108 results on '"Nguyen, Julia"'
Search Results
2. MASP-2 and MASP-3 inhibitors block complement activation, inflammation, and microvascular stasis in a murine model of vaso-occlusion in sickle cell disease.
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Belcher, John D., Nguyen, Julia, Chen, Chunsheng, Abdulla, Fuad, Conglin, Ruan, Ivy, Zalaya K., Cummings, Jason, Dudler, Thomas, and Vercellotti, Gregory M.
- Abstract
Patients with sickle cell disease (SCD) have ongoing hemolysis that promotes endothelial injury, complement activation, inflammation, vaso-occlusion, ischemia-reperfusion pathophysiology, and pain. Complement activation markers are increased in SCD in steady-state and further increased during vaso-occlusive crisis (VOC). However, the mechanisms driving complement activation in SCD have not been completely elucidated. Ischemia-reperfusion and heme released from hemoglobin during hemolysis, events that characterize SCD pathophysiology, can activate the lectin pathway (LP) and alternative pathway (AP), respectively. Here we evaluated the role of LP and AP in Townes sickle (SS) mice using inhibitory monoclonal antibodies (mAb) to mannose binding lectin (MBL)-associated serine protease (MASP)-2 or MASP-3, respectively. Townes SS mice were pretreated with MASP-2 mAb, MASP-3 mAb, isotype control mAb, or PBS before they were challenged with hypoxia-reoxygenation or hemoglobin. Pretreatment of SS mice with MASP-2 or MASP-3 mAb, markedly reduced Bb fragments, C4d and C5a in plasma and complement deposition in the liver, kidneys, and lungs collected 4 hours after challenge compared to control mAb-treated mice. Consistent with complement inhibition, hepatic inflammation markers NF-ĸB phospho-p65, VCAM-1, ICAM-1, and E-selectin were significantly reduced in SS mice pretreated with MASP-2 or MASP-3 mAb. Importantly, MASP-2 or MASP-3 mAb pretreatment significantly inhibited microvascular stasis (vaso-occlusion) induced by hypoxia-reoxygenation or hemoglobin. These studies suggest that the LP and the AP are both playing a role in promoting inflammation and vaso-occlusion in SCD. Inhibiting complement activation via the LP or the AP might inhibit inflammation and prevent VOC in SCD patients. [ABSTRACT FROM AUTHOR]
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- 2022
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3. Augmentation of MS/MS Libraries with Spectral Interpolation for Improved Identification.
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King, Ethan, Overstreet, Richard, Nguyen, Julia, and Ciesielski, Danielle
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- 2022
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4. Augmentation of MS/MS Libraries with Spectral Interpolation for Improved Identification
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King, Ethan, Overstreet, Richard, Nguyen, Julia, and Ciesielski, Danielle
- Abstract
Tandem mass spectrometry (MS/MS) is a primary tool for the identification of small molecules and metabolites where resultant spectra are most commonly identified by matching them with spectra in MS/MS reference libraries. The high degree of variability in MS/MS spectrum acquisition techniques and parameters creates a significant challenge for building standardized reference libraries. Here we present a method to improve the usefulness of existing MS/MS libraries by augmenting available experimental spectra data sets with statistically interpolated spectra at unreported collision energies. We find that highly accurate spectral approximations can be interpolated from as few as three experimental spectra and that the interpolated spectra will be consistent with true spectra gathered from the same instrument as the experimental spectra. Supplementing existing spectral databases with interpolated spectra yields consistent improvements to identification accuracy on a range of instruments and precursor types. Applying this method yields significant improvements (∼10% more spectra correctly identified) on large data sets (2000–10 000 spectra), indicating this is a quick yet adept tool for improving spectral matching in situations where available reference libraries are not yet sufficient. We also find improvements of matching spectra across instrument types (between an Agilent Q-TOF and an Orbitrap Elite), at high collision energies (50–90 eV), and with smaller data sets available through MassBank.
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- 2022
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5. Changes in Vestibular Function in Patients With Head-and-Neck Cancer Undergoing Chemoradiation
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Hülse, Roland, Stuck, Boris A., Hörmann, Karl, Rotter, Nicole, Nguyen, Julia, Aderhold, Christoph, and Schell, Angela
- Abstract
Introduction: While the cochleotoxicity of cisplatin has been well investigated, less is known about the effects of platinum-based chemotherapy on the vestibular system. In particular, there is a lack of prospective studies using modern laboratory vestibular testing that examine the effects of cisplatin on the semicircular canals and on the otolith organs. The aim of the present study was, therefore, to investigate the vestibulotoxic effect of cisplatin in patients with head and neck tumors who are undergoing chemoradiation.Methods: Forty-five patients undergoing cisplatin-based chemoradiation for head and neck cancer received a vestibular assessment consisting of anamnesis, a horizontal video head impulse test (vHIT), ocular and cervical vestibular evoked myogenic potential testing, as well as pure tone audiometry. This assessment was performed before therapy, 6 weeks after therapy, and 3 months after therapy.Results: Video head impulse test showed a significantly reduced median gain 6 weeks after chemoradiation. In addition, significantly more refixational saccades could be detected after therapy. Vestibular evoked myogenic potential testing results also revealed significant changes, whereas pure tone audiometry did not. None of the patients mentioned “dizziness” during the follow-up examinations.Conclusion: We demonstrated a vestibulotoxic effect of cisplatin-based chemoradiation in patients with head and neck cancer. Future studies are needed to better understand cisplatin-induced vestibulotoxicity and to identify possible vestibuloprotective substances. Still, before and after chemoradiation, patients should undergo not only auditory testing but also vestibular testing in order to detect potential vestibular loss as soon as possible and to quickly initiate vestibular physiotherapy.
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- 2022
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6. QC-GN2oMS2: a Graph Neural Net for High Resolution Mass Spectra Prediction
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Overstreet, Richard, King, Ethan, Clopton, Grady, Nguyen, Julia, and Ciesielski, Danielle
- Abstract
Predicting the mass spectrum of a molecular ion is often accomplished via three generalized approaches: rules-based methods for bond breaking, deep learning, or quantum chemical (QC) modeling. Rules-based approaches are often limited by the conditions for different chemical subspaces and perform poorly under chemical regimes with few defined rules. QC modeling is theoretically robust but requires significant amounts of computational time to produce a spectrum for a given target. Among deep learning techniques, graph neural networks (GNNs) have performed better than previous work with fingerprint-based neural networks in mass spectra prediction. To explore this technique further, we investigate the effects of including quantum chemically derived information as edge features in the GNN to increase predictive accuracy. The models we investigated include categorical bond order, bond force constants derived from extended tight-binding (xTB) quantum chemistry, and acyclic bond dissociation energies. We evaluated these models against a control GNN with no edge features in the input graphs. Bond dissociation enthalpies yielded the best improvement with a cosine similarity score of 0.462 relative to the baseline model (0.437). In this work we also apply dynamic graph attention which improves performance on benchmark problems and supports the inclusion of edge features. Between implementations, we investigate the nature of the molecular embedding for spectra prediction and discuss the recognition of fragment topographies in distinct chemistries for further development in tandem mass spectrometry prediction.
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- 2024
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7. Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease
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Sparkenbaugh, Erica M., Chen, Chunsheng, Brzoska, Tomasz, Nguyen, Julia, Wang, Shaobin, Vercellotti, Gregory M., Key, Nigel S., Sundd, Prithu, Belcher, John D., and Pawlinski, Rafal
- Abstract
Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.
