Publications by authors named "Julia Nguyen"

52 Publications

Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Front Immunol 2020 19;11:613278. Epub 2021 Jan 19.

Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN, United States.

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS- had similar complete blood counts and serum chemistries as SS- mice. However, SS- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS- mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS- livers, SS- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS- or SS- bone marrow into AA- or AA- recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA- . These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.
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http://dx.doi.org/10.3389/fimmu.2020.613278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851052PMC
January 2021

Corrigendum to "Interaction between CIEDs and modern radiotherapy techniques: Flattening filter free-VMAT, dose-rate effects, scatter radiation, and neutron-generating energies" [Radiother Oncol 152 (2020) 196-202].

Radiother Oncol 2021 Jan 15;154:291. Epub 2021 Jan 15.

First Department of Medicine, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Germany; DZHK (German Centre for Cardiovascular Research) partner site, Mannheim, Germany.

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http://dx.doi.org/10.1016/j.radonc.2020.12.023DOI Listing
January 2021

Nimodipine after aneurysmal subarachnoid hemorrhage: Fourteen-day course for patients that meet criteria for early hospital discharge.

Clin Neurol Neurosurg 2021 Jan 8;200:106299. Epub 2020 Oct 8.

Department of Neurosurgery, University of Virginia, Charlottesville, VA, United States. Electronic address:

Background: Randomized-controlled trials and meta-analyses showed nimodipine use after aneurysmal subarachnoid hemorrhage (aSAH) leads to reduction in incidence of cerebral infarction, persistent neurological deficits, and poor outcomes. Trials administered it for 21 days; however, we assessed whether a shorter duration might be reasonable for a subset of patients.

Methods: We performed a retrospective single-center study to compare outcomes between patients who received ≤14 days, 15-20 days or ≥21 days of nimodipine. Primary outcome was defined as rate of good functional outcome at final follow-up, assessed using dichotomized modified Rankin Score (mRS). Secondary outcomes included median mRS at follow-up, discharge disposition, and readmission for stroke or vasospasm.

Results: 195 patients were included: 101 patients received nimodipine for ≤14 days, 72 patients for 15-20 days, and 22 patients for ≥21 days. There were differences in baseline characteristics of the groups. The shorter duration groups had higher admission GCS score (GCS 15 for ≤14 days, GCS 13 for 15-20 days, GCS 8 for ≥21 days, p = 0.003) and lower Hunt-Hess grade (2 for ≤14 days, 3 for 15-20 days, 4 for ≥21 days, p = 0.001). Of the group of patients that received ≤14 days of nimodipine, 3 patients (3%) were readmitted for concerns for possible stroke or vasospasm, but they did not experience worsening of their functional status related to this.

Conclusion: Our data suggests a more limited 14-day course of nimodipine therapy after aSAH may be reasonable and efficacious in patients with higher GCS and lower Hunt-Hess grade on presentation.
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http://dx.doi.org/10.1016/j.clineuro.2020.106299DOI Listing
January 2021

Changes in Vestibular Function in Patients With Head-and-Neck Cancer Undergoing Chemoradiation.

Ear Nose Throat J 2020 Sep 14:145561320949482. Epub 2020 Sep 14.

Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Mannheim, Germany.

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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http://dx.doi.org/10.1177/0145561320949482DOI Listing
September 2020

Heme Causes Pain in Sickle Mice Toll-Like Receptor 4-Mediated Reactive Oxygen Species- and Endoplasmic Reticulum Stress-Induced Glial Activation.

Antioxid Redox Signal 2021 02 24;34(4):279-293. Epub 2020 Aug 24.

Vascular Biology Center, Division of Hematology, Oncology & Transplantation, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA.

