explore the dose-response relationship of SGN-30 in Hodgkin’s Disease. Further evaluation of this novel immunotherapy in additional HD patients is ongoing.
Phase I/II, Open-Label, Dose-Escalating Study of MDX-060 Administered Weekly for 4 Weeks in Subjects with Refractory/Relapsed CD30 Positive Lymphoma.
Authors: Ansell Stephen M. , Byrd John C. , Horwitz Steven M. , Borchmann Peter , Engert Andreas , Strair Roger , Khan Khuda D. , Fischkoff Steven A. , Yellin Michael
Published: April 2020

Background: MDX-060 is a fully human anti-CD30 IgG1k monoclonal antibody that induces Fc-receptor mediated killing of CD30 expressing cell lines and inhibits the growth of CD30-expressing tumor cells in a murine xenograft model. We previously reported data from the first 31 patients in a Phase I/II open-label, dose escalation study of MDX-060 in patients with relapsed or refractory CD30+ lymphomas. The purpose of the study is to determine the safety and tolerability profile of the drug, and to preliminarily evaluate efficacy. We now update the findings of this study, including reporting the results of a new higher dose cohort.

Methods: In the Phase I portion of the study, MDX-060 was administered intravenously at dose levels of 0.1, 1, 5, or 10 mg/kg weekly for 4 weeks to cohorts of 3–6 patients. In the Phase II portion of the study additional patients were administered MDX-060 intravenously at 10 (n=10) or 15 (n=17) mg/kg weekly for 4 weeks. Responses were assessed at month 2.

Results: To date, MDX-060 has been administered to 48 patients; 40 with Hodgkin’s disease (HD), 6 with anaplastic large cell lymphoma (ALCL) and 2 with other CD30 positive lymphomas. MDX-060 has been well tolerated, and a maximum tolerated dose has not been identified. There were 2 drug-related serious adverse events, 1 patient in the 1 mg/kg dosing cohort had Grade 3 elevated liver transaminase, and 1 patient in the 15 mg/kg cohort had Grade 3 pneumonia/Grade 4 ARDS. Objective clinical responses have been observed in 5 patients. Two patients had complete responses; 1 patient with ALCL in the 1 mg/kg cohort lasting 100 days, and 1 patient with HD in the 15 mg/kg cohort lasting 117 days. Retreatment of the ALCL patient with MDX-060 resulted in a second complete response. Three patients had partial responses; 1 patient with HD in the 5 mg/kg cohort lasting 92 days, and 1 patient with HD in the 15 mg/kg cohort, which is continuing at 176 days. In addition, 1 patient with ALCL in the 15 mg/kg cohort had a partial response. Stable disease was observed in 17 patients following MDX-060 administration with a median duration of 132 days (range:15–387 days). Baseline soluble CD30 levels in the plasma were evaluated, but did not appear to correlate with clinical outcome.

Conclusions: Preliminary evidence indicates MDX-060 to be well tolerated in heavily pre-treated patients with CD30+ lymphomas, with minimal toxicity, and with clinical activity observed at 1 mg/kg, 5 mg/kg and 15 mg/kg. Additional patients are being accrued at 1 mg/kg and 5 mg/kg to more accurately assess efficacy at these dose levels.
CTLA-4 and PD-1 Receptors Inhibit T-Cell Activation by Distinct Mechanisms.
Authors: Chemnitz Jens M. , Riley James L. , Frauwirth Kenneth A. , Braunstein Inbal , Kobayashi Sumire V. , Linsley Peter S. , Thompson Craig B. , Parry Richard V.
Published: April 2020

CTLA-4 and PD-1 are receptors that negatively regulate T cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28 mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4 mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28 mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/Atk activation was dependent upon the ITSM located in its cytoplasmic tail. Lastly, PD-1 ligation is more effective in suppressing CD3/28 induced changes in the T cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T cell activation through distinct and potentially synergistic mechanisms.
Accelerated FDA Approval of Drugs Used To Treat Hematologic Malignancies: Are Policy Changes Needed?.
Authors: Lyons Elizabeth A. , McKoy June M. , Belknap Steven , Carson Kenneth R. , Bennett Charles L.
Published: April 2020

Background: In 1992, the FDA extended the accelerated approval program from HIV and AIDs, to include drugs for other diseases that are serious or life-threatening, appear to provide benefit over available therapy, or for which no other therapy is available. Approval is based on a surrogate endpoint, and pharmaceutical companies are required to confirm safety and efficacy by conducting clinical trials with clinically relevant outcomes. Since 1995, eight drugs used to treat hematologic malignancies have received accelerated approval.

