Introduction: Preeclampsia is a hypertensive disorder of pregnancy which is associated with subst... more Introduction: Preeclampsia is a hypertensive disorder of pregnancy which is associated with substantial morbidity for the mother and fetus. Reduced nitric oxide bioavailability and oxidative stress are key in the maternal and fetal pathophysiology of preeclampsia. Method: In this study, we assessed the potential therapeutic effect and mechanisms of action of a novel dual-function nitric oxide donor/redox modulator, AKT-1005, in an animal model of Ad.sFlt1-induced hypertension, proteinuria, and glomerular endotheliosis. Ad.sFlt1-overexpressing CD1 mice were treated via minipump with AKT-1005 (20 mg/kg) or vehicle (n=15 per group). Results: Treatment with AKT-1005 for 7 days significantly reduced sFlt1-induced hypertension and endotheliosis of the kidney glomeruli. Kidney function was also assessed by cystatin C ELISA, which showed significant reduction in AKT-1005 treated mice compared to controls. AKT-1005 treatment increased NO bioavailabilty as assessed by Griess method. The incre...
Suppl. Table 4. Log2 Fold Changes (FC) of some genes differentially expressed in PTC wih BRAFV600... more Suppl. Table 4. Log2 Fold Changes (FC) of some genes differentially expressed in PTC wih BRAFV600E and hTERT mutation vs BRAFWT-PTC
Suppl. Table 2. Log2 Fold Changes (FC) of some genes differentially expressed in BRAFV600E-PTC vs... more Suppl. Table 2. Log2 Fold Changes (FC) of some genes differentially expressed in BRAFV600E-PTC vs BRAFWT-PTC
Suppl. Figure 1 Drug dose-effect analysis in BRAFWT/V600E-KTC1 cells Suppl.Figure 2 Dose-effect a... more Suppl. Figure 1 Drug dose-effect analysis in BRAFWT/V600E-KTC1 cells Suppl.Figure 2 Dose-effect analysis of vemurafenib and sorafenib on pericyte viability. Suppl. Figure 3 BRAFWT/WT-TPC1 cells viability assay. Suppl. Figure 4 ELISA analysis of cytokines secretion in pericytes, and PTC cells harboring the heterozygous BRAFWT/V600E mutation or with BRAFWT/WT. Suppl. Figure 5 Effects of pericyte secretome in BRAFWT/WT-TPC1 cells. Suppl. Figure 6 Analysis of secreted TSP-1 levels in PTC cells harboring the heterozygous BRAFWT/V600E mutation or with BRAFWT/WT treated with shTSP-1 or shGFP (control) pericyte secretome. Suppl. Figure 7 Effects of shTSP-1 pericyte secretome in BRAFWT/WT-TPC1 cells. Suppl. Figure 8 Viability assay in the presence of SRI31277 in PTC cells and pericytes. Suppl. Figure 9 Effects of the small molecule SRI31277 in PTC cells with BRAFWT/WT. Suppl. Figure 10 THBS1 (TSP-1) mRNA expression levels in PTC samples harboring both BRAFWT/V600E and hTERT mutations compare...
Purpose:The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, ... more Purpose:The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown.Experimental Design:We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment.Results:Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib...
Background Bioavailable 25-hydroxyvitamin D (25OHD) may be a better indicator of vitamin D suffic... more Background Bioavailable 25-hydroxyvitamin D (25OHD) may be a better indicator of vitamin D sufficiency than total 25OHD. This report describes a novel assay for measuring serum bioavailable 25OHD. Methods We developed an assay for 25OHD % bioavailability based on competitive binding of 25OHD tracer between vitamin D-binding protein (DBP)-coated affinity chromatography beads and serum DBP. Bioavailable 25OHD, total 25OHD, albumin, and DBP protein concentrations were measured in 89 samples from hospitalized patients and 42 healthy controls to determine how the DBP binding assay responds to differences in concentrations of DBP and compares to calculated bioavailable 25OHD values. Results DBP binding assay showed a linear relationship between DBP-bound 25OHD tracer recovered from bead supernatant and DBP calibrator concentrations (y = 0.0017x +0.731, R2 = 0.9961, p<0.001). Inversion of this relationship allowed interpolation of DBP binding equivalents based upon 25OHD tracer recovere...