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- 2020
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8. Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease
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Sparkenbaugh, Erica M., Chen, Chunsheng, Brzoska, Tomasz, Nguyen, Julia, Wang, Shaobin, Vercellotti, Gregory M., Key, Nigel S., Sundd, Prithu, Belcher, John D., and Pawlinski, Rafal
- Abstract
Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.
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- 2020
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9. Effect of Integrin α 4β 1-VCAM-1 Interactions in JAK2V617FMyeloproliferative Neoplasm Pro-Thrombotic Interactions
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DaSilva, Angelica, Beckman, Joan D, Abdullah, Maya, Aronovich, Elena, Nguyen, Aithanh Viet, Nguyen, Julia, and Wood, David
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Introduction: The JAK2V617Fmutation, which occurs in up to 95% of polycythemia vera (PV) myeloproliferative neoplasm (MPN) patients, increases thrombosis risk 6-fold. A recent study in PV mice demonstrated that blocking JAK2V617F+leukocyte-mediated β1/β2 integrin interactions with anti-VCAM-1 antibodies decreases thrombosis. Interestingly, hydroxyurea (HU), a common PV treatment, reduces leukocyte and red cell α4β1 integrin expression. Overall, JAK2V617F+mouse models show inconsistent thrombosis and bleeding phenotype, hindering pre-clinical study reproducibility; therefore, we have developed an in vitroendothelialized microfluidic model to assess MPN-flow behavior. We hypothesizedthat in our microfluidic model, disrupting α4β1 integrin expression on JAK2V617F+leukocytes would reduce pro-adhesive interactions. We tested this hypothesis by assessing JAK2V617F+MPN blood flow in untreated and hydroxyurea treated subjects and through addition of a monoclonal antibody against α4β1 integrin, natalizumab.
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- 2023
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10. Histone Deacetylase 6 Inhibition Prevents Vaso-Occlusion and Pain in Sickle Cell Mice
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Nguyen, Aithanh, Juliette, Joseph P, Wong, Alison, Aronovich, Elena, Abdullah, Maya, Nguyen, Julia, Abdulla, Fuad, Chen, Chunsheng, Khasabova, Iryna A, Belcher, John D, Vercellotti, Gregory M., Simone, Donald, and Beckman, Joan D
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- 2022
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11. Cold Exposure Induces Vaso-Occlusion and Hyperalgesia in Mice with Sickle Cell Disease That Is Dependent on Complement Activation
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Ivy, Zalaya K, Belcher, John D, Khasabova, Iryna A, Chen, Chunsheng, Juliette, Joseph P, Abdulla, Fuad, Ruan, Conglin, Allen, Kaje, Nguyen, Julia, Rogness, Victoria M, Khasabov, Sergey, Gupta, Kalpna, Simone, Donald, and Vercellotti, Gregory M.
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- 2022
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12. Histone Deacetylase 6 Inhibition Prevents Vaso-Occlusion and Pain in Sickle Cell Mice
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Nguyen, Aithanh, Juliette, Joseph P, Wong, Alison, Aronovich, Elena, Abdullah, Maya, Nguyen, Julia, Abdulla, Fuad, Chen, Chunsheng, Khasabova, Iryna A, Belcher, John D, Vercellotti, Gregory M., Simone, Donald, and Beckman, Joan D
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- 2022
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13. Cold Exposure Induces Vaso-Occlusion and Hyperalgesia in Mice with Sickle Cell Disease That Is Dependent on Complement Activation
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Ivy, Zalaya K, Belcher, John D, Khasabova, Iryna A, Chen, Chunsheng, Juliette, Joseph P, Abdulla, Fuad, Ruan, Conglin, Allen, Kaje, Nguyen, Julia, Rogness, Victoria M, Khasabov, Sergey, Gupta, Kalpna, Simone, Donald, and Vercellotti, Gregory M.
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- 2022
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14. The functional organization of cortical feedback inputs to primary visual cortex
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Marques, Tiago, Nguyen, Julia, Fioreze, Gabriela, and Petreanu, Leopoldo
- Abstract
Cortical feedback is thought to mediate cognitive processes like attention, prediction, and awareness. Understanding its function requires identifying the organizational logic of feedback axons relaying different signals. We measured retinotopic specificity in inputs from the lateromedial visual area in mouse primary visual cortex (V1) by mapping receptive fields in feedback boutons and relating them to those of neurons in their vicinity. Lateromedial visual area inputs in layer 1 targeted, on average, retinotopically matched locations in V1, but many of them relayed distal visual information. Orientation-selective axons overspread around the retinotopically matched location perpendicularly to their preferred orientation. Direction-selective axons were biased to visual areas shifted from the retinotopically matched position along the angle of their antipreferred direction. Our results show that feedback inputs show tuning-dependent retinotopic specificity. By targeting locations that would be activated by stimuli orthogonal to or opposite to a cell’s own tuning, feedback could potentially enhance visual representations in time and space. The authors measured the organization of cortical feedback inputs in mouse primary visual cortex. They found that the locations in visual cortex targeted by feedback axons relate to their tuning properties according to a simple geometrical rule.
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- 2018
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15. Prioritizing Happiness From Teaching History to Grantmaking.
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Nguyen, Julia
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COLLEGE teachers ,GRANTMAKING foundations ,EMPLOYEES - Abstract
The article presents a personal narrative by the author who talks about transitioning her career from a history professor to a program officer in Grant-making at the National Endowment for the Humanities in Washington, DC.
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- 2017
16. Cold exposure induces vaso-occlusion and pain in sickle mice that depend on complement activation
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Ivy, Zalaya K., Belcher, John D., Khasabova, Iryna A., Chen, Chunsheng, Juliette, Joseph P., Abdulla, Fuad, Ruan, Conglin, Allen, Kaje, Nguyen, Julia, Rogness, Victoria M., Beckman, Joan D., Khasabov, Sergey G., Gupta, Kalpna, Taylor, Ronald P., Simone, Donald A., and Vercellotti, Gregory M.
- Abstract
•Cold exposure induces complement activation and vaso-occlusive pain crisis in sickle mice.•Inhibition of C5a generation with anti-C5 or signaling with anti-C5aR antibody inhibits cold-induced VOE and hyperalgesia in sickle mice.
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- 2023
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17. Single-Cell Analysis of [18F]Fluorodeoxyglucose Uptake by Droplet Radiofluidics.
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Türkcan, Silvan, Nguyen, Julia, Vilalta, Marta, Bin Shen, Chin, Frederick T., Pratx, Guillem, and Abbyad, Paul
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- 2015
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18. Student Internship with Veterinarians Without Borders Canada -- Kenya 2016.
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White, Katharine and Nguyen, Julia
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ANIMAL welfare ,INTERNSHIP programs ,FARMERS - Abstract
The article provides information on a student internship and service project which aims to help improve dairy cow management, productivity, and animal welfare for small-holder dairy farmers in Mukarwe-ini, Kenya.
