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Key Words. Marrow stromal cells ? Calcitonin gene–related peptide ? Adenovirus ? Gene expression ? Differentiation ? Gene therapy @. s, a" \# c0 M* b: H/ i
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Correspondence: Philip J. Kadowitz, Ph.D., Department of Pharmacology, SL83, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA. Telephone: 504-584-2637; Fax: 504-588-5283; e-mail: pkad-owi@tulane.edu
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! U& |1 H3 ?* ?7 |ABSTRACT6 t- D* {: e/ R* k
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Calcitonin gene–related peptide (CGRP), a 37-amino-acid neuropeptide derived from alternative splicing of RNA from the calcitonin gene , is an important molecule with multiple biological effects, including a potent vasodilator activity . CGRP is extensively localized in the perivascular or periadventitia nerves throughout the body, and its release from nerve endings is associated with the dilation of blood vessels . CGRP exerts its vasodilator effect through interaction with CGRP receptors that are present on both endothelial cells and vascular smooth muscle cells via endothelium-dependent or endothelium-independent mechanisms, depending on vessel type and species . CGRP deficiency has been suggested to be involved in cardiovascular diseases such as pulmonary hypertension , erectile dysfunction , and cerebral vasospasm after subarachnoid hemorrhage (SAH) . Therefore, the enhancement of local CGRP delivery is a promising approach for the treatment of these cardiovascular disorders.6 f; Y9 X& w4 b: s
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Intravenous, intracavernosal, or intrathecal administration of exogenous CGRP has been shown to have a beneficial effect in pulmonary hypertension , erectile dysfunction , and cerebral vasospasm after SAH . However, the half-life of CGRP in plasma is only 10 minutes, and chronic infusion of CGRP to patients is not feasible . An alternative approach such as a gene therapy strategy should be developed to deliver CGRP over long periods of time. Although direct in vivo injection of adenovirus containing prepro-calcitonin gene-related peptide (prepro-CGRP) has been shown to be effective for the treatment of pulmonary hypertension , erectile dysfunction , and cerebral vasospasm , major disadvantages, such as inflammation and random transgene expression in almost all cell types, would limit the clinical application of this strategy in human diseases, and an improved therapy should be developed .2 p( w" v- x' M+ i
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Gene-engineered ex vivo–expanded adult stem cells are attractive for use in gene therapy, because some disadvantages associated with direct in vivo delivery of viral vectors, nonviral vectors, or gene-modified ex vivo–expanded differentiated cells are avoided . Marrow stromal cells (MSCs) are nonhematopoietic adult stem cells from bone marrow, are relatively easy to isolate and expand ex vivo, and have multipotential differentiation capability . These cells can be used as a vehicle for gene delivery in an adult stem cell–based gene therapy strategy . For therapeutic gene transfer, adenoviral vectors have major advantages over other vectors such as high-level transgene expression, a broad host range, the ability to infect quiescent cells, and ease of preparation of high titer viral stock . Therefore, the aim of this study was to determine whether ex vivo–expanded rat MSCs (rMSCs) can be transduced with adenovirus containing CGRP and to ascertain whether the cells still retain their multipotentiality after adenoviral-mediated gene transfer.
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In this study, data are presented showing that adenoviral vectors can be used to gene engineer ex vivo–expanded rMSCs and that high-level functional CGRP secretion by adult stem cells can be achieved, pointing out the potential clinical application of this novel method for adult stem cell–based cell and gene therapy.
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MATERIALS AND METHODS
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Adenoviral Transduction of ExVivo–Expanded rMSCs
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The effectiveness of adenoviral-mediated gene transfer into ex vivo–expanded rMSCs was examined using rMSCs transduced with AdntlacZ at MOI 50, 100, 150, 200, and 300. After 48 hours, the expression of nuclear-targeted ?-galactosidase was assessed by X-gal staining, and, as shown in Figure 1, transduction efficiency was observed to be >40% at MOI 50 and >90% at MOI 300. Cell viability was determined to be >95% at all MOIs studied.2 B: j6 Q' ?7 \, ~8 @
0 d3 w2 n. v; Z# DFigure 1. (A–D): Photomicrographs showing ?-galactosidase–positive blue nuclei in AdntlacZ-transduced rMSCs. (A): Control rMSCs. (B): rMSCs transduced at MOI 50. (C): rMSCs transduced at MOI 150. (D): rMSCs transduced at MOI 300. Magnification, x250. (E): Efficiency of adenoviral-mediated ntlacZ gene transfer into rMSCs. rMSCs were transduced with AdntlacZ at the indicated MOI for 48 hours. The cells were X-gal stained for the nuclear-targeted ?-galactosidase activity, and the transduction efficiency was determined. Each value represents mean ± standard error of the mean (n = 3). Abbreviations: MOI, multiplicity of infection; rMSC, rat marrow stromal cell., b/ Z' u" @' i+ X) c) I4 F A
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Differentiation of AdntlacZ-Transduced rMSCs In Vitro3 r v" E1 x1 I
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To ascertain whether rMSCs retain multipotential differentiation capability after adenoviral-mediated ntlacZ gene transfer, rMSCs were first transduced with AdntlacZ at MOI 300 for 48 hours and then incubated in the presence of differentiation media. As seen in Figures 2A–2D, after exposure to osteogenic medium, mineral deposition was observed. Moreover, after exposure to adipogenic medium, the cells exhibited lipid droplets. Therefore, the osteogenic potential and adipogenic potential of AdntlacZ-transduced rMSCs were retained.