Lifelong pain is a hallmark feature of sickle cell disease (SCD). How sickle pathobiology evokes pain remains unknown. We hypothesize that increased cell-free heme due to ongoing hemolysis activates toll-like receptor 4 (), leading to the formation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Together, these processes lead to spinal microglial activation and neuroinflammation, culminating in acute and chronic pain. Spinal heme levels, TLR4 transcripts, oxidative stress, and ER stress were significantly higher in sickle mice than controls. , TLR4 inhibition in spinal cord microglial cells attenuated heme-induced ROS and ER stress. Heme treatment led to a time-dependent increase in the characteristic features of sickle pain (mechanical and thermal hyperalgesia) in both sickle and control mice; this effect was absent in TLR4-knockout sickle and control mice. TLR4 deletion in sickle mice attenuated chronic and hypoxia/reoxygenation (H/R)-evoked acute hyperalgesia. Sickle mice treated with the TLR4 inhibitor resatorvid; selective small-molecule inhibitor of TLR4 (TAK242) had significantly reduced chronic hyperalgesia and had less severe H/R-evoked acute pain with quicker recovery. Notably, reducing ER stress with salubrinal ameliorated chronic hyperalgesia in sickle mice. Our findings demonstrate the causal role of free heme in the genesis of acute and chronic sickle pain and suggest that TLR4 and/or ER stress are novel therapeutic targets for treating pain in SCD. Heme-induced microglial activation TLR4 in the central nervous system contributes to the initiation and maintenance of sickle pain ER stress in SCD. . 34, 279-293.
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http://dx.doi.org/10.1089/ars.2019.7913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821434PMC
February 2021

Identification of a Heme Activation Site on the MD-2/TLR4 Complex.

Front Immunol 2020 30;11:1370. Epub 2020 Jun 30.

Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN, United States.

Myeloid differentiation factor-2 (MD-2) binds lipopolysaccharide (LPS) and initiates toll-like receptor-4 (TLR4) pro-inflammatory signaling. Heme also activates TLR4 signaling, but it is unknown if heme interacts with MD-2. Therefore, we examined MD-2 for a potential heme activation site. Heme-agarose and biotin-heme/streptavidin-agarose pulled down recombinant MD-2, which was inhibited by excess free heme. UV/visible spectroscopy confirmed MD-2-heme binding. To determine whether MD-2 was required for heme-mediated TLR4 signaling, HEK293 cells were transfected with MD-2, TLR4, CD14, and an NF-κB luciferase reporter, and then stimulated with heme or LPS. Heme or LPS treatment elicited robust reporter activity. Absence of MD-2, TLR4 or CD14 plasmid abolished NF-κB reporter responses to heme or LPS. analysis identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44. Heme-induced NF-κB activity was reduced by 39 and 78% in HEK293 cells transfected with MD-2 mutants W23A and Y34A, respectively, compared to WT-MD-2. NF-κB activation by LPS was not affected by the same mutants. Biotinyl-heme/streptavidin-agarose pulled down 68% less W23A and 80% less W23A/S33A/Y34A mutant MD-2 than WT-MD-2. In contrast, at the Y36/C37/I44 MD-2 site, heme-induced NF-κB activity was significantly increased by mutants Y36A (191% of WT-MD-2) and unchanged by mutants C37A and I44A (95 and 92%, respectively, of WT-MD-2). In conclusion, these data suggest that heme binds and activates TLR4 signaling at amino acids W23 and Y34 on MD-2.
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http://dx.doi.org/10.3389/fimmu.2020.01370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338675PMC
June 2020

Association of Childhood Trauma Exposure with Inflammatory Biomarkers Among Midlife Women.

J Womens Health (Larchmt) 2020 Dec 4;29(12):1540-1546. Epub 2020 May 4.

Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Childhood abuse has been associated with poor health outcomes in adulthood. However, the physiologic pathways by which abuse is linked to health are not fully elucidated. Inflammation plays a significant role in the pathophysiology of multiple chronic diseases. We tested whether childhood trauma exposure was related to increased systemic inflammation in midlife women. Participants were 304 nonsmoking perimenopausal and postmenopausal women aged 40 to 60 years and free of cardiovascular disease. They completed questionnaires assessing psychosocial and behavioral factors, including childhood trauma, anthropometric measures, wrist actigraphy sleep measurements, and a fasting blood draw for inflammatory markers high-sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6). Associations between childhood trauma and inflammatory markers were tested in linear regression models controlling for age, race/ethnicity, education, body mass index, anti-inflammatory medication use, and alcohol consumption. Other covariates considered included sleep continuity and depressive symptoms. A total of 44.8% of the sample experienced at least one type of childhood abuse/neglect. Women with a history of emotional abuse had higher IL-6 levels than women without this history in multivariate models ( = 0.077, standard error = 0.032,  = 0.017). Results were not accounted for by covariates and persisted additionally controlling for depressive symptoms and sleep. Childhood abuse/neglect was not related to hsCRP. Childhood emotional abuse was associated with higher levels of IL-6 in midlife women. Assessing childhood trauma exposure along with inflammatory markers may be important for the development of prevention strategies at midlife to prevent chronic diseases later in life.
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http://dx.doi.org/10.1089/jwh.2019.7779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757571PMC
December 2020

Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease.