Methods: Information available to the public under the Freedom of Information Act was surveyed for new drug applications and supplements for new uses approved by the Division of Oncology Products from January 1, 1995 to August 31, 2003. The package insert and corresponding published literature for the approval of each drug were reviewed for indication, time from drug submission to approval, surrogate endpoint, number of patients in trial, percent response and status of full approval.

Results: Of the eight drugs which received accelerated FDA approval, approval was granted within 6 months for three (these also received priority review), 1 was approved within 6–12 months; and 4 were approved after 1 year of FDA review. Approvals were based on the surrogate clinical outcome of response rate. Pivotal studies were often small, with < 100 patients (n=3) and 100–250 patients (n=4). The overall response rate was < 33% for four drugs. Only one of the approvals has been converted to full approval (imatinib). Post-marketing studies have identified three potentially fatal adverse drug reactions related to two drugs (gemtuzumab associated veno-occlusive diesease, gemtuzumab associated severe infusion reactions and imatinib associated upper GI bleeds).

Conclusion: To date, accelerated FDA approval for eight hematologic oncology drugs has been granted based on pivotal trials with small sample sizes and low response rates (with the exception of imatinib). Conversion to full approval has rarely occurred (N=1) and post-marketing identification of severe adverse drug reactions, in what were initially off-label settings, has occurred for two of the drugs. Modification of the accelerated approval process for hematologic oncology drugs should be considered.

FDA approval information for accelerated approval drugs used to treat hematologic malignancies.

Drug Name Indication Time to Approval (Months) N in Trial Overall Response Rate (%) Alemtuzumab CLL 16.5 93 33 Bortezomib Myeloma 3.7 188 14 Cytarabine liposomal Lymphomatous meningitis 5.5 17 41 Denileukin diftitox T-cell lymphoma 14 206 16 Gemtuzumab CD33 positive AML 7 142 26 Ibritumomab tiuxetan Low-grade NHL 15.6 157 62 Imatinib mesylate CML 3 1027 66 Tositumomab NHL 48 40 63
Membrane Type 1-Matrix Metalloproteinase Is Directly Involved in G-CSF Induced Human Hematopoietic Stem and Progenitor Cell Mobilization.
Authors: Avigdor Abraham , Schwartz Shimon , Goichberg Polina , Petit Isabelle , Hardan Izhar , Resnick Igor , Slavin Shimon , Nagler Arnon , Lapidot Tsvee
Published: April 2020

G-CSF induced hematopoietic stem/progenitor cell (HSC/HPC) mobilization is regulated by a complex interplay between extracellular matrix (ECM), cytokines/chemokines, adhesion molecules, and proteases, which affect stem cell retention and proliferation in the bone marrow microenvironment. Several studies suggest that membrane type 1-matrix metalloproteinase (MT1-MMP), expressed on the surface of various cell types, is a key enzyme for normal cell motility and tumor cell migration and invasion. We found that enriched human CD34+ cells express various surface MT1-MMP levels, depending on the cell source and G-CSF treatment. CD34+ cells obtained from BM of healthy donors treated with G-CSF were found to have the highest mean fluorescence intensity (>900 arbitrary units), while level of expression was lower in CD34+ cells derived from G-CSF mobilized peripheral blood from healthy donors (159±40), human steady-state (SS) BM (80±19), and human cord blood (41±4). Following 48 hr incubation of human SS-BM CD34+ cells with G-CSF, the expression of MT1-MMP increased 2-fold compared to untreated cells, whereas treatment with other cytokines, such as SCF, SDF-1 and IL-6, had only a minimal impact.

Immunocytochemical analysis of human cord blood CD34+ enriched cells plated on fibronectin or hyaluronate-coated cover slips revealed that in response to SDF-1, MT1-MMP changes its localization in the polarized and spreading cells, suggesting a role in the process of HPCs directional migration. Indeed, incubation with neutralizing antihuman MT1-MMP Ab, targeting its catalytic site, significantly reduced human progenitor migration towards a gradient of SDF-1 in transwells. Interestingly, low concentrations of tissue inhibitor of the metalloproteinase-2 (TIMP-2) enhanced SDF-1 induced transwell migration, while higher concentrations hampered this process. More importantly, 5 daily injections of G-CSF to NOD/SCID mice previously engrafted with human cells, up-regulated MT1-MMP expression on CD45+ and CD34+ human cells in the BM and peripheral blood of the mobilized chimeric mice compared to untreated, control chimeras. Treatment of chimera mice with antihuman MT1-MMP Ab on days 3–5 of G-CSF induced mobilization, significantly reduced the number of CD45+ and CD34+ human cells in peripheral blood. In summary, based on our data we suggest that following G-CSF treatment, increased levels of MT1-MMP on the surface of human progenitors in the BM facilitates their mobilization most probably due to pericellular ECM degradation and/or activation of other regulatory molecules, pointing to the essential role of MT1-MMP in G-CSF induced mobilization.