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- 2017
19. Engaging Vietnamese Americans in research on aging and dementia.
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Meyer, Oanh L, Nguyen, Julia Phuong, Nam, Bora, Nguyen, Khue, Zhu, Jeanette, Vuong, Quyen, Bang, Joon, Hinton, Ladson, Tsoh, Janice Y, and Park, Van Ta
- Abstract
Background: Vietnamese Americans are a large Asian American group in the U.S. and at high risk for health disparities, partially due to their limited English proficiency, low socioeconomic status, history of war and trauma, and a lack of culturally and linguistically appropriate care. As this relatively recent refugee population continues to age in the U.S., it is important to engage them in research around Alzheimer's disease and related dementias (ADRD). Yet few studies have examined their perceptions of aging, dementia, and ADRD research participation. This qualitative study examined the perceptions of Vietnamese in Northern and Southern California, the state with the largest population of Vietnamese Americans in the U.S. Method: We conducted three focus groups with a total of 20 Vietnamese individuals in both Vietnamese and English. The focus group guide assessed participants' attitudes and experiences with research participation, perceptions of ADRD and aging, and insights into registry outreach and recruitment. There were nine women, and the mean age was 40 years, SD = 17.74 (range 18‐73). Participants consisted of key stakeholders including community leaders, ADRD caregivers, and community members. Result: Several themes emerged from the analyses, including (1) Motivations to participate in research to gain knowledge: (a) for oneself, (b) for family's benefit, and (c) for the Vietnamese American community as a whole; (2) The necessity of trustworthy and credible individuals/spokespersons to promote the research initiative; (3) Recruitment strategies that are age‐specific and culturally appropriate, and (4) Importance of monetary incentives. Conclusion: Results highlight key issues that are important to Vietnamese Americans' participation in aging and dementia research. Practical implications of these results will be discussed. [ABSTRACT FROM AUTHOR]
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- 2021
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20. Quantitative real-time polymerase chain reaction (qRT-PCR) restriction fragment length polymorphism (RFLP) method for monitoring highly conserved transgene expression during gene therapy.
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Bruzzone, Carol M., Belcher, John D., Schuld, Nathan J., Newman, Kristal A., Vineyard, Julie, Nguyen, Julia, Chen, Chunsheng, Beckman, Joan D., Steer, Clifford J., and Vercellotti, Gregory M.
- Abstract
Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, the total HO-1 mRNA was enriched 2-fold relative to control cells, and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and was injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate the transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery, and monitoring of gene-therapy vectors. [Copyright &y& Elsevier]
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- 2008
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21. The establishment of murine blood outgrowth endothelial cells and observations relevant to gene therapy.
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Somani, Arif, Nguyen, Julia, Milbauer, Liming C., Solovey, Anna, Sajja, Suchitra, and Hebbel, Robert P.
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Endothelial cells are an attractive vehicle for gene therapy because they may be used in an autologous fashion and may allow for direct exposure of the gene product into the intravascular space. To explore this future potential, a reproducible system was developed for the culture of murine blood outgrowth endothelial cells. These cells demonstrated acetylated low-density lipoprotein (LDL) incorporation, matrigel tube formation, and specific endothelial staining characteristics, namely P1H12, VeCAD, vascular cell adhesion molecule (VCAM), vWF, platelet endothelial cell adhesion molecule (PECAM-1), and vascular endothelial growth factor receptor-2 (VEGFR2). They were also negative for smooth muscle actin and monocytic markers CD11b, CD14, and CD16. Moreover, these cells were amendable to gene transfer with red fluorescent and green fluorescent expression vectors as well as human Factor VIII (hFVIII) while maintaining endothelial characteristics. Both source- and gene-introduced cells also manifested excellent proliferative potential. Furthermore, murine blood outgrowth endothelial cells (BOECs) demonstrated persistent in vivo seeding in the liver, lung, spleen, and bone morrow of recipient mice. [Copyright &y& Elsevier]
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- 2007
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22. Polypharmacy as a risk factor for adverse drug reactions in geriatric nursing home residents.
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Nguyen, Julia K., Fouts, Michelle M., Kotabe, Sharon E., and Lo, Eunice
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PHARMACODYNAMICS ,NURSING home patients ,OLDER people ,DRUG side effects - Abstract
Abstract: Objective:: Polypharmacy is a well-known risk factor for adverse drug reactions (ADRs). The objective of this study was to determine the relationship between the use of ≥9 different scheduled medications and the occurrence of ADRs in geriatric nursing home residents. Methods:: This was a retrospective cohort study conducted in a 1200-bed, county-owned and -operated, longterm care skilled nursing facility Participants were 335 subjects aged ≥65 years who were present at the facility during the index month of October 1998. Hospice, respite care, and rehabilitation patients were excluded. Use of ≥9 different scheduled medications was defined a priori as routinely administered medications, excluding as-needed agents, topical agents, 1-time administration, and vaccinations. ADRs were identified by voluntary reporting and by chart review during a 12-month period. ADRs were assessed individually by 2 clinical pharmacists applying the Naranjo ADR probability scale. Results:: A total of 207 ADRs were identified. The cohort receiving ≥9 scheduled medications (n = 43) experienced 53 ADRs, compared with 154 ADRs in the control group receiving <9 medications (n = 292). The demographic distribution was similar in both cohorts, with white as the dominant ethnicity; 45% were white in the control group and 51% were white in the cohort group receiving ≥9 scheduled medications. The sex distribution was also similar, with women outnumbering men in both cohorts: 60% and 81% were women in the control and cohort groups, respectively. The mean age was 72 years (range, 65–100 years). After the data were adjusted for the number of days each subject was at risk for experiencing an ADR, subjects using ≥9 different scheduled medications were 2.33 times more likely than controls to experience an ADR (95% CI, 1.54–3.52; P < 0.001). Conclusion:: A positive correlation between the use of ≥9 different scheduled medications and ADRs was found among these geriatric nursing home residents. [Copyright &y& Elsevier]
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- 2006
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23. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease
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Belcher, John D., Chen, Chunsheng, Nguyen, Julia, Milbauer, Liming, Abdulla, Fuad, Alayash, Abdu I., Smith, Ann, Nath, Karl A., Hebbel, Robert P., and Vercellotti, Gregory M.
- Abstract
Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4β1, or αVβ3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4−/− mice transplanted with TLR4+/+ sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4−/− ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.
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- 2014
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24. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease
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Belcher, John D., Chen, Chunsheng, Nguyen, Julia, Milbauer, Liming, Abdulla, Fuad, Alayash, Abdu I., Smith, Ann, Nath, Karl A., Hebbel, Robert P., and Vercellotti, Gregory M.
- Abstract
Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4β1, or αVβ3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4−/−mice transplanted with TLR4+/+sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4−/−ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.
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- 2014
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25. MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a modulator of HO-1, inflammation, and vaso-occlusion in transgenic sickle mice
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Belcher, John D., Young, Mark, Chen, Chunsheng, Nguyen, Julia, Burhop, Kenneth, Tran, Phuc, and Vercellotti, Gregory M.