Blood 2020 05;135(20):1783-1787

UNC Blood Research Center, Division of Hematology/Oncology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC.

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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http://dx.doi.org/10.1182/blood.2019003543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225686PMC
May 2020

Interaction between CIEDs and modern radiotherapy techniques: Flattening filter free-VMAT, dose-rate effects, scatter radiation, and neutron-generating energies.

Radiother Oncol 2020 Nov 20;152:196-202. Epub 2020 Jan 20.

First Department of Medicine, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Germany; DZHK (German Centre for Cardiovascular Research) partner site, Mannheim, Germany.

Background And Purpose: Providing evidence for radiotherapy (RT)-induced effects on cardiac implantable electric devices (CIEDs) with focus on flattening filter free-volumetric modulated arc therapy (FFF-VMAT) at 6 and 10 MV as well as 3D-conformal radiotherapy (3D-CRT) at 18 MV.

Materials And Methods: 68 CIEDs (64 implantable cardioverter-defibrillators (ICDs) and 4 cardiac pacemakers (PMs)) were located on the left chest position on a slab phantom and irradiated under telemetrical surveillance either directly, or distant to 3D-CRT or FFF-VMAT, dose-rate 2500 cGy/min, and target dose of 150 Gy. Devices were placed within, close by (2.5 cm and 5 cm), and distant (35 cm) to the radiation field. Scatter radiation (SR) and photon neutrons (PN) were recorded. CIEDs were investigated in following groups: 1a) 18 MV 3D-CRT - 4 ICDs/4 PMs out of radiation field, 1b) 18 MV open field - 4 ICDs/4 PMs within radiation field, 2) 6 MV FFF-VMAT, 15 ICDs in 35 cm distance to VMAT, 3) 10 MV-FFF VMAT, 15 ICDs in 35 cm distance to VMAT, 4) 6 MV FFF-VMAT, 15 ICDs in 2.5 cm distance to VMAT, 5) 10 MV FFF-VMAT, 15 ICDs in 2.5 cm distance to VMAT.

Results: No incidents occurred at 6 MV FFF. 10 MV FFF-VMAT and 18 MV 3D-CRT resulted in data loss, reset, and erroneous sensing with inhibition of pacing (leading to inadequate defibrillation) in 8/34 ICDs and 2/4 PMs which were not located within radiation. Direct radiation triggered instantaneous defibrillation in 3/4 ICDs.

Conclusions: 6 MV FFF-VMAT is safe even at high dose-rates of 2500 cGy/min, regardless whether CIEDs are located close (2.5 cm) or distant (35 cm) to the radiation beam. CIEDs should never be placed within radiation and energy should always be limited to 6 MV. At 6 MV, VMAT at high dose-rates can be used to treat tumors, which are located close to CIEDs.
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http://dx.doi.org/10.1016/j.radonc.2019.12.007DOI Listing
November 2020

Decreased erythrocyte binding of Siglec-9 increases neutrophil activation in sickle cell disease.

Blood Cells Mol Dis 2020 03 20;81:102399. Epub 2019 Dec 20.

Division of Hematology, Oncology and Transplantation, University of Minnesota School of Medicine, Minneapolis, MN, United States of America. Electronic address:

Oxidative stress and inflammation promote vaso-occlusion in sickle cell disease (SCD). CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids inhibit innate immune cell functions. We have shown that Siglec-9 on human neutrophils interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactive oxygen species (ROS). We hypothesized that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and thus a decreased neutrophil oxidative burst. SS erythrocytes express significantly more sialic acid than AA erythrocytes (p = 0.02). SS erythrocytes displayed significantly less Siglec-9-Fc binding 39% ± 11 (mean ± SEM) compared to AA erythrocytes 78% ± 5 (p = 0.009). Treatment of AA erythrocytes with sialidase to remove sialic acid decreased binding to 3% ± 7.9 (p ≤ 0.001). When freshly isolated neutrophils were incubated with AA erythrocytes, neutrophils achieved 16% ± 6 of the oxidative burst exhibited by a stimulated neutrophil without erythrocytes. In contrast, neutrophils incubated with SS erythrocytes achieved 47% ± 6 of the oxidative burst (AA versus SS, p = 0.03). Stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation while with SS erythrocytes NETs increased. SS erythrocytes are deficient in binding to neutrophil Siglec-9 which may contribute to the increased oxidative stress in SCD.
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http://dx.doi.org/10.1016/j.bcmd.2019.102399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002293PMC
March 2020

A novel, highly potent and selective phosphodiesterase-9 inhibitor for the treatment of sickle cell disease.