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9 l J" v2 J- G( H" S, i! A* `Figure 2. Photomicrographs showing that AdntlacZ-transduced rMSCs retain multipotentiality in culture and the differentiated cells express ?-galactosidase. rMSCs were transduced with AdntlacZ at a multiplicity of infection of 300 for 2 days. The cells were then induced to differentiate into osteoblast or adipocyte lineage for 21 days and stained with Alizarin red S for mineral deposition, Oil red O for lipid droplet formation, or X-gal for the nuclear-targeted ?-galactosidase activity. (A):AdntlacZ-transduced rMSCs stained with Alizarin red S. (B):AdntlacZ-transduced rMSCs first treated with osteogenic medium for 21 days and then stained with Alizarin red S. (C):AdntlacZ-transduced rMSCs stained with Oil red O. (D):AdntlacZ-transduced rMSCs first treated with adipogenic medium for 21 days and then stained with Oil red O. (E): AdntlacZ-transduced rMSCs first treated with osteogenic medium for 21 days and then X-gal stained for ?-galactosidase activity. (F): AdntlacZ-transduced rMSCs first treated with adipogenic medium for 21 days and then X-gal stained for ?-galactosidase activity. Arrows indicate ntlacZ blue differentiated osteoblasts, and arrowheads indicate ntlacZ blue differentiated adipocytes. Magnification, x 500. Abbreviation: rMSC, rat marrow stromal cell.! b' ^8 D z( Q5 p' t* U; {
( Q* ^. k" [1 eThe percentage of differentiated cells in both control rMSCs and AdntlacZ-transduced rMSCs was counted. The percentage of differentiated osteoblasts in control rMSCs and AdntlacZ-transduced rMSCs was 59 ± 2% and 57 ± 2% (mean ± SEM, n = 3; p > .05, t-test), respectively. The percentage of differentiated adipocytes in control rMSCs and AdntlacZ-transduced rMSCs was 37 ± 1% and 39 ± 1% (mean ± SEM, n = 3; p > .05, t-test), respectively. Therefore, there is no significant difference between the differentiation potential of control rMSCs and AdntlacZ-transduced rMSCs either in osteogenic medium or adipogenic medium.
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The expression of ntlacZ in these differentiated cells was also assessed. The percentage of ?-galactosidase–positive cells at day 21 in osteogenic medium–treated cells was 6 ± 1% (mean ± SEM, n = 3). The percentage of ?-galactosidase–positive cells at day 21 in adipogenic medium–treated cells was 29 ± 1% (mean ± SEM, n = 3). As seen in Figures 2E and 2F, the differentiated osteoblasts and adipocytes were still positive for ?-galactosidase.0 k% ^+ Y* F3 E# D7 b
0 B$ o/ t6 u9 o( r# S7 Y! f; wPersistence of Adenoviral-Mediated ntlacZ Transgene Expression In Vitro8 W8 i% ]5 q, A" W+ u
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To study the persistence of ntlacZ transgene expression in vitro, rMSCs were transduced with AdntlacZ at MOI 300 for 48 hours. The virus-containing culture medium was then removed. The cells were washed with PBS three times and additionally incubated in low-serum medium (-MEM with 2% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, and 2 mM L-glutamine) for 21 days. The low-serum medium was changed every 3 days, and ntlacZ transgene expression was assessed at various time intervals after transduction. As shown in Figure 3, the number of cells expressing ?-galactosidase was >90% at day 2 and >50% at day 21.! B' Z8 ] U. y4 |5 ^+ N
# v# m4 G: L/ f" q! M& F7 z# A& QFigure 3. (A–D): Photomicrographs showing AdntlacZ-transduced rMSCs at various time intervals after transduction. (A): Control at day 2. (B): MOI 300 at day 2. (C): Control at day 21. (D): MOI 300 at day 21. Magnification, x 250. (E): Effect of time on ntlacZ transgene expression in AdntlacZ-transduced rMSCs. rMSCs were transduced with AdntlacZ at MOI 300 for 2 days. The virus-containing culture medium was removed, and the cells were washed with phosphate-buffered saline three times and further incubated in low-serum medium. The low-serum medium was changed every 3 days until day 21. The cells were X-gal stained for ntlacZ expression at days 2, 4, 7, 14, and 21, and the percentage of cells expressing ntlacZ was quantified. Each value represents mean ± standard error of the mean (n = 3). *p > .05 versus day 2. **p > .05 versus day 2 or day 4. ***p .05 versus day 14 (analysis of variance). Abbreviations: MOI, multiplicity of infection; rMSC, rat marrow stromal cell.