- Abstract
Transgenic sickle mice expressing βS hemoglobin have activated vascular endothelium in multiple organs that exhibits enhanced expression of NF-ĸB and adhesion molecules and promotes microvascular stasis in sickle, but not normal, mice in response to hypoxia/reoxygenation (H/R), or heme. Induction of heme oxygenase-1 (HO-1) or administration of its products, carbon monoxide (CO) or biliverdin, inhibits microvascular stasis in sickle mice. Infusion of human hemoglobin conjugated with polyethylene glycol and saturated with CO (MP4CO) markedly induced hepatic HO-1 activity and inhibited NF-ĸB activation and H/R-induced microvascular stasis in sickle mice. These effects were mediated by CO; saline or MP4 saturated with O2 (MP4OX) had little to no effect on H/R-induced stasis, though unmodified oxyhemoglobin exacerbated stasis. The HO-1 inhibitor, tin protoporphyrin, blocked MP4CO protection, consistent with HO-1 involvement in the protection afforded by MP4CO. MP4CO also induced nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), an important transcriptional regulator of HO-1 and other antioxidant genes. In a heterozygous (hemoglobin-AS) sickle mouse model, intravenous hemin induced cardiovascular collapse and mortality within 120 minutes, which was significantly reduced by MP4CO, but not MP4OX. These data demonstrate that MP4CO induces cytoprotective Nrf2 and HO-1 and decreases NF-ĸB activation, microvascular stasis, and mortality in transgenic sickle mouse models.
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- 2013
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26. Mast cell activation contributes to sickle cell pathobiology and pain in mice
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Vincent, Lucile, Vang, Derek, Nguyen, Julia, Gupta, Mihir, Luk, Kathryn, Ericson, Marna E., Simone, Donald A., and Gupta, Kalpna
- Abstract
Sickle cell anemia (SCA) is an inherited disorder associated with severe lifelong pain and significant morbidity. The mechanisms of pain in SCA remain poorly understood. We show that mast cell activation/degranulation contributes to sickle pain pathophysiology by promoting neurogenic inflammation and nociceptor activation via the release of substance P in the skin and dorsal root ganglion. Mast cell inhibition with imatinib ameliorated cytokine release from skin biopsies and led to a correlative decrease in granulocyte-macrophage colony-stimulating factor and white blood cells in transgenic sickle mice. Targeting mast cells by genetic mutation or pharmacologic inhibition with imatinib ameliorates tonic hyperalgesia and prevents hypoxia/reoxygenation-induced hyperalgesia in sickle mice. Pretreatment with the mast cell stabilizer cromolyn sodium improved analgesia following low doses of morphine that were otherwise ineffective. Mast cell activation therefore underlies sickle pathophysiology leading to inflammation, vascular dysfunction, pain, and requirement for high doses of morphine. Pharmacological targeting of mast cells with imatinib may be a suitable approach to address pain and perhaps treat SCA.
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- 2013
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27. Mast cell activation contributes to sickle cell pathobiology and pain in mice
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Vincent, Lucile, Vang, Derek, Nguyen, Julia, Gupta, Mihir, Luk, Kathryn, Ericson, Marna E., Simone, Donald A., and Gupta, Kalpna
- Abstract
Sickle cell anemia (SCA) is an inherited disorder associated with severe lifelong pain and significant morbidity. The mechanisms of pain in SCA remain poorly understood. We show that mast cell activation/degranulation contributes to sickle pain pathophysiology by promoting neurogenic inflammation and nociceptor activation via the release of substance P in the skin and dorsal root ganglion. Mast cell inhibition with imatinib ameliorated cytokine release from skin biopsies and led to a correlative decrease in granulocyte-macrophage colony-stimulating factor and white blood cells in transgenic sickle mice. Targeting mast cells by genetic mutation or pharmacologic inhibition with imatinib ameliorates tonic hyperalgesia and prevents hypoxia/reoxygenation-induced hyperalgesia in sickle mice. Pretreatment with the mast cell stabilizer cromolyn sodium improved analgesia following low doses of morphine that were otherwise ineffective. Mast cell activation therefore underlies sickle pathophysiology leading to inflammation, vascular dysfunction, pain, and requirement for high doses of morphine. Pharmacological targeting of mast cells with imatinib may be a suitable approach to address pain and perhaps treat SCA.
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- 2013
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28. Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids
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Kohli, Divyanshoo R., Li, Yunfang, Khasabov, Sergey G., Gupta, Pankaj, Kehl, Lois J., Ericson, Marna E., Nguyen, Julia, Gupta, Vinita, Hebbel, Robert P., Simone, Donald A., and Gupta, Kalpna
- Abstract
Sickle cell disease causes severe pain. We examined pain-related behaviors, correlative neurochemical changes, and analgesic effects of morphine and cannabinoids in transgenic mice expressing human sickle hemoglobin (HbS). Paw withdrawal threshold and withdrawal latency (to mechanical and thermal stimuli, respectively) and grip force were lower in homozygous and hemizygous Berkley mice (BERK and hBERK1, respectively) compared with control mice expressing human hemoglobin A (HbA-BERK), indicating deep/musculoskeletal and cutaneous hyperalgesia. Peripheral nerves and blood vessels were structurally altered in BERK and hBERK1 skin, with decreased expression of μ opioid receptor and increased calcitonin gene-related peptide and substance P immunoreactivity. Activators of neuropathic and inflammatory pain (p38 mitogen-activated protein kinase, STAT3, and mitogen-activated protein kinase/extracellular signal-regulated kinase) showed increased phosphorylation, with accompanying increase in COX-2, interleukin-6, and Toll-like receptor 4 in the spinal cord of hBERK1 compared with HbA-BERK. These neurochemical changes in the periphery and spinal cord may contribute to hyperalgesia in mice expressing HbS. In BERK and hBERK1, hyperalgesia was markedly attenuated by morphine and cannabinoid receptor agonist CP 55940. We show that mice expressing HbS exhibit characteristics of pain observed in sickle cell disease patients, and neurochemical changes suggestive of nociceptor and glial activation. Importantly, cannabinoids attenuate pain in mice expressing HbS.
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- 2010
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29. Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids
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Kohli, Divyanshoo R., Li, Yunfang, Khasabov, Sergey G., Gupta, Pankaj, Kehl, Lois J., Ericson, Marna E., Nguyen, Julia, Gupta, Vinita, Hebbel, Robert P., Simone, Donald A., and Gupta, Kalpna
- Abstract
Sickle cell disease causes severe pain. We examined pain-related behaviors, correlative neurochemical changes, and analgesic effects of morphine and cannabinoids in transgenic mice expressing human sickle hemoglobin (HbS). Paw withdrawal threshold and withdrawal latency (to mechanical and thermal stimuli, respectively) and grip force were lower in homozygous and hemizygous Berkley mice (BERK and hBERK1, respectively) compared with control mice expressing human hemoglobin A (HbA-BERK), indicating deep/musculoskeletal and cutaneous hyperalgesia. Peripheral nerves and blood vessels were structurally altered in BERK and hBERK1 skin, with decreased expression of µ opioid receptor and increased calcitonin gene-related peptide and substance P immunoreactivity. Activators of neuropathic and inflammatory pain (p38 mitogen-activated protein kinase, STAT3, and mitogen-activated protein kinase/extracellular signal-regulated kinase) showed increased phosphorylation, with accompanying increase in COX-2, interleukin-6, and Toll-like receptor 4 in the spinal cord of hBERK1 compared with HbA-BERK. These neurochemical changes in the periphery and spinal cord may contribute to hyperalgesia in mice expressing HbS. In BERK and hBERK1, hyperalgesia was markedly attenuated by morphine and cannabinoid receptor agonist CP 55940. We show that mice expressing HbS exhibit characteristics of pain observed in sickle cell disease patients, and neurochemical changes suggestive of nociceptor and glial activation. Importantly, cannabinoids attenuate pain in mice expressing HbS.