Haematologica 2020 03 30;105(3):623-631. Epub 2019 May 30.

INSERM UMR 1163, CNRS ERL 8254, Imagine Institute, Laboratory of Excellence GR-Ex, Paris Descartes - Sorbonne Paris Cité University, Paris, France.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated using a two-phase liquid culture of CD34 progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD.
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http://dx.doi.org/10.3324/haematol.2018.213462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049346PMC
March 2020

Sexual dysfunction in female cancer survivors: A narrative review.

Gen Hosp Psychiatry 2019 Sep - Oct;60:141-147. Epub 2019 Apr 18.

University of Pittsburgh School of Medicine, United States of America.

Objectives: Due to improvements in earlier detection and expansions in available treatments, the number of individuals surviving with cancer is steadily increasing. Sexual dysfunction is a common and often persistent complication for cancer survivors, affecting >60% of women diagnosed with cancer. Although highly prevalent, issues related to sexual health are often not addressed among survivors, with women reporting less discussion with providers compared to men.

Methods: In this narrative review, we present a case series of three women seen in a psycho-oncology clinic who experienced sexual dysfunction following a cancer diagnosis. We then review existing literature on the presentation and management of sexual issues associated with cancer and its treatment.

Results: The three cases highlight different mechanisms of sexual dysfunction after cancer, including anatomic changes, hormonal alterations, psychiatric conditions and medication side effects. The literature review includes discussion of the prevalence and course of sexual dysfunction in female cancer survivors. Tools for screening and assessment are then reviewed, as well as contributing factors and common presenting symptoms. We conclude with a discussion of both pharmacologic and non-pharmacologic approaches to management.

Conclusions: Despite its high prevalence and considerable impact on quality of life, the complication of sexual dysfunction after cancer diagnosis and treatment is still under recognized and undertreated. Improving awareness, communication, and screening, as well as appropriate referral to treatment, could have a profound impact on the ever growing number of women surviving with cancer with sexual health concerns.
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http://dx.doi.org/10.1016/j.genhosppsych.2019.04.003DOI Listing
April 2020

Nickel-catalyzed anti-Markovnikov hydroarylation of alkenes.

Chem Sci 2019 Mar 13;10(11):3231-3236. Epub 2019 Feb 13.

Department of Chemistry , University of Washington , Seattle , Washington 98195 , USA . Email:

We have developed a nickel-catalyzed hydroarylation of alkenes using aryl halides as coupling partners. Excellent anti-Markovnikov selectivity is achieved with aryl-substituted alkenes and enol ethers. We also show that hydroarylation occurs with alkyl substituted alkenes to yield linear products. Preliminary examination of the reaction mechanism suggests irreversible hydrometallation as the selectivity determining step of the hydroarylation.
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http://dx.doi.org/10.1039/c8sc05445bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429615PMC
March 2019

Mast Cells Induce Blood Brain Barrier Damage in SCD by Causing Endoplasmic Reticulum Stress in the Endothelium.

Front Cell Neurosci 2019 19;13:56. Epub 2019 Feb 19.

Vascular Biology Center, Division of Hematology, Oncology and Transplantation, Department of Medicine, Medical School, University of Minnesota, Minneapolis, MN, United States.

Endothelial dysfunction underlies the pathobiology of cerebrovascular disease. Mast cells are located in close proximity to the vasculature, and vasoactive mediators released upon their activation can promote endothelial activation leading to blood brain barrier (BBB) dysfunction. We examined the mechanism of mast cell-induced endothelial activation endoplasmic reticulum (ER) stress mediated P-selectin expression in a transgenic mouse model of sickle cell disease (SCD), which shows BBB dysfunction. We used mouse brain endothelial cells (mBECs) and mast cells-derived from skin of control and sickle mice to examine the mechanisms involved. Compared to control mouse mast cell conditioned medium (MCCM), mBECs incubated with sickle mouse MCCM showed increased, structural disorganization and swelling of the ER and Golgi, aggregation of ribosomes, ER stress marker proteins, accumulation of galactose-1-phosphate uridyl transferase, mitochondrial dysfunction, reactive oxygen species (ROS) production, P-selectin expression and mBEC permeability. These effects of sickle-MCCM on mBEC were inhibited by Salubrinal, a reducer of ER stress. Histamine levels in the plasma, skin releasate and in mast cells of sickle mice were higher compared to control mice. Compared to control BBB permeability was increased in sickle mice. Treatment of mice with imatinib, Salubrinal, or P-selectin blocking antibody reduced BBB permeability in sickle mice. Mast cells induce endothelial dysfunction ER stress-mediated P-selectin expression. Mast cell activation contributes to ER stress mediated endothelial P-selectin expression leading to increased endothelial permeability and impairment of BBB. Targeting mast cells and/or ER stress has the potential to ameliorate endothelial dysfunction in SCD and other pathobiologies.
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http://dx.doi.org/10.3389/fncel.2019.00056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389721PMC
February 2019