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5 H6 X7 m! S5 R" ZWestern Blot Analysis for CGRP
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8 {$ R9 a$ B: k7 ^To determine whether rMSCs can be gene engineered with CGRP, rMSCs were transduced with Adprepro-CGRP at MOI 300. CGRP expression was assessed in the whole-cell lysate from control rMSCs, AdntlacZ-transduced rMSCs, and Adprepro-CGRP–transduced rMSCs using Western blot analysis. No constitutive CGRP expression was detected in either control rMSCs or AdntlacZ-transduced rMSCs. Expression of CGRP was detected in Adprepro-CGRP–transduced rMSCs (Fig. 4A). The culture supernatants from control rMSCs, AdntlacZ-transduced rMSCs, and Adprepro-CGRP–transduced rMSCs were also analyzed for CGRP secretion. As seen in Figure 4A, high-level CGRP secretion in culture supernatant from Adprepro-CGRP–transduced rMSCs was detected, whereas there was no constitutive CGRP secretion from either control rMSCs or AdntlacZ-transduced rMSCs.$ S4 E2 F2 w) }9 C) B4 K% L8 _
2 `) L! ~; \) S& a2 e% yFigure 4. (A): Western blot analysis for the expression and secretion of CGRP by Adprepro-CGRP–transduced rMSCs. rMSCs were transduced with Adprepro-CGRP (MOI 300) or AdntlacZ (MOI 300) for 48 hours, and the whole-cell lysates were used for the analysis of CGRP transgene expression. Lane 1: control rMSCs; lane 2: AdntlacZ-transduced rMSCs; lane 3: Adprepro-CGRP–transduced rMSCs. Furthermore, after being transduced with either Adprepro-CGRP (MOI 300) or AdntlacZ (MOI 300) for 2 days, the cells were washed with PBS three times and incubated in fresh culture medium for 48 hours. The culture supernatant was then collected and analyzed for CGRP secretion by Western blot analysis. Lane 4: supernatant from control rMSCs; lane 5: supernatant from AdntlacZ-transduced rMSCs; lane 6: supernatant from Adprepro-CGRP–transduced rMSCs. The analysis for ?-tubulin was carried out to ensure that sample loading was similar in all lanes. (B): EIA analysis for CGRP secretion by Adprepro-CGRP–transduced rMSCs. rMSCs were transduced with Adprepro-CGRP (MOI 50, 150, and 300) or AdntlacZ (MOI 300) for 2 days, the virus-containing culture medium was removed, and the cells were washed with PBS three times and further incubated in fresh culture medium for 48 hours. The culture supernatant was then collected and analyzed for CGRP secretion by EIA. Each value represents mean ± standard error of the mean (n = 3). Abbreviations: CGRP, calcitonin gene-related peptide; EIA, enzyme immunoassay; MOI, multiplicity of infection; PBS, phosphate-buffered saline; rMSC, rat marrow stromal cell.. U5 j" j2 J+ M' \- Z1 e; J
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Enzyme Immunoassay for CGRP o7 a) w5 ~9 r5 L9 ^
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CGRP concentration in the culture supernatant from control rMSCs, AdntlacZ-transduced rMSCs, and Adprepro-CGRP–transduced rMSCs was measured using the EIA. As seen in Figure 4B, there was no constitutive secretion of CGRP to culture medium by either control rMSCs or AdntlacZ-transduced rMSCs. However, as much as 9.5 ± 0.4 pmol CGRP/1 x 106 cells/48 hours (mean ± SEM, n = 3) was secreted to culture medium by Adprepro-CGRP–transduced rMSCs at MOI 300, and the secretion of CGRP is dose dependent.