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- 2010
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30. The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice
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Hebbel, Robert P., Vercellotti, Gregory M., Pace, Betty S., Solovey, Anna N., Kollander, Rahn, Abanonu, Chine F., Nguyen, Julia, Vineyard, Julie V., Belcher, John D., Abdulla, Fuad, Osifuye, Shadé, Eaton, John W., Kelm, Robert J., and Slungaard, Arne
- Abstract
The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.
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- 2010
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31. The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice
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Hebbel, Robert P., Vercellotti, Gregory M., Pace, Betty S., Solovey, Anna N., Kollander, Rahn, Abanonu, Chine F., Nguyen, Julia, Vineyard, Julie V., Belcher, John D., Abdulla, Fuad, Osifuye, Shadé, Eaton, John W., Kelm, Robert J., and Slungaard, Arne
- Abstract
The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.
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- 2010
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32. ML-0207/ASP8731: A Novel BACH1 Inhibitor That Induces Fetal Hemoglobin in Treatment of Sickle Cell Disease
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Nataraja, Selva, Singh, Maneet, Demes, Shilpa, Olson, Lyndsay, Stanwix, Jeff, Biddle, Margaret, Clarke, Emer, Huang, Yongzhao, Nguyen, Julia, Chen, Chunsheng, Zhang, Ping, Abdulla, Fuad, Vercellotti, Gregory M., and Belcher, John D
- Abstract
Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the β-globin subunit resulting in hemoglobin S (HbS). Following deoxygenation of red blood cells, HbS forms polymers that can promote hemolysis and the release of free heme that cause pro-oxidative and pro-inflammatory stress, vaso-occlusive pain crises, and ischemia-reperfusion pathophysiology. Heme also functions as an intracellular activator of antioxidant and globin gene expression. Heme binds to the transcriptional repressor BTB and CNC homolog 1 (BACH1), which relieves BACH1's repression of gene transcription. The release of BACH1 repression increases the binding of nuclear factor erythroid 2-related factor 2 (NRF2) to antioxidant response elements (ARE) and the cell-specific transcription of antioxidant genes such as heme oxygenase-1 (HMOX1), glutathione reductase (GR), solute carrier family 7 member 11 (SLC7A11), and NAD(P)H dehydrogenase [quinone] 1 (NQO1). We have previously shown that pharmacologic activation of the NRF2 pathway in SCD mice provides protection against heme-induced vascular occlusion, is anti-inflammatory, and decreases hepatic necrosis. NRF2 activation also promotes erythroid expression of the A-gamma (HBG1) and G-gamma (HBG2) globins, which are subunits of hemoglobin F (HbF) that replace β S-globins and thus increase HbF and decrease HbS in red blood cells. Thus, BACH1 inhibitors have the potential to increase expression of antioxidant and HbF genes and prevent or reduce SCD-related pathophysiology, resulting in reduction of hemolysis, inflammation, and vaso-occlusive pain crises. Mitobridge is currently developing ML-0207/ASP8731, a highly potent, selective small molecule inhibitor of BACH1 capable of activating the Nrf2 pathway in human and murine models and investigated the ability of ML-0207 to modulate antioxidant and anti-inflammatory genes and induce HbF in human translational cellular models and a preclinical murine model of SCD.
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- 2021
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33. ML-0207/ASP8731: A Novel BACH1 Inhibitor That Induces Fetal Hemoglobin in Treatment of Sickle Cell Disease
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Nataraja, Selva, Singh, Maneet, Demes, Shilpa, Olson, Lyndsay, Stanwix, Jeff, Biddle, Margaret, Clarke, Emer, Huang, Yongzhao, Nguyen, Julia, Chen, Chunsheng, Zhang, Ping, Abdulla, Fuad, Vercellotti, Gregory M., and Belcher, John D
- Abstract
Nataraja: Mitobridge: Current Employment. Singh: Mitobridge: Current Employment. Demes: Astellas: Current Employment. Olson: Mitobridge: Current Employment. Stanwix: Mitobridge: Current Employment. Biddle: Rheos Medicine: Current Employment. Vercellotti: Mitobridge, an Astellas Company: Consultancy, Research Funding; CSL Behring: Research Funding. Belcher: Mitobridge/Astellas: Consultancy, Research Funding; CSL Behring: Research Funding.
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- 2021
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34. Morphine induces mesangial cell proliferation and glomerulopathy via κ-opioid receptors
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Weber, Marc L., Farooqui, Mariya, Nguyen, Julia, Ansonoff, Michael, Pintar, John E., Hebbel, Robert P., and Gupta, Kalpna
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Morphine sulfate (MS) stimulates mesangial cell (MC) proliferation, a process central to development of glomerular disease. The purpose of this study was to examine whether specific opioid receptors (OR) and signal transducer and activators of transcription 3 (STAT3) signaling are associated with MS-induced MC proliferation. C57Bl/6J and OR-specific knockout (KO) mice were treated for up to 6 wk with PBS, MS (0.7–2.14 mg/kg), naloxone (equimolar to MS), or MS+naloxone (n= 6 per group). Glomerular volume and expression of PCNA, Thy1, and ED1/CD68 were analyzed in kidney sections. Cell proliferation and STAT3 phosphorylation were analyzed by bromodeoxyuridine (BrdU) ELISA and Western blot, respectively, in MCs in vitro. MS treatment led to enlarged kidneys and glomerulopathy and naloxone reversed these effects. MS treatment increased glomerular volume in both μ-OR (MOR) KO and δ-OR (DOR) KO mice, but not in κ-OR (KOR) KO mice. To ascertain that MS-induced glomerulopathy in vivo was due to MC proliferation, we further examined the OR-specific effects of MS in MCs in vitro. MS-induced MC proliferation in vitro was inhibited by KOR-specific nor-BNI, but not by DOR or MOR-specific antagonists naltrindol or CTOP, respectively. KOR-specific agonist U50488H stimulated proliferation of MCs, but DOR-specific agonist DPDPE and MOR-specific agonist DAMGO did not. MS failed to stimulate proliferation of MCs from KOR KO mice. MS and KOR agonists induced STAT3 phosphorylation, and STAT3 inhibitor blocked KOR agonist-induced MC proliferation. We show that MS stimulates glomerulopathy and MC proliferation via KOR and STAT3 signaling.
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- 2008
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35. Transgenic sickle mice have vascular inflammation
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Belcher, John D., Bryant, Christopher J., Nguyen, Julia, Bowlin, Paul R., Kielbik, Miroslaw C., Bischof, John C., Hebbel, Robert P., and Vercellotti, Gregory M.
- Abstract
Inflammation may play an essential role in vaso-occlusion in sickle cell disease. Sickle patients have high white counts and elevated levels of serum C-reactive protein (CRP), cytokines, and adhesion molecules. In addition, circulating endothelial cells, leukocytes, and platelets are activated. We examined 4 transgenic mouse models expressing human α- and sickle β-globin genes to determine if they mimic the inflammatory response seen in patients. These mouse models are designated NY-S, Berk-SAntilles, NY-S/SAntilles(NY-S × Berk-SAntilles), and Berk-S. The mean white counts were elevated 1.4- to 2.1-fold (P≤ .01) in the Berk-SAntilles, NY-S/SAntilles, and Berk-S mice, but not in the NY-S mice compared with controls. Serum amyloid P-component (SAP), an acute-phase response protein with 60% to 70% sequence homology to CRP, was elevated 8.5- to 12.1-fold (P≤ .001) in transgenic sickle mice. Similarly, serum interleukin-6 (IL-6) was elevated 1.6- to 1.9-fold (P≤ .05). Western blots, confirming immunohistochemical staining, showed vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and platelet-endothelial cell adhesion molecule (PECAM) were up-regulated 3- to 5-fold (P≤ .05) in the lungs of sickle mice. Ribonuclease protection assays (RPAs) demonstrated VCAM mRNA also was elevated in sickle mice 1.2- to 1.4-fold (P≤ .01). Nuclear factor κB (NF-κB), a transcription factor critical for the inflammatory response, was elevated 1.9-fold (P≤ .006) in NY-S sickle mouse lungs. We conclude that transgenic sickle mice are good models to study vascular inflammation and the potential benefit of anti-inflammatory therapies to prevent vaso-occlusion in sickle cell disease.
