Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions

2010 ◽  
Vol 108 (5) ◽  
pp. 1241-1249 ◽  
Author(s):  
Suping Li ◽  
Quanwei Shi ◽  
Guanglei Liu ◽  
Weilin Zhang ◽  
Zhicheng Wang ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Surface Expression ◽  
Filamin A ◽  
Modeled Microgravity ◽  
Functional Changes ◽  
Cytoskeleton Reorganization ◽  
Intracellular Ca ◽  
Thrombotic Diseases ◽  
Platelet Functions

Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbα was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

Effects of microgravity and hypergravity on platelet functions

2009 ◽  
Vol 101 (05) ◽  
pp. 902-910 ◽  
Author(s):  
Yuedan Wang ◽  
Rong Yan ◽  
Quanwei Shi ◽  
Zhicheng Wang ◽  
Yanhong Yuan ◽  
...  
Keyword(s):  
Simulated Microgravity ◽  
Surface Expression ◽  
Gravity Change ◽  
Thrombotic Diseases ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
The Brain ◽  
Platelet Functions

SummaryMany serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to micro-gravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of –8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibα surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions.

The Mechanisms for Platelet Hyperactivity Induced by Hypergravity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5059-5059
Author(s):  
Suping Li ◽  
Quanwei Shi ◽  
Zhicheng Wang ◽  
Rong Yan ◽  
Changgeng Ruan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Actin Cytoskeleton ◽  
Platelet Activation ◽  
Surface Expression ◽  
Gravity Change ◽  
Human Beings ◽  
Cytoskeleton Reorganization ◽  
Significant Difference ◽  
Thrombotic Diseases ◽  
Platelet Functions

Abstract Abstract 5059 Many gravity change-related thrombotic and hemorrhagic diseases or fatalities have been reported to occur in human beings, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Because platelets, as anucleated blood cell, are the primary players in both thrombosis and hemostasis, function change of platelets in altered gravity conditions might offer some insights into the mechanisms leading to these gravity change-related diseases. We have demonstrated that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, which reveals a novel mechanism for gravity change-related hemorrhagic and thrombotic diseases. The mechanisms, however, remain to be further investigated. Here, firstly we investigated the effect of hypergravity (acceleration centrifugation) on platelet functions. Platelet rich plasma (PRP) or washed platelets were exposed to hypergravity at 8 G for different time points (2 minutes, 10 minutes, 15 minutes, 20 minutes), and then platelet aggregation was measured at each time point. No platelet aggregation was induced by 8 G alone, whereas ristocetin or collagen-induced platelet aggregation was time-dependently increased by hypergravity stimulus. The effect was maximal at 15 minutes after hypergravity treatment. Spreading assay shows that the number of platelet adhesion to immobilized fibrinogen and the mean platelet area spreading on von Willbrand factor (VWF) matrix were obviously increased by hypergravity. To further explore the effect of hypergravity on platelet activity state, the markers of platelet activations (integrin αaIIbβ3 activation, P-selectin surface expression) were assessed by flow cytometry. αaIIbβ3 was activated, in part, in 8 G-exposed platelets as detected by PAC-1 binding, but no significant difference was observed in P-selectin surface expression compared with 1 G controls. Next, we investigated the effects of hypergravity stimulus on actin distribution and intracellular Ca2+ ([Ca2+]i) in isolated human platelets. Hypergravity at 8 G induced actin cytoskeleton reorganization in platelets, and the release of Ca2+ from internal stores, but not Ca2+ influx via mechanosensitive Ca2+ channels in the plasma membrane. Pretreatment of platelets with intercellular Ca2+ chelator BAPTA/AM had no effect on hypergravity-induced changes in cytoskeleton, but significantly reduced platelet aggregation induced by hypergravity/ristocetin. In addition, using two different platelet activation inhibitors acetylsalicylic acid (aspirin) and tirofiban, which are widely used as antithrombotic drugs, we assessed the effects of the drugs on hypergravity-induced platelet activation in mice. Treatment of mice with aspirin or tirofiban significantly reduced hypergravity-induced P-selectin expression on platelet surface, prolonged tail bleeding time in vivo and reduced death rate in hypergravity-treated mice. Thus, our results indicate that hypergravity leads to human platelet hyperactivity, but fails to incur essential platelet activation events. Actin cytoskeleton reorganization and [Ca2+]i play important roles in regulation of platelet functions under hypergravity condition. The reorganization of actin cytoskeleton may be upstream or independent of [Ca2+]i, suggesting that hypergravity would affect various intercellular signals leading to platelet hyperactivity. In addition, antithrombotic agents such as aspirin and tirofiban have the therapeutic potential for hypergravity-induced thrombotic diseases. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circadian modulation of neurons and astrocytes controls synaptic plasticity in hippocampal area CA1