Critical role of C5a in sickle cell disease.

Am J Hematol 2019 03 3;94(3):327-337. Epub 2019 Jan 3.

Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, Minnesota.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.
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http://dx.doi.org/10.1002/ajh.25384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986874PMC
March 2019

Oral carbon monoxide therapy in murine sickle cell disease: Beneficial effects on vaso-occlusion, inflammation and anemia.

PLoS One 2018 11;13(10):e0205194. Epub 2018 Oct 11.

University of Minnesota, Department of Medicine, Vascular Research Center, Division of Hematology, Oncology and Transplantation, Minneapolis, MN, United States of America.

Carbon monoxide (CO) at low, non-toxic concentrations has been previously demonstrated to exert anti-inflammatory protection in murine models of sickle cell disease (SCD). However CO delivery by inhalation, CO-hemoglobin infusion or CO-releasing molecules presents problems for daily CO administration. Oral administration of a CO-saturated liquid avoids many of these issues and potentially provides a platform for self-administration to SCD patients. To test if orally-delivered CO could modulate SCD vaso-occlusion and inflammation, a liquid CO formulation (HBI-002) was administered by gavage (10 ml/kg) once-daily to NY1DD and Townes-SS transgenic mouse models of SCD. Baseline CO-hemoglobin (CO-Hb) levels were 1.6% and 1.8% in NY1DD and Townes-SS sickle mice and 0.6% in Townes-AS control mice. CO-Hb levels reached 5.4%, 4.7% and 3.0% within 5 minutes in NY1DD, SS and AS mice respectively after gavage with HBI-002. After ten treatments, each once-daily, hemoglobin levels rose from 5.3g/dL in vehicle-treated Townes-SS mice to 6.3g/dL in HBI-002-treated. Similarly, red blood cell (RBC) counts rose from 2.36 x 106/μL in vehicle-treated SS mice to 2.89 x 106/μL in HBI-002-treated mice. In concordance with these findings, hematocrits rose from 26.3% in vehicle-treated mice to 30.0% in HBI-002-treated mice. Reticulocyte counts were not significantly different between vehicle and HBI-002-treated SS mice implying less hemolysis and not an increase in RBC production. White blood cell counts decreased from 29.1 x 103/μL in vehicle-treated versus 20.3 x 103/μL in HBI-002-treated SS mice. Townes-SS mice treated with HBI-002 had markedly increased Nrf2 and HO-1 expression and decreased NF-κB activation compared to vehicle-treated mice. These anti-inflammatory effects were examined for the ability of HBI-002 (administered orally once-daily for up to 5 days) to inhibit vaso-occlusion induced by hypoxia-reoxygenation. In NY1DD and Townes-SS sickle mice, HBI-002 decreased microvascular stasis in a duration-dependent manner. Collectively, these findings support HBI-002 as a useful anti-inflammatory agent to treat SCD and warrants further development as a therapeutic.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205194PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181332PMC
March 2019

Molecular detection of inflammation in cell models using hyperpolarized C-pyruvate.

Theranostics 2018 23;8(12):3400-3407. Epub 2018 May 23.

Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, California, USA.