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- 2003
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36. Transgenic sickle mice have vascular inflammation
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Belcher, John D., Bryant, Christopher J., Nguyen, Julia, Bowlin, Paul R., Kielbik, Miroslaw C., Bischof, John C., Hebbel, Robert P., and Vercellotti, Gregory M.
- Abstract
Inflammation may play an essential role in vaso-occlusion in sickle cell disease. Sickle patients have high white counts and elevated levels of serum C-reactive protein (CRP), cytokines, and adhesion molecules. In addition, circulating endothelial cells, leukocytes, and platelets are activated. We examined 4 transgenic mouse models expressing human α- and sickle β-globin genes to determine if they mimic the inflammatory response seen in patients. These mouse models are designated NY-S, Berk-SAntilles, NY-S/SAntilles (NY-S × Berk-SAntilles), and Berk-S. The mean white counts were elevated 1.4- to 2.1-fold (P ≤ .01) in the Berk-SAntilles, NY-S/SAntilles, and Berk-S mice, but not in the NY-S mice compared with controls. Serum amyloid P-component (SAP), an acute-phase response protein with 60% to 70% sequence homology to CRP, was elevated 8.5- to 12.1-fold (P ≤ .001) in transgenic sickle mice. Similarly, serum interleukin-6 (IL-6) was elevated 1.6- to 1.9-fold (P ≤ .05). Western blots, confirming immunohistochemical staining, showed vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and platelet-endothelial cell adhesion molecule (PECAM) were up-regulated 3- to 5-fold (P ≤ .05) in the lungs of sickle mice. Ribonuclease protection assays (RPAs) demonstrated VCAM mRNA also was elevated in sickle mice 1.2- to 1.4-fold (P ≤ .01). Nuclear factor κB (NF-κB), a transcription factor critical for the inflammatory response, was elevated 1.9-fold (P ≤ .006) in NY-S sickle mouse lungs. We conclude that transgenic sickle mice are good models to study vascular inflammation and the potential benefit of anti-inflammatory therapies to prevent vaso-occlusion in sickle cell disease.
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- 2003
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37. Microvascular Stasis Inhibition By Hemopexin in the Townes Mouse Model of Sickle Cell Disease
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Gentinetta, Thomas, Vercellotti, Gregory M., Chen, Chunsheng, Nguyen, Julia, Abdulla, Fuad, Zhang, Ping, Kato, Gregory J, Brinkman, Nathan, Zuercher, Adrian, and Belcher, John D
- Abstract
Polymerization of hemoglobin-S (HbS) in the deoxy conformation shortens the lifespan of sickle red blood cells and promotes intravascular and extravascular hemolysis. When sickle red blood cells are lysed intravascularly, HbS is released into the vascular space where it can consume nitric oxide and be oxidized to higher oxidative forms. During these reactions, ferric (Fe3+) HbS is formed, which readily releases heme. The released heme can activate the innate immune pattern recognition receptor toll-like receptor 4 (TLR4) on endothelium, leading to P-selectin expression, NF-ĸB activation, and microvascular stasis in sickle cell disease (SCD) mice with implanted dorsal skin-fold chambers (DSFCs). Heme-induced TLR4 signalling and stasis are blocked by administering hemopexin with the heme.
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- 2020
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38. Ruxolitinib Reduces Endothelial Pro-Adhesive Interactions: Implications for JAK2V617+MPN Thrombosis
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DaSilva, Angelica, Aronovich, Elena, Nguyen, Aithanh, Nguyen, Julia, Reynolds, David, Doak, Geneva D., Vercellotti, Gregory M., Wood, David K., and Beckman, Joan D.
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Introduction
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- 2020
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39. Microvascular Stasis Inhibition By Hemopexin in the Townes Mouse Model of Sickle Cell Disease
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Gentinetta, Thomas, Vercellotti, Gregory M., Chen, Chunsheng, Nguyen, Julia, Abdulla, Fuad, Zhang, Ping, Kato, Gregory J, Brinkman, Nathan, Zuercher, Adrian, and Belcher, John D
- Abstract
Gentinetta: CSL Behring: Current Employment. Vercellotti:CSL Behring: Research Funding. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment. Belcher:Mitobridge-Astellas: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Hillhurst Biopharmaceuticals: Consultancy.
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- 2020
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40. Deletion of TLR4 in Townes SS Mice Prevents Microvascular Stasis in Response to Hemin, LPS and Hypoxia/Reoxygenation: Role of Inflammation
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Belcher, John D, Abdulla, Fuad, Chen, Chunsheng, Zhang, Ping, Rivera-Rodriguez, Dormarie, Kirchner, Rachel, Nguyen, Julia, Kiser, Zachary Monroe, Nachbor, Kristine, Lloyd, Jacob, and Vercellotti, Gregory M
- Abstract
We have previously shown that heme, a damage-associated molecular pattern (DAMP), released from sickle RBCs interacts with the innate immune toll-like receptor 4 (TLR4) on endothelium and blood cells to activate cell signaling. This activates the pro-inflammatory transcription factor NF-κB leading to the production of cytokines and adhesion molecules that promote inflammation, coagulation, and vaso-occlusion (VO). Previous studies in our lab have demonstrated that inhibition of TLR4 in Townes-SS mice with TAK242 reduces microvascular stasis in response to hemin, hypoxia/reoxygenation and LPS. To further delineate the role of TLR4 in sickle cell disease (SCD), we bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. These Tlr-/- Townes mice were backcrossed 10 generations to homogenize their genetic background with our Tlr4+/+ Townes colony. Townes-SS Tlr4-/- mice developed less microvascular stasis than Townes-SS Tlr4+/+ mice in response to challenges with heme infusion (4% vs 30% at 1 hr, p<0.05, Fig 1A); LPS infusion (13% vs 36% at 1 hr, p<0.05, Fig 1B); and hypoxia/reoxygenation (3% vs 20% at 1 hr, p<0.05, Fig 1C).
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- 2019
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41. Deletion of TLR4 in Townes SS Mice Prevents Microvascular Stasis in Response to Hemin, LPS and Hypoxia/Reoxygenation: Role of Inflammation
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Belcher, John D, Abdulla, Fuad, Chen, Chunsheng, Zhang, Ping, Rivera-Rodriguez, Dormarie, Kirchner, Rachel, Nguyen, Julia, Kiser, Zachary Monroe, Nachbor, Kristine, Lloyd, Jacob, and Vercellotti, Gregory M
- Abstract
Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.
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- 2019
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42. Identification of a Heme Activation Site on the MD-2/TLR4 Complex
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Belcher, John D, Zhang, Ping, Nguyen, Julia, Kiser, Zachary Monroe, Trent, John O, and Vercellotti, Gregory M
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Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.