2019 ◽  
Author(s):  
John P. McCauley ◽  
Maurice A. Petroccione ◽  
Lianna Y. D’Brant ◽  
Gabrielle C. Todd ◽  
Nurat Affinnih ◽  
...  
Keyword(s):  
Cognitive Processing ◽  
Temporal Dynamics ◽  
Rhythmic Activity ◽  
Surface Expression ◽  
Receptor Activation ◽  
Cellular Mechanisms ◽  
Functional Changes ◽  
Area Ca1 ◽  
Hippocampal Area

SummaryMost animal species operate according to a 24-hour period set by the suprachiasmatic nucleus (SCN) of the hypothalamus. The rhythmic activity of the SCN is known to modulate hippocampal-dependent memory processes, but the molecular and cellular mechanisms that account for this effect remain largely unknown. Here, we show that there are cell-type specific structural and functional changes that occur with circadian rhythmicity in neurons and astrocytes in hippocampal area CA1. Pyramidal neurons change the surface expression of NMDA receptors, whereas astrocytes change their proximity to synapses. Together, these phenomena alter glutamate clearance, receptor activation and integration of temporally clustered excitatory synaptic inputs, ultimately shaping hippocampal-dependent learningin vivo. We identify corticosterone as a key contributor to changes in synaptic strength. These findings identify important mechanisms through which neurons and astrocytes modify the molecular composition and structure of the synaptic environment, contribute to the local storage of information in the hippocampus and alter the temporal dynamics of cognitive processing.

scholarly journals Pharmacological Inhibition of Soluble Epoxide Hydrolase Ameliorates Diet-Induced Metabolic Syndrome in Rats

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Abishek Iyer ◽  
Kathleen Kauter ◽  
Md. Ashraful Alam ◽  
Sung Hee Hwang ◽  
Christophe Morisseau ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Epoxide Hydrolase ◽  
Therapeutic Potential ◽  
Body Weight Gain ◽  
Oral Treatment ◽  
Soluble Epoxide Hydrolase ◽  
Endothelial Damage ◽  
Metabolic Dysfunction ◽  
Functional Changes

The signs of metabolic syndrome following chronic excessive macronutrient intake include body weight gain, excess visceral adipose deposition, hyperglycaemia, glucose and insulin intolerances, hypertension, dyslipidaemia, endothelial damage, cardiovascular hypertrophy, inflammation, ventricular contractile dysfunction, fibrosis, and fatty liver disease. Recent studies show increased activity of soluble epoxide hydrolase (sEH) during obesity and metabolic dysfunction. We have tested whether sEH inhibition has therapeutic potential in a rat model of diet-induced metabolic syndrome. In these high-carbohydrate, high-fat-fed rats, chronic oral treatment withtrans-4-[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid (t-AUCB), a potent sEH inhibitor, alleviated the signs of metabolic syndromein vivoincluding glucose, insulin, and lipid abnormalities, changes in pancreatic structure, increased systolic blood pressure, cardiovascular structural and functional abnormalities, and structural and functional changes in the liver. The present study describes the pharmacological responses to this selective sEH inhibitor in rats with the signs of diet-induced metabolic syndrome.

Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-α

2003 ◽  
Vol 284 (3) ◽  
pp. H960-H969 ◽  
Author(s):  
Andrzej M. Janczewski ◽  
Toshiaki Kadokami ◽  
Bonnie Lemster ◽  
Carole S. Frye ◽  
Charles F. McTiernan ◽  
...  
Keyword(s):  
Contractile Function ◽  
Left Ventricular ◽  
Mrna Levels ◽  
Functional Changes ◽  
Tnf Α ◽  
Intracellular Ca ◽  
And Gender ◽  
Factor Α ◽  
And Function

Transgenic (TG) TNF1.6 mice, which cardiac specifically overexpress tumor necrosis factor-α (TNF-α), exhibit heart failure (HF) and increased mortality, which is markedly higher in young (<20 wk) males (TG-M) than females (TG-F). HF in this model may be partly caused by remodeling of the extracellular matrix and/or structure/function alterations at the single myocyte level. We studied left ventricular (LV) structure and function using echocardiography and LV myocyte morphometry, contractile function, and intracellular Ca2+ (Ca[Formula: see text]) handling using cell edge detection and fura 2 fluorescence, respectively, in 12-wk-old TG-M and TG-F mice and their wild-type (WT) littermates. TG-F mice showed LV hypertrophy without dilatation and only a small reduction of basal fractional shortening (FS) and response to isoproterenol (Iso). TG-M mice showed a large LV dilatation, higher mRNA levels of β-myosin heavy chain and atrial natriuretic factor versus TG-F mice, reduced FS relative to both WT and TG-F mice, and minimal response to Iso. TG-F and TG-M myocytes were similarly elongated (by ≈20%). The amplitude of Ca[Formula: see text] transients and contractions and the response to Iso were comparable in WT and TG-F myocytes, whereas the time to 50% decline (TD50%) of the Ca[Formula: see text]transient, an index of the rate of sarcoplasmic reticulum Ca2+ uptake, was prolonged in TG-F myocytes. In TG-M myocytes, the amplitudes of Ca[Formula: see text] transients and contractions were reduced, TD50% of the Ca[Formula: see text] transient was prolonged, and the inotropic effect of Iso on Ca[Formula: see text] transients was reduced approximately twofold versus WT myocytes. Protein expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2 and phospholamban was unaltered in TG versus WT hearts, suggesting functional origins of impaired Ca2+ handling in the former. These results indicate that cardiac-specific overexpression of TNF-α induces myocyte hypertrophy and gender-dependent alterations in Ca[Formula: see text] handling and contractile function, which may at least partly account for changes in LV geometry and in vivo cardiac function in this model.

scholarly journals Combination Therapy with STAT3 Inhibitor Enhances SERCA2a-Induced BMPR2 Expression and Inhibits Pulmonary Arterial Hypertension

2021 ◽  
Vol 22 (17) ◽  
pp. 9105
Author(s):  
Malik Bisserier ◽  
Michael G. Katz ◽  
Carlos Bueno-Beti ◽  
Agnieszka Brojakowska ◽  
Shihong Zhang ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Therapeutic Potential ◽  
Combined Therapy ◽  
Pulmonary Arteries ◽  
Combination Therapies ◽  
Functional Changes ◽  
Beneficial Effects ◽  
Pulmonary Arterial

Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by the progressive obstruction of the distal pulmonary arteries (PA). Structural and functional alteration of pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) contributes to PA wall remodeling and vascular resistance, which may lead to maladaptive right ventricular (RV) failure and, ultimately, death. Here, we found that decreased expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) in the lung samples of PAH patients was associated with the down-regulation of bone morphogenetic protein receptor type 2 (BMPR2) and the activation of signal transducer and activator of transcription 3 (STAT3). Our results showed that the antiproliferative properties of SERCA2a are mediated through the STAT3/BMPR2 pathway. At the molecular level, transcriptome analysis of PASMCs co-overexpressing SERCA2a and BMPR2 identified STAT3 amongst the most highly regulated transcription factors. Using a specific siRNA and a potent pharmacological STAT3 inhibitor (STAT3i, HJC0152), we found that SERCA2a potentiated BMPR2 expression by repressing STAT3 activity in PASMCs and PAECs. In vivo, we used a validated and efficient model of severe PAH induced by unilateral left pneumonectomy combined with monocrotaline (PNT/MCT) to further evaluate the therapeutic potential of single and combination therapies using adeno-associated virus (AAV) technology and a STAT3i. We found that intratracheal delivery of AAV1 encoding SERCA2 or BMPR2 alone or STAT3i was sufficient to reduce the mean PA pressure and vascular remodeling while improving RV systolic pressures, RV ejection fraction, and cardiac remodeling. Interestingly, we found that combined therapy of AAV1.hSERCA2a with AAV1.hBMPR2 or STAT3i enhanced the beneficial effects of SERCA2a. Finally, we used cardiac magnetic resonance imaging to measure RV function and found that therapies using AAV1.hSERCA2a alone or combined with STAT3i significantly inhibited RV structural and functional changes in PNT/MCT-induced PAH. In conclusion, our study demonstrated that combination therapies using SERCA2a gene transfer with a STAT3 inhibitor could represent a new promising therapeutic alternative to inhibit PAH and to restore BMPR2 expression by limiting STAT3 activity.