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* X* d- F* I9 \) kCGRP Secreted by Adprepro-CGRP-Transduced rMSCs Is Biologically Active
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1 A' }: x& D. L' nTo determine whether CGRP secreted by Adprepro-CGRP-transduced rMSCs is biologically active, rat PASMCs were treated with supernatant from control rMSCs, AdntlacZ-transduced rMSCs, or Adprepro-CGRP–transduced rMSCs, and intracellular cAMP levels were measured. As shown in Figure 5, culture supernatant from Adprepro-CGRP–transduced rMSCs increased intracellular cAMP levels in PASMCs, whereas culture supernatant from control rMSCs or AdntlacZ-transduced rMSCs did not increase intracellular cAMP levels in PASMCs. Therefore, CGRP secreted by Adprepro-CGRP–transduced rMSCs is biologically active .
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5 f' q# z6 Y% w' V) S; @; C6 fFigure 5. Effect of culture supernatant from Adprepro-CGRP–transduced rMSCs on intracellular cAMP levels of rat PASMCs. rMSCs were transduced with Adprepro-CGRP (MOI 300) or AdntlacZ (MOI 300) for 2 days. The virus-containing culture medium was removed, and the cells were washed with phosphate-buffered saline three times. The cells were further incubated in fresh culture medium for 48 hours, and the culture supernatant was collected. Rat PASMCs were then treated with culture supernatant from control rMSCs, AdntlacZ-transduced rMSCs, orAdprepro-CGRP–transduced rMSCs, and intracellular cAMP levels of the cells were measured. Each value represents mean ± standard error of the mean (n = 3). *p > .05 versus supernatant from control rMSCs. **p 3 M% m5 E4 [" M
2 ^6 H7 T1 E6 Y8 I9 G( ?* Y: p/ PPersistence of Adenoviral-Mediated CGRP Transgene Expression In Vitro' N3 T3 x& i; L" d$ Z! y
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To study the persistence of CGRP transgene expression in vitro, rMSCs were transduced with Adprepro-CGRP at MOI 300 for 48 hours. The virus-containing culture medium was removed, and the cells were washed with PBS three times. The cells were cultured in fresh culture medium for 48 hours, and the culture supernatant was collected. The cells were further incubated in low-serum medium (-MEM with 2% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, and 2 mM L-glutamine), and the low-serum medium was changed every 2–3 days until day 21. The culture supernatant at various time intervals after transduction was collected and analyzed for CGRP secretion by EIA. As shown in Figure 6, Adprepro-CGRP–transduced rMSCs (MOI 300) secreted 9.5 ± 0.4 and 0.02 ± 0.02 pmol CGRP/1 x 106 cells/48 hours (mean ± SEM, n = 3) to culture medium at days 2 and 21, respectively.
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Y3 i2 c% D* J. J0 x1 fFigure 6. Effect of time on CGRP secretion by Adprepro-CGRP–transduced rMSCs in culture. rMSCs were transduced with Adprepro-CGRP at a multiplicity of infection of 300 for 2 days. The virus-containing culture medium was removed, and the cells were washed with phosphate-buffered saline three times. The cells were cultured in fresh culture medium for 48 hours, and the culture supernatant was collected. The cells were further incubated in low-serum medium, and the low-serum medium was changed every 2–3 days until day 21. The culture supernatant at various time intervals was collected and analyzed for CGRP secretion by enzyme immunoassay. CGRP secretion by Adprepro-CGRP–transduced rMSCs in culture at days 2, 4, 7, 14, and 21 was then determined. Each value represents mean ± standard error of the mean (n = 3). *p .05 versus day 7. ****p .05 versus day 7 or day 14 (analysis of variance). Abbreviations: CGRP, calcitonin gene-related peptide; rMSC, rat marrow stromal cell.
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* ]5 r) B, m4 R1 P& `Differentiation of Adprepro-CGRP–Transduced rMSCs In Vitro
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9 L, q) I+ a5 ~1 qTo ascertain whether rMSCs retain multipotentiality after adenoviral-mediated CGRP gene transfer, rMSCs were transduced with Adprepro-CGRP at MOI 300 for 48 hours and additionally cultured in the presence of differentiation medium for osteoblast or adipocyte lineage. After exposure to osteogenic medium for 21 days, the cells were stained with Alizarin red S and mineral deposition was observed. After exposure to adipogenic medium for 21 days, the cells were stained with Oil red O and lipid droplet formation was confirmed. Thus, both the osteogenic potential and adipogenic potential of Adprepro-CGRP–transduced rMSCs were retained.