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- 2019
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43. Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells
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Zhang, Ping, Belcher, John D, Nguyen, Julia, Abdulla, Fuad, and Vercellotti, Gregory M
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Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.
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- 2019
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44. Bach1 Inhibitors Reduce Stasis in Townes Sickle Cell Mouse Model
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Belcher, John D, Biddle, Margaret, Stickens, Domi, Olson, Lyndsay, Bell, Eric, Chen, Chunsheng, Abdulla, Fuad, Nguyen, Julia, Kiser, Zachary Monroe, Lloyd, Jacob, and Vercellotti, Gregory M
- Abstract
Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Biddle:Mitobrige, an Astellas Company: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Stickens:Mitobridge, an Astellas Company: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Olson:Mitobridge, an Astellas Company: Employment. Bell:Mitobridge, an Astellas Company: Employment. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.
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- 2019
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45. Bach1 Inhibitors Reduce Stasis in Townes Sickle Cell Mouse Model
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Belcher, John D, Biddle, Margaret, Stickens, Domi, Olson, Lyndsay, Bell, Eric, Chen, Chunsheng, Abdulla, Fuad, Nguyen, Julia, Kiser, Zachary Monroe, Lloyd, Jacob, and Vercellotti, Gregory M
- Abstract
In sickle cell disease (SCD) excessive release of Hb and heme into the vasculature can activate innate immune toll-like receptor 4 (TLR4) signaling in the vasculature leading to inflammation and vaso-occlusion. Heme-mediated TLR4 signaling can be interrupted by inducing heme oxygenase 1 (HO-1) to degrade heme or ferritin heavy chain (FTH1) to sequester iron. Globin synthesis, HO-1 expression, heme/iron homeostasis, and antioxidant levels are regulated in part by antioxidant response genes, which is a set of hundreds of genes that possess antioxidant response elements (ARE) in their promoter regions. Bach1 complexes bind to ARE sites and dominantly repress Nrf2 transcriptional activity. This transcriptional repression can be inactivated by heme binding to Bach1. We have discovered small molecule BACH1 inhibitors that increase HO-1 and FTH1 mRNA in HepG2 cells. Additionally, they restore glutathione levels and inhibit VCAM-1 expression in human primary aortic endothelial cells upon hemin or TNFα challenge. To investigate if Bach1 inhibitors could have an effect on vaso-occlusion in vivo, microvascular stasis was examined in Townes-SS mice with implanted dorsal skin-fold chambers. Townes-SS mice were orally gavaged with vehicle or Bach1 inhibitors at different doses once daily for 8 days. Microvascular stasis was determined in 20-25 preselected flowing subcutaneous venules in response to intravenous infusion of hemin (3.2 μmol/kg). Stasis was significantly inhibited by Bach1 inhibitors in a dose responsive manner. After stasis measurement, blood was collected and the F-cells were stained using the Kleihauer-Betke Test. F-cell % was significantly increased by Bach1 inhibitor treatment at all doses tested.
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- 2019
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46. Identification of a Heme Activation Site on the MD-2/TLR4 Complex
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Belcher, John D, Zhang, Ping, Nguyen, Julia, Kiser, Zachary Monroe, Trent, John O, and Vercellotti, Gregory M
- Abstract
Lipopolysaccharide (LPS), the first-identified TLR4 agonist, binds myeloid differentiation factor-2 (MD-2) in association with TLR4 to initiate TLR4 signaling. LPS binds to a large hydrophobic pocket in MD-2 and directly bridges the MD-2/TLR4 heterodimer. The MD-2/TLR4 complex also recognizes a diverse number of endogenous molecules released from injured cells called damage-associated molecular patterns or DAMPs. One such DAMP is heme. Large amounts of heme can be released intravascularly by trauma, sepsis, malaria and red blood cell disorders such as sickle cell disease (SCD). Recent studies underscore the importance of heme-mediated MD-2/TLR4 activation in inflammation, vessel occlusion, lethality and pulmonary injury in SCD. Therefore, we examined human MD-2 for potential heme activation sites. Recombinant MD-2 (rMD-2) was produced by transfecting Chinese hamster ovary (CHO) cells with human MD-2 plasmids. After 72 hours, Western blots of the CHO-conditioned media demonstrated soluble rMD-2 was present. Heme was shown to bind rMD-2 using pull-down assays utilizing heme-agarose or biotin-heme with streptavidin-agarose coupled with MD-2 Western blots of the pellet. These pull-down assays of rMD2 were inhibited by excess heme, indicating specific binding of heme to rMD-2. UV/visible scanning spectroscopy (250 - 550 nm) of purified rMD-2 in the presence or absence of heme, confirmed specific rMD-2-heme binding. In silico analyses combining both structure and sequence-based methods, identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44 (Figure 1). To determine whether MD-2 mutations at these two sites affect heme-MD-2/TLR4 signaling, HEK293 cells were transfected with plasmids encoding human MD-2, TLR4, CD14 and an NF-κB luciferase reporter. After 24 hours, transfected cells were stimulated with heme (10 μM) or LPS (10 ng/ml) for 6 hours and NF-κB luciferase reporter activity was measured. Heme or LPS treatment elicited robust luciferase activity. The addition of both heme and LPS had an additive effect on NF-κB luciferase activity. Absence of an MD-2, TLR4 or CD14 plasmid abolished NF-κB luciferase reporter responses to heme and/or LPS. When plasmids encoding MD-2 point mutants W23A or Y34A were introduced into MD-2, heme-induced NF-κB luciferase activity was inhibited 91-92% compared to WT-MD-2. The S33A MD-2 mutant stimulated NF-κB luciferase activity by 40%. NF-κB activation by LPS was marginally affected by the same mutants. Biotin-heme/streptavidin-agarose pulled down 68% less W23A mutant MD-2 and 80% less W23A/S33A/Y34A mutant MD-2 than WT-MD-2. In contrast, at the other potential heme binding site, heme-induced NF-κB luciferase activity was increased in mutants Y36A (120%), C37A (121%) and I44A (230%) compared to WT-MD-2. These data suggest that amino acids W23 and Y34 on MD-2 are specific for heme binding and TLR4 signaling. This heme activation site was targeted for potential inhibitors using virtual screening. The virtual screen identified 60 potential inhibitors for screening in heme-stimulated primary human umbilical vein endothelial cells (HUVEC) and a human U-937 monocyte cell line. Four of these molecules inhibited Weibel-Palade body P-selectin and von Willebrand factor expression in HUVEC and IL-8 secretion by U-937 cells stimulated with heme. We conclude that heme activates MD-2/TLR4 signaling at residues W23 and Y34 on MD-2, which might be a drugable target in SCD and other hemolytic diseases.