EXTH-28. NLGN4X TCR TRANSGENIC T CELLS TARGETING EXPERIMENTAL GLIOMAS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi169-vi169
Author(s):  
Christopher Krämer ◽  
Michael Kilian ◽  
Rainer Will ◽  
Khwab Sanghvi ◽  
Edward Green ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Size ◽  
Therapeutic Potential ◽  
Phase 1 ◽  
Glioma Cells ◽  
Surface Expression ◽  
Target Antigen ◽  
T Cell Therapy

Abstract BACKGROUND The application of personalized vaccines has shown to be effective in patients with newly diagnosed glioblastoma in phase 1 clinical trials. Responses of CD8 T cells directed against the glioma-associated antigen Neuroligin-4, X-linked (NLGN4X) were reported in multipeptide vaccine trials in patients with glioblastoma. Here, we characterized the functional status of NLGN4X TCR transgenic T cells in vitro and assessed their therapeutic capacity in vivo. METHODS TCR encoding sequences were delivered by lentiviral transduction to activated T cells from healthy donors. After confirmation of TCR surface expression T cells were used for a functional in vitro characterization. For in vivo assessment of NLGN4X-specific TCR transgenic T cells, NLGN4X-expressing U87 glioma cells were injected into the flank of NSG MHCI/MHC II knockout mice, which do not develop graft versus host disease. TCR transgenic T cells were injected intravenously on day 11 and day 18 and tumor size was monitored. RESULTS TCR transgenic T cells depicted stable surface expression for at least 11 days in vitro after transduction. Thereby, murine TCR beta constant region positive T cells featured a polyfunctional phenotype demonstrated by a significant increase of Interferon-γ and TNF-α and remained reactive to the NLGN4X epitope for at least 7 days. Additionally, NLGN4X TCR transgenic T cells showed significantly increased antigen-specific production of the cytolytic protein granzyme B and elevated levels of perforin. In a novel xenograft mouse model NLGN4X TCR transgenic T cells slowed the tumor growth compared to the initial tumor size until day 25 after tumor inoculation. DISCUSSION We demonstrate that NLGN4X TCR transgenic T cells specifically and consistently recognize their corresponding immunogenic sequence and target antigen-overexpressing glioma cells. We present first evidence of in vivo reactivity, while further experiments are required to assess the full therapeutic potential of NLGN4X-TCR-transgenic T cell therapy for glioma patients.

scholarly journals Tetrandrine alleviates podocyte injury via calcium-dependent calpain-1 signaling blockade

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yin Ding ◽  
Xuanli Tang ◽  
Yuhui Wang ◽  
Dongrong Yu ◽  
Caifeng Zhu ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Therapeutic Potential ◽  
Kidney Diseases ◽  
In Vivo Studies ◽  
Chinese Drug ◽  
Calpain Activity ◽  
Podocyte Injury ◽  
Intracellular Ca