Elevated circulating sFLT-1 (soluble fms-like tyrosine kinase) and low levels of its ligand, PlGF... more Elevated circulating sFLT-1 (soluble fms-like tyrosine kinase) and low levels of its ligand, PlGF (placental growth factor), are key characteristics of preeclampsia. However, it is unclear if the low levels of plasma PlGF noted during preeclampsia are due to decreased placental production of PlGF or due to binding of PlGF by increased circulating sFLT-1. Here, we describe a biochemical procedure to dissociate PlGF-sFLT-1 complex ex vivo and when used in conjunction with an immunoassay platform, demonstrate a method to measure total and free PlGF in human blood samples. Using this method, we noted that plasma free PlGF levels were significantly lower in preeclampsia (N=22) than in nonhypertensive controls (N=24; mean, 314 versus 686 pg/mL, P <0.05), but total PlGF levels were not different (mean, 822 versus 800 pg/mL, P =0.49). In contrast, total sFLT-1 levels were significantly higher in preeclampsia than in nonhypertensive controls (mean, 16 957 versus 3029 pg/mL, P <0.01) an...
Background: Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of all pregnant wom... more Background: Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of all pregnant women, and there is no effective treatment available. Early abnormal placental development is associated with oxidative stress and the release of reactive oxygen species in the placenta. This phenomenon leads to downstream signaling and production of anti-angiogenic factors, which cause endothelial and trophoblast dysfunction and the cardinal features of PE, i.e., hypertension, proteinuria, and in severe cases, eclampsia. Our group developed a novel series of mitochondria-targeted antioxidants and sought to test if these compounds can effectively reduce placental oxidative stress and mitigate PE symptoms in vitro. Methods: We induced in vitro oxidant stress in human trophoblast (HTR8/SVneo) cells with hydrogen peroxide (H 2 O 2 ) and assessed whether augmenting cell-redox function with the proposed antioxidant compounds reduced (i) cell injury (cell cytotoxicity assay), (ii) mitochondrial s...
Human cyotsolic malate dehydrogenase (MDH1) is important in transporting NADH equivalents across ... more Human cyotsolic malate dehydrogenase (MDH1) is important in transporting NADH equivalents across the mitochondrial membrane, controlling tricarboxylic acid (TCA) cycle pool size and providing contractile function. Cellular localization studies indicate that MDH1 mRNA expression has a strong tissue‐specific distribution, being expressed primarily in cardiac and skeletal muscle and in the brain, at intermediate levels in the spleen, kidney, intestine, liver, and testes and at low levels in lung and bone marrow. The observed MDH1 localizations reflect the role of NADH in the support of a variety of functions in different organs. These functions are primarily related to aerobic energy production for muscle contraction, neuronal signal transmission, absorption/resorption functions, collagen‐supporting functions, phagocytosis of dead cells, and processes related to gas exchange and cell division. During neonatal development, MDH1 is expressed in human embryonic heart as early as the 3rd m...
Suppl. Table 3. Log2 Fold Changes (FC) of some genes differentially expressed in PTC with BRAFV60... more Suppl. Table 3. Log2 Fold Changes (FC) of some genes differentially expressed in PTC with BRAFV600E and hTERT mutation vs BRAFV600E-PTC
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized b... more Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin-homing malignant T cells that express high level of chemokine receptor CCR4, which is associated with their skin-homing capacity. CCR4 is also highly expressed on T-regulatory cells (Tregs) that can migrate to several different types of chemotactic ligand CCL17- and CCL22-secreting tumors to facilitate tumor cell evasion from immune surveillance. Thus, its high-level expression on CTCL cells and Tregs makes CCR4 a potential ideal target for antibody-based immunotherapy for CTCL and other types of solid tumors. Here, we conducted humanization and affinity optimization of a murine anti-CCR4 monoclonal antibody (mAb), mAb1567, that recognizes both the N-terminal and extracellular domains of CCR4 with high affinity and inhibits chemotaxis of CCR4+ CTCL cells. In a mouse CTCL tumor model, mAb1567 exhibited a potent antitumor effect and in vitro mechanistic studies ...