The detection and treatment monitoring of inflammatory states remain challenging in part due to the multifactorial mechanisms of immune activation and spectrum of clinical manifestations. Currently, diagnostic strategies tend to be subjective and limited quantitative tools exist to monitor optimal treatment strategies. Pro-inflammatory M1 polarized macrophages exhibit a distinct metabolic glycolytic phenotype compared to the continuum of M2 polarization states. In the present study, the distinct metabolic phenotypes of resting and activated macrophages were successfully characterized and quantified using hyperpolarized carbon-13 (C) labeled pyruvate and its metabolic products, i.e. lactate, as a biomarker of resting, disease and treated states. : Mouse macrophage J774A.1 cells were used as a model system in an NMR compatible bioreactor to facilitate dynamic hyperpolarized C measurements. The glycolytic metabolism of the cells in the quiescent or resting state were compared with macrophages stimulated by lipopolysaccharide, a classical M1 activator using hyperpolarized C labeled pyruvate. Additionally, the activated macrophages were also treated with a non-steroidal anti-inflammatory drug to assess the changes in hyperpolarized lactate signal. The hyperpolarized lactate signals were then correlated using biochemical and molecular assays. : We first validated our model system of inflammatory cells by the hallmarks of M1 polarization using steady state metabolic profiling with high resolution NMR in conjunction with nitric oxide Greiss assay, enzyme activity, and mRNA expression. Subsequently, we clearly showed that the cutting edge technology of hyperpolarized C NMR can be used to detect elevated lactate levels in M1 polarized macrophages in comparison to control and non-steroidal anti-inflammatory drug treated M2 states. Hyperpolarized C lactate has the potential to serve as a biomarker to non-invasively detect and quantify pro-inflammatory state of immune regulatory cells and its response to therapy.
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http://dx.doi.org/10.7150/thno.24322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010986PMC
August 2019

Haptoglobin and hemopexin inhibit vaso-occlusion and inflammation in murine sickle cell disease: Role of heme oxygenase-1 induction.

PLoS One 2018 25;13(4):e0196455. Epub 2018 Apr 25.

Department of Medicine, Division of Hematology, Oncology and Transplantation, Vascular Biology Center, University of Minnesota, Minneapolis, Minnesota, United States of America.

During hemolysis, hemoglobin and heme released from red blood cells promote oxidative stress, inflammation and thrombosis. Plasma haptoglobin and hemopexin scavenge free hemoglobin and heme, respectively, but can be depleted in hemolytic states. Haptoglobin and hemopexin supplementation protect tissues, including the vasculature, liver and kidneys. It is widely assumed that these protective effects are due primarily to hemoglobin and heme clearance from the vasculature. However, this simple assumption does not account for the consequent cytoprotective adaptation seen in cells and organs. To further address the mechanism, we used a hyperhemolytic murine model (Townes-SS) of sickle cell disease to examine cellular responses to haptoglobin and hemopexin supplementation. A single infusion of haptoglobin or hemopexin (± equimolar hemoglobin) in SS-mice increased heme oxygenase-1 (HO-1) in the liver, kidney and skin several fold within 1 hour and decreased nuclear NF-ĸB phospho-p65, and vaso-occlusion for 48 hours after infusion. Plasma hemoglobin and heme levels were not significantly changed 1 hour after infusion of haptoglobin or hemopexin. Haptoglobin and hemopexin also inhibited hypoxia/reoxygenation and lipopolysaccharide-induced vaso-occlusion in SS-mice. Inhibition of HO-1 activity with tin protoporphyrin blocked the protections afforded by haptoglobin and hemopexin in SS-mice. The HO-1 reaction product carbon monoxide, fully restored the protection, in part by inhibiting Weibel-Palade body mobilization of P-selectin and von Willebrand factor to endothelial cell surfaces. Thus, the mechanism by which haptoglobin and hemopexin supplementation in hyperhemolytic SS-mice induces cytoprotective cellular responses is linked to increased HO-1 activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196455PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919001PMC
July 2018

The functional organization of cortical feedback inputs to primary visual cortex.

Nat Neurosci 2018 05 16;21(5):757-764. Epub 2018 Apr 16.

Champalimaud Research, Champalimaud Center for the Unknown, Lisbon, Portugal.

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.
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http://dx.doi.org/10.1038/s41593-018-0135-zDOI Listing
May 2018

The Influence of Solid Microneedles on the Transdermal Delivery of Selected Antiepileptic Drugs.

Pharmaceutics 2016 Nov 15;8(4). Epub 2016 Nov 15.

Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844, USA.