Abstract Background Podocytes have become a crucial target for interventions in proteinuric kidney diseases. Many studies have reported that overexpression of transient receptor potential cation channel protein 6 (TRPC6) in podocyte injury upregulates intracellular Ca2+ influx and stimulates Ca2+-dependent protease calpain-1 signaling. The traditional Chinese drug, tetrandrine, a nonselective Ca2+ channel blocker, has long been used to treat chronic kidney disease. This research aimed to explore the possible mechanisms underlying the anti-proteinuric properties of tetrandrine. Methods We investigated the involvement of tetrandrine in Ca2+ dependent calpain-1 signaling in mouse podocytes and adriamycin-induced nephropathy rats. Cyclosporine A (CsA) and U73122 were used as positive controls. Cell viability, cytotoxicity, Ca2+ concentration, calpain activity, and mRNA and protein expression levels of calpain-1 signaling pathways were examined. The clinical and pathological changes were measured. Results Tetrandrine decreased intracellular Ca2+ influx in cultured TRPC6-overexpressing podocytes. In both in vitro and in vivo studies, the administration of tetrandrine downregulated calpain activity and the expression of calpain-1 and restored the expression of downstream Talin-1 and nephrin. Compared to CsA, tetrandrine treatment exhibited superior inhibitory effects on calpain activity and calpain-1 expression. Conclusions Tetrandrine has therapeutic potential in podocyte damage by blocking Ca2+-dependent activation of the calpain-1 signaling pathway. Tetrandrine reduced proteinuria, improved renal function, and alleviate renal pathological damage.

Hypoxia induces hypersensitivity and hyperreactivity to thromboxane receptor agonist in neonatal pulmonary arterial myocytes

2006 ◽  
Vol 290 (2) ◽  
pp. L375-L384 ◽  
Author(s):  
M. Hinton ◽  
L. Mellow ◽  
A. J. Halayko ◽  
A. Gutsol ◽  
S. Dakshinamurti
Keyword(s):  
Pulmonary Arteries ◽  
Hypoxic Exposure ◽  
Functional Changes ◽  
Newborn Piglets ◽  
Pathway Activation ◽  
Intracellular Ca ◽  
Pulmonary Arterial ◽  
Altered Gene

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd–6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (FiO2 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.

Preclinical In Vitro and In Vivo Evidence Support a Therapeutic Role for the CD70 Directed Monoclonal Antibody (SGN-70) in Waldenström’s Macroglobulinemia (WM).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2490-2490 ◽  
Author(s):  
Evdoxia Hatjiharissi ◽  
Allen W. Ho ◽  
Lian Xu ◽  
Kelly E. O’Connor ◽  
Zachary R. Hunter ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Disease Progression ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Induction Of Apoptosis ◽  
Direct Induction

Abstract Introduction: WM is a B-cell disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells (LPC), along with excess mast cells (MC) which support the growth and survival of BM LPC through multiple TNF-family ligands including CD40L, APRIL and BLyS/BAFF. Importantly, BM LPC stimulate cell surface expression of TNF-family ligands through release of sCD27 which induces CD70 on MC. We therefore have sought the development of agents which could target CD27-CD70 interactions. As such, we examined the therapeutic potential of directly targeting CD70 using the fully humanized monoclonal antibody SGN-70 (Seattle Genetics, Inc., Bothell WA). Methods-Results: As part of these studies, we used flow cytometric analysis to evaluate the expression of CD70 on primary WM patient BM LPC and MC, as well as 2 WM cell lines (BCWM.1 and WM-WSU). These studies demonstrated cell surface expression of CD70 on BM LPC and MC from 20/26 (77%) and 10/11 (90%) WM patients, respectively. We next assessed the ability of the SGN-70 antibody to eradicate primary WM LPC (n=5) and WM cell lines by assessing for direct induction of apoptosis, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) as well as induction of TNF family ligands on primary WM MC and the LAD2 MC line. Following incubation of WM LPC with SGN-70 (0.01–20 μg/ml), no direct induction of apoptosis or CDC activity was observed. However, SGN-70 mediated significant dose-dependent ADCC against WM LPC and MC at concentrations of 0.1–20 ug/ml. Importantly, SGN-70 blocked sCD27-induced expression of CD40L and APRIL on primary WM MC and LAD2 MC. To further evaluate the therapeutic potential of SGN-70 in an in vivo model, SCID-hu mice bearing BCWM.1 WM cells were treated with SGN-70 (1 mg/kg, i.p., qOD) Serum human IgM and sCD27 levels were measured by ELISA to monitor for tumor engraftment and disease progression. SGN-70 initiated 6 weeks following tumor engraftment blocked tumor growth in 12/12 treated mice, whereas all 5 untreated mice demonstrated disease progression. The results of these studies provide the framework for clinical trials to examine the therapeutic potential of the SGN-70 monoclonal antibody in WM.

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