Introduction: Preeclampsia is a hypertensive disorder of pregnancy which is associated with subst... more Introduction: Preeclampsia is a hypertensive disorder of pregnancy which is associated with substantial morbidity for the mother and fetus. Reduced nitric oxide bioavailability and oxidative stress are key in the maternal and fetal pathophysiology of preeclampsia. Method: In this study, we assessed the potential therapeutic effect and mechanisms of action of a novel dual-function nitric oxide donor/redox modulator, AKT-1005, in an animal model of Ad.sFlt1-induced hypertension, proteinuria, and glomerular endotheliosis. Ad.sFlt1-overexpressing CD1 mice were treated via minipump with AKT-1005 (20 mg/kg) or vehicle (n=15 per group). Results: Treatment with AKT-1005 for 7 days significantly reduced sFlt1-induced hypertension and endotheliosis of the kidney glomeruli. Kidney function was also assessed by cystatin C ELISA, which showed significant reduction in AKT-1005 treated mice compared to controls. AKT-1005 treatment increased NO bioavailabilty as assessed by Griess method. The incre...
Suppl. Table 4. Log2 Fold Changes (FC) of some genes differentially expressed in PTC wih BRAFV600... more Suppl. Table 4. Log2 Fold Changes (FC) of some genes differentially expressed in PTC wih BRAFV600E and hTERT mutation vs BRAFWT-PTC
Suppl. Table 2. Log2 Fold Changes (FC) of some genes differentially expressed in BRAFV600E-PTC vs... more Suppl. Table 2. Log2 Fold Changes (FC) of some genes differentially expressed in BRAFV600E-PTC vs BRAFWT-PTC
Suppl. Figure 1 Drug dose-effect analysis in BRAFWT/V600E-KTC1 cells Suppl.Figure 2 Dose-effect a... more Suppl. Figure 1 Drug dose-effect analysis in BRAFWT/V600E-KTC1 cells Suppl.Figure 2 Dose-effect analysis of vemurafenib and sorafenib on pericyte viability. Suppl. Figure 3 BRAFWT/WT-TPC1 cells viability assay. Suppl. Figure 4 ELISA analysis of cytokines secretion in pericytes, and PTC cells harboring the heterozygous BRAFWT/V600E mutation or with BRAFWT/WT. Suppl. Figure 5 Effects of pericyte secretome in BRAFWT/WT-TPC1 cells. Suppl. Figure 6 Analysis of secreted TSP-1 levels in PTC cells harboring the heterozygous BRAFWT/V600E mutation or with BRAFWT/WT treated with shTSP-1 or shGFP (control) pericyte secretome. Suppl. Figure 7 Effects of shTSP-1 pericyte secretome in BRAFWT/WT-TPC1 cells. Suppl. Figure 8 Viability assay in the presence of SRI31277 in PTC cells and pericytes. Suppl. Figure 9 Effects of the small molecule SRI31277 in PTC cells with BRAFWT/WT. Suppl. Figure 10 THBS1 (TSP-1) mRNA expression levels in PTC samples harboring both BRAFWT/V600E and hTERT mutations compare...
Purpose:The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, ... more Purpose:The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown.Experimental Design:We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment.Results:Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib...
Background Bioavailable 25-hydroxyvitamin D (25OHD) may be a better indicator of vitamin D suffic... more Background Bioavailable 25-hydroxyvitamin D (25OHD) may be a better indicator of vitamin D sufficiency than total 25OHD. This report describes a novel assay for measuring serum bioavailable 25OHD. Methods We developed an assay for 25OHD % bioavailability based on competitive binding of 25OHD tracer between vitamin D-binding protein (DBP)-coated affinity chromatography beads and serum DBP. Bioavailable 25OHD, total 25OHD, albumin, and DBP protein concentrations were measured in 89 samples from hospitalized patients and 42 healthy controls to determine how the DBP binding assay responds to differences in concentrations of DBP and compares to calculated bioavailable 25OHD values. Results DBP binding assay showed a linear relationship between DBP-bound 25OHD tracer recovered from bead supernatant and DBP calibrator concentrations (y = 0.0017x +0.731, R2 = 0.9961, p<0.001). Inversion of this relationship allowed interpolation of DBP binding equivalents based upon 25OHD tracer recovere...