The aim of this project was to examine the effect of microneedle rollers on the percutaneous penetration of tiagabine hydrochloride and carbamazepine across porcine skin in vitro. Liquid chromatography-mass spectrometric analysis was carried out using an Agilent 1200 Series HPLC system coupled to an Agilent G1969A TOF-MS system. Transdermal flux values of the drugs were determined from the steady-state portion of the cumulative amount versus time curves. Following twelve hours of microneedle roller application, there was a 6.74-fold increase in the percutaneous penetration of tiagabine hydrochloride (86.42 ± 25.66 µg/cm²/h) compared to passive delivery (12.83 ± 6.30 µg/cm²/h). For carbamazepine in 20% ethanol, passive transdermal flux of 7.85 ± 0.60 µg/cm²/h was observed compared to 10.85 ± 0.11 µg/cm²/h after microneedle treatment. Carbamazepine reconstituted in 30% ethanol resulted in only a 1.19-fold increase in drug permeation across porcine skin (36.73 ± 1.83 µg/cm²/h versus 30.74 ± 1.32 µg/cm²/h). Differences in flux values of untreated and microneedle-treated porcine skin using solid microneedles for the transdermal delivery of tiagabine were statistically significant. Although there were 1.38- and 1.19-fold increases in transdermal flux values of carbamazepine when applied as 20% and 30% ethanol solutions across microneedle-treated porcine skin, respectively, the increases were not statistically significant.
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http://dx.doi.org/10.3390/pharmaceutics8040033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198017PMC
November 2016

Sustained treatment of sickle cell mice with haptoglobin increases HO-1 and H-ferritin expression and decreases iron deposition in the kidney without improvement in kidney function.

Br J Haematol 2016 Nov 10;175(4):714-723. Epub 2016 Aug 10.

Department of Hematology, Oncology, and Transplantation, University of Minnesota Medical School, Minneapolis, MN, USA.

There is growing evidence that extracellular haemoglobin and haem mediate inflammatory and oxidative damage in sickle cell disease. Haptoglobin (Hp), the scavenger for free haemoglobin, is depleted in most patients with sickle cell disease due to chronic haemolysis. Although single infusions of Hp can ameliorate vaso-occlusion in mouse models of sickle cell disease, prior studies have not examined the therapeutic benefits of more chronic Hp dosing on sickle cell disease manifestations. In the present study, we explored the effect of Hp treatment over a 3-month period in sickle mice at two dosing regimens: the first at a moderate dose of 200 mg/kg thrice weekly and the second at a higher dose of 400 mg/kg thrice weekly. We found that only the higher dosing regimen resulted in increased haem-oxygenase-1 and heavy chain ferritin (H-ferritin) expression and decreased iron deposition in the kidney. Despite the decreased kidney iron deposition following Hp treatment, there was no significant improvement in kidney function. However, there was a nearly significant trend towards decreased liver infarction.
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http://dx.doi.org/10.1111/bjh.14280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118172PMC
November 2016

Hepatic Overexpression of Hemopexin Inhibits Inflammation and Vascular Stasis in Murine Models of Sickle Cell Disease.

Mol Med 2016 Sep 19;22:437-451. Epub 2016 Jul 19.

Division of Hematology, Oncology and Transplantation, Department of Medicine, 420 Delaware St SE, MMC 480, University of Minnesota, Minneapolis, Minnesota, USA.

Sickle cell disease (SCD) patients have low serum hemopexin (Hpx) levels due to chronic hemolysis. We hypothesize that in SCD mice, hepatic overexpression of hemopexin will scavenge the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis. To examine the protective role of Hpx in SCD, we transplanted bone marrow from NY1DD SCD mice into Hpx or Hpx C57BL/6 mice. Dorsal skin fold chambers were implanted in week 13 post-transplant and microvascular stasis (% non-flowing venules) evaluated in response to heme infusion. Hpx sickle mice had significantly greater microvascular stasis in response to heme infusion than Hpx sickle mice (p<0.05), demonstrating the protective effect of Hpx in SCD. We utilized Sleeping Beauty (SB) transposon-mediated gene transfer to overexpress wild-type rat Hpx (wt-Hpx) in NY1DD and Townes-SS SCD mice. Control SCD mice were treated with lactated Ringer's solution (LRS) or a luciferase (Luc) plasmid. Plasma and hepatic Hpx were significantly increased compared to LRS and Luc controls. Microvascular stasis in response to heme infusion in NY1DD and Townes-SS mice overexpressing wt-Hpx had significantly less stasis than controls (p<0.05). Wt-Hpx overexpression markedly increased hepatic nuclear Nrf2 expression, HO-1 activity and protein, the heme-Hpx binding protein and scavenger receptor, CD91/LRP1 and decreased NF-κB activation. Two missense (ms)-Hpx SB-constructs that bound neither heme nor the Hpx receptor, CD91/LRP1, did not prevent heme-induced stasis. In conclusion, increasing Hpx levels in transgenic sickle mice via gene transfer activates the Nrf2/HO-1 anti-oxidant axis and ameliorates inflammation and vaso-occlusion.
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http://dx.doi.org/10.2119/molmed.2016.00063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082291PMC
September 2016

Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors.