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% V% j- V0 e6 JThe percentage of differentiated cells in both control rMSCs and Adprepro-CGRP–transduced rMSCs was also counted. The percentage of differentiated osteoblasts in control rMSCs and Adprepro-CGRP–transduced rMSCs was 66 ± 5% and 69 ± 9% (mean ± SEM, n = 3; p > .05, t-test), respectively. The percentage of differentiated adipocytes in control rMSCs and Adprepro-CGRP–transduced rMSCs was 22 ± 2% and 22 ± 4% (mean ± SEM; n = 3; p > .05, t-test), respectively. These data indicate that there is no significant difference between the differentiation potential of control rMSCs and Adprepro-CGRP–transduced rMSCs in either osteogenic or adipogenic medium.
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The secretion of CGRP by the differentiated Adprepro-CGRP–transduced rMSCs was also assessed. The cells treated with osteogenic medium for 21 days secreted 0.02 ± 0.01 pmol CGRP/1 x 106 cells/48 hours (mean ± SEM, n = 3) in culture. The cells treated with adipogenic medium for 21 days secreted 1.47 ± 0.03 pmol CGRP/1 x 106 cells/48 hours (mean ± SEM, n = 3) in culture./ L: ~: p2 Z# s( _4 K* y ~1 y
; P7 n. ]5 l% w( J2 \2 }' m5 }Proliferation and Viability of rMSCs during and afterAdenoviral Infection
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; b A0 }! \: g, D/ u+ A8 G& A+ JTo determine whether adenoviral transduction can alter the proliferation and viability of rMSCs during adenoviral infection, rMSCs were transduced with AdntlacZ or Adprepro-CGRP at MOI 300 for 48 hours. The proliferation and viability of transduced rMSCs were then compared with control rMSCs. As seen in Figure 7A, there is no significant difference among control rMSCs, AdntlacZ-transduced rMSCs, and Adprepro-CGRP–transduced rMSCs for either cell proliferation or viability. To ascertain whether adenoviral transduction alters proliferation and viability of rMSCs after adenoviral infection, rMSCs were transduced with AdntlacZ or Adprepro-CGRP at MOI 300 for 48 hours. The virus-containing culture medium was then removed and the cells were washed with PBS three times and additionally incubated in fresh culture medium for 48 hours. The proliferation and viability of transduced rMSCs were then compared with control rMSCs. As shown in Figure 7B, there is no significant difference among control rMSCs, AdntlacZ-transduced rMSCs, and Adprepro-CGRP–transduced rMSCs for either cell proliferation or viability. Therefore, the proliferation and viability of rMSCs were not altered by either AdntlacZ or Adprepro-CGRP transduction at MOI 300.3 y$ |) J' I- K
4 L2 _" `. _4 Y; {Figure 7. Lack of influence of AdntlacZ or Adprepro-CGRP transduction at MOI 300 on the proliferation and viability of rMSCs. (A): AdntlacZ or Adprepro-CGRP transduction at MOI 300 does not alter the proliferation and viability of rMSCs during adenoviral infection. rMSCs were transduced with AdntlacZ or Adprepro-CGRP at MOI 300 for 48 hours. Cell proliferation was assessed by counting the cells and expressing the data as percentage of control, and cell viability was determined by trypan blue exclusion method. (B):AdntlacZ or Adprepro-CGRP transduction at MOI 300 does not alter the proliferation and viability of rMSCs after adenoviral infection. rMSCs were transduced with AdntlacZ or Adprepro-CGRP at MOI 300 for 48 hours. The virus-containing culture medium was removed, and the cells were washed three times with phosphate-buffered saline and further incubated in fresh culture medium for 2 days. Cell proliferation was assessed by counting the cells and expressing the data as percentage of control, and cell viability was determined by trypan blue exclusion method. For A and B, each value represents mean ± standard error of the mean (n = 3) and p > .05 by analysis of variance. Abbreviations: CGRP, calcitonin gene-related peptide; MOI, multiplicity of infection; rMSC, rat marrow stromal cell." }+ f5 M6 F; I' Z
/ y( n# W, q) j' |$ pDISCUSSION
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% m: o) n9 }/ a- m% ?& fThis work was supported by NIH NHLBI grant HL-62000 and NCI grant CA-65600.
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