Elevated circulating sFLT-1 (soluble fms-like tyrosine kinase) and low levels of its ligand, PlGF... more Elevated circulating sFLT-1 (soluble fms-like tyrosine kinase) and low levels of its ligand, PlGF (placental growth factor), are key characteristics of preeclampsia. However, it is unclear if the low levels of plasma PlGF noted during preeclampsia are due to decreased placental production of PlGF or due to binding of PlGF by increased circulating sFLT-1. Here, we describe a biochemical procedure to dissociate PlGF-sFLT-1 complex ex vivo and when used in conjunction with an immunoassay platform, demonstrate a method to measure total and free PlGF in human blood samples. Using this method, we noted that plasma free PlGF levels were significantly lower in preeclampsia (N=22) than in nonhypertensive controls (N=24; mean, 314 versus 686 pg/mL, P <0.05), but total PlGF levels were not different (mean, 822 versus 800 pg/mL, P =0.49). In contrast, total sFLT-1 levels were significantly higher in preeclampsia than in nonhypertensive controls (mean, 16 957 versus 3029 pg/mL, P <0.01) an...
Background: Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of all pregnant wom... more Background: Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of all pregnant women, and there is no effective treatment available. Early abnormal placental development is associated with oxidative stress and the release of reactive oxygen species in the placenta. This phenomenon leads to downstream signaling and production of anti-angiogenic factors, which cause endothelial and trophoblast dysfunction and the cardinal features of PE, i.e., hypertension, proteinuria, and in severe cases, eclampsia. Our group developed a novel series of mitochondria-targeted antioxidants and sought to test if these compounds can effectively reduce placental oxidative stress and mitigate PE symptoms in vitro. Methods: We induced in vitro oxidant stress in human trophoblast (HTR8/SVneo) cells with hydrogen peroxide (H 2 O 2 ) and assessed whether augmenting cell-redox function with the proposed antioxidant compounds reduced (i) cell injury (cell cytotoxicity assay), (ii) mitochondrial s...
Human cyotsolic malate dehydrogenase (MDH1) is important in transporting NADH equivalents across ... more Human cyotsolic malate dehydrogenase (MDH1) is important in transporting NADH equivalents across the mitochondrial membrane, controlling tricarboxylic acid (TCA) cycle pool size and providing contractile function. Cellular localization studies indicate that MDH1 mRNA expression has a strong tissue‐specific distribution, being expressed primarily in cardiac and skeletal muscle and in the brain, at intermediate levels in the spleen, kidney, intestine, liver, and testes and at low levels in lung and bone marrow. The observed MDH1 localizations reflect the role of NADH in the support of a variety of functions in different organs. These functions are primarily related to aerobic energy production for muscle contraction, neuronal signal transmission, absorption/resorption functions, collagen‐supporting functions, phagocytosis of dead cells, and processes related to gas exchange and cell division. During neonatal development, MDH1 is expressed in human embryonic heart as early as the 3rd m...
Suppl. Table 3. Log2 Fold Changes (FC) of some genes differentially expressed in PTC with BRAFV60... more Suppl. Table 3. Log2 Fold Changes (FC) of some genes differentially expressed in PTC with BRAFV600E and hTERT mutation vs BRAFV600E-PTC
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized b... more Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin-homing malignant T cells that express high level of chemokine receptor CCR4, which is associated with their skin-homing capacity. CCR4 is also highly expressed on T-regulatory cells (Tregs) that can migrate to several different types of chemotactic ligand CCL17- and CCL22-secreting tumors to facilitate tumor cell evasion from immune surveillance. Thus, its high-level expression on CTCL cells and Tregs makes CCR4 a potential ideal target for antibody-based immunotherapy for CTCL and other types of solid tumors. Here, we conducted humanization and affinity optimization of a murine anti-CCR4 monoclonal antibody (mAb), mAb1567, that recognizes both the N-terminal and extracellular domains of CCR4 with high affinity and inhibits chemotaxis of CCR4+ CTCL cells. In a mouse CTCL tumor model, mAb1567 exhibited a potent antitumor effect and in vitro mechanistic studies ...
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