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- 2019
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47. Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells
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Zhang, Ping, Belcher, John D, Nguyen, Julia, Abdulla, Fuad, and Vercellotti, Gregory M
- Abstract
Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide, resulting from a mutation in the beta globin gene. SCD has significant pathophysiological consequences – hemolysis, inflammation, oxidative stress, hypercoagulability, endothelial dysfunction and painful vaso-occlusive crises. The latter can be precipitated by infection or other metabolic stressors. Hemolysis chronically exposes endothelial cells, leukocytes, and platelets to hemoglobin and heme that promote pro-inflammatory and prothrombotic phenotypes. We previously demonstrated that toll-like receptor 4 (TLR4) signaling is required for microvascular stasis induced by hemoglobin, heme, or lipopolysaccharide (LPS) in sickle mice. MD-2 is a glycoprotein, co-expressed with TLR4 at the surface of various cell types, principally myeloid and endothelial lineages. MD-2 also exists as a soluble plasma protein (sMD-2), mainly as a large disulfide-bound multimeric glycoprotein, as well as oligomers and monomers. sMD-2 binds LPS and confers TLR4 sensitivity to LPS . A marked increase in sMD-2 has been reported in plasma from patients with sepsis and rheumatoid arthritis. sMD-2 in SCD plasma has not been studied. Since SCD has a pro-inflammatory phenotype, we hypothesized that sMD-2 is increased in SCD plasma and promotes pro-inflammatory signaling of endothelial cells. We assessed plasma levels of sMD-2 by Western blot and found that sMD-2 was increased 1.7-fold in SS human plasma (n=8) compared to healthy AA plasma (p<0.05, n=7). In mice, plasma sMD-2 was increased 7.6-fold in Townes-SS sickle mice (n=9) compared to control Townes-AA mice (p<0.0002, n=7). In contrast, plasma CD14, another required component of LPS-TLR4 signaling, was not significantly different in SS humans (n=8) and SS mice (n=9) compared to AA controls (p<0.05). The liver is one potential source of sMD-2 in plasma. In mice, hepatic MD-2 mRNA was increased 2.1-fold in SS compared to AA (p<0.05, n=6). Activated vascular endothelium is another potential source and target of sMD-2 in plasma. It has been reported by other groups and confirmed by us that LPS induces sMD-2 secretion by human umbilical vein endothelial cells (HUVEC). To determine whether heme can induce sMD-2 secretion from endothelial cells, we treated HUVEC with heme (0-30 μM) for 18 hours and found heme increased sMD-2 in media in a dose-responsive manner.
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- 2019
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48. Mast Cells Contribute to Brain Microvascular Permeability in Sickle Cell Disease
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Mittal, Aditya M, Tran, Huy, Sagi, Varun, Nguyen, Aithanh, Luk, Kathryn, Nguyen, Julia, Lei, Jianxun, and Gupta, Kalpna
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Gupta: Novartis: Honoraria; Tau tona: Consultancy.
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- 2018
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49. Thrombin-Mediated Activation of PAR-1 Contributes to Microvascular Stasis in Mouse Models of Sickle Cell Disease Via Increased Endothelial Expression of P-Selectin and VWF
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Sparkenbaugh, Erica, Chen, Chunsheng, Nguyen, Julia, Wang, Shaobin, Vercellotti, Gregory M., Belcher, John D, and Pawlinski, Rafal
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Vercellotti: CSL Behring: Research Funding. Belcher:CSL Behring: Research Funding.
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- 2018
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50. Targeting Elastase with Alpha1 Anti-Trypsin Reduces Hyperalgesia in Mice with Sickle Cell Disease
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Nguyen, Aithanh, Pantano, Sophia, Song-Naba, Waogwende Leonce, Jha, Ritu, Nguyen, Julia, and Gupta, Kalpna
- Abstract
Sickle cell disease (SCD) is associated with increased activity of proteases including tryptase and elastase, which can contribute to a wide variety of pathologies including endothelial activation and injury to the neural system. In turn, these effects of proteases contribute to pain, a major comorbidity of SCD (Vincent et al., Blood 2013; Vicuña et al., Nature Med 2015). The enzyme activity of proteases released from granulocytes including neutrophils is regulated by serine protease inhibitors (Serpins). Transcriptomic analysis revealed downregulation of Serpins in the dorsal root ganglion (DRG) of sickle mice (Paul et al., Sci Data 2017). Increased neutrophil activation has been observed in patients with SCD and in mouse models of SCD (Zhang et al., Blood. 2016).Therefore, we hypothesized that increased neutrophil elastase activity contributes to pain in SCD and that a Serpin, a1anti-trypsin (A1AT) would inhibit elastase activity and reduce pain in SCD. We used transgenic homozygous HbSS-BERK (sickle) and HbAA-BERK (control) mice expressing human sickle hemoglobin and normal human hemoglobin A, respectively, and Prolastin C (Grifols) an A1AT, a Food and Drug Administration (FDA) approved drug, to examine our hypothesis. We observed a significant increase in elastase activity in the plasma, lungs and DRG of sickle mice compared to control mice (p<0.002, 0.05 & 0.05, respectively). Treatment with 80 mg/Kg/day for 3 days with intraperitoneal (ip) dose of A1AT led to a significant decrease in elastase activity in the plasma, lungs and DRG of sickle mice compared to vehicle treatment (p<0.02, 0.001 & 0.05, respectively). A1AT had no significant effect on elastase activity in any of these tissues in control mice, perhaps due to constitutively low activity of elastase in control mice. DRG neurons transmit nociceptive action potentials to the dorsal horn of spinal cord where signals are further processed by the activation of neuromodulators and signaling pathways. One of the key pathways is p38 mitogen activated protein kinase (MAPK), which is activated in chronic pain and has been found to be significantly activated in the spinal cords of BERK sickle mice compared to control mice. A1AT treatment resulted in a decrease in the phosphorylation of p38 MAPK in the dorsal horn of the spinal cord of sickle mice, suggesting that inhibition of elastase attenuates the central mechanisms of chronic pain in sickle mice. Next, we examined the effect of A1AT on hyperalgesia in male sickle mice using 3 different doses of 40, 80 and 200 mg/kg injected ip on day 1 of a 3-day cycle for 3 - 5 cycles. A significant decrease for heat hyperalgesia with 40 mg/kg A1AT was observed on the 3rdday of Cycles 1, 2 and 3 (p < 0.01). The 80 mg/kg dose also reduced heat hyperalgesia on the 3rdday of Cycles 1 and 2 (p < 0.005) but at 24 hours after the injection for Cycle 3 (p < 0.05). This reduction remained significant through the end of Cycle 5. A significant difference for cold hyperalgesia with 40 mg/kg (p < 0.005) was observed after the first injection for Cycle 1. This reduction remained significant through the end of Cycle 3 (p < 0.01). Similar, but more sustained effect was noted with 80 and 200 mg/Kg dose. Thus, an optimum dose of 80 mg/Kg A1AT reduced thermal hyperalgesia significantly, which was sustained over a period of 15 days without leading to tolerance. However, none of the doses of A1AT had a significant effect on mechanical or deep tissue hyperalgesia in male sickle. Different nociceptors and channels on C-fibers mediate thermal sensitivity. It is likely that A1AT inhibition of proteases targets thermal but not mechanoreceptors. Nonetheless, it is noteworthy that cold hyperalgesia, a characteristic feature of sickle pain, is ameliorated with A1AT. In conclusion, our data demonstrate a novel role for A1AT, an FDA approved agent, to reduce thermal sensitivity in SCD. Since A1AT deficiency is known to adversely influence lung function, A1AT may even provide benefit in ameliorating lung injury observed in SCD. We speculate that proteases may be treatable targets and provide a therapeutic effect on sickle pathobiology in addition to ameliorating pain. Since several drugs containing A1AT including Prolastin C used by us are FDA approved for clinical use, our observations have the potential for developing these agents for the treatment of pain in SCD following clinical trials.
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- 2018
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