Proc Natl Acad Sci U S A 2016 Apr 31;113(16):4464-9. Epub 2016 Mar 31.

Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University, Stanford, CA 94305; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.
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http://dx.doi.org/10.1073/pnas.1600007113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843455PMC
April 2016

Control of Oxidative Stress and Inflammation in Sickle Cell Disease with the Nrf2 Activator Dimethyl Fumarate.

Antioxid Redox Signal 2017 05 30;26(14):748-762. Epub 2016 Mar 30.

1 Division of Hematology, Oncology and Transplantation, Department of Medicine, Vascular Biology Center, University of Minnesota , Minneapolis, Minnesota.

Aims: Heme derived from hemolysis is pro-oxidative and proinflammatory and promotes vaso-occlusion in murine models of sickle cell disease (SCD), suggesting that enhanced detoxification of heme may be beneficial. Nuclear factor erythroid-2-related factor-2 (Nrf2) transcription pathway is the principal cellular defense system responding to pro-oxidative and proinflammatory stress. Dimethyl fumarate (DMF), a drug approved for treatment of multiple sclerosis, provides neuroprotection by activating Nrf2-responsive genes. We hypothesized that induction of Nrf2 with DMF would be beneficial in murine SCD models.

Results: DMF (30 mg/kg/day) or vehicle (0.08% methyl cellulose) was administered for 3-7 days to NY1DD and HbSS-Townes SCD mice. Vaso-occlusion, a hallmark of SCD, measured in sickle mice with dorsal skinfold chambers, was inhibited by DMF. The inhibitory effect of DMF was abrogated by the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin. DMF increased nuclear Nrf2 and cellular mRNA of Nrf2-responsive genes in livers and kidneys. DMF increased heme defenses, including HO-1, haptoglobin, hemopexin, and ferritin heavy chain, although plasma hemoglobin and heme levels were unchanged. DMF decreased markers of inflammation, including nuclear factor-kappa B phospho-p65, adhesion molecules, and toll-like receptor 4. DMF administered for 24 weeks to HbSS-Townes mice decreased hepatic necrosis, inflammatory cytokines, and irregularly shaped erythrocytes and increased hemoglobin F, but did not alter hematocrits, reticulocyte counts, lactate dehydrogenase, plasma heme, or spleen weights, indicating that the beneficial effects of DMF were not attributable to decreased hemolysis.

Innovation: These studies identify Nrf2 activation as a new therapeutic target for the treatment of SCD.

Conclusion: DMF activates Nrf2, enhances antioxidant defenses, and inhibits inflammation and vaso-occlusion in SCD mice. Antioxid. Redox Signal. 26, 748-762.
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http://dx.doi.org/10.1089/ars.2015.6571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421647PMC
May 2017

Influence of polymer molecular weight on in vitro dissolution behavior and in vivo performance of celecoxib:PVP amorphous solid dispersions.

Eur J Pharm Biopharm 2016 Apr 17;101:145-51. Epub 2016 Feb 17.

Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz, D-55128 Mainz, Germany.

In this study, the influence of the molecular weight of polyvinylpyrrolidone (PVP) on the non-sink in vitro dissolution and in vivo performance of celecoxib (CCX):PVP amorphous solid dispersions were investigated. The dissolution rate of CCX from the amorphous solid dispersions increased with decreasing PVP molecular weight and crystallization inhibition was increased with increasing molecular weight of PVP, but reached a maximum for PVP K30. This suggested that the crystallization inhibition was not proportional with molecular weight of the polymer, but rather there was an optimal molecular weight where the crystallization inhibition was strongest. Consistent with the findings from the non-sink in vitro dissolution tests, the amorphous solid dispersions with the highest molecular weight PVPs (K30 and K60) resulted in significantly higher in vivo bioavailability (AUC0-24h) compared with pure amorphous and crystalline CCX. A linear relationship between the in vitro and in vivo parameter AUC0-24h indicated that the simple non-sink in vitro dissolution method used in this study could be used to predict the in vivo performance of amorphous solid dispersion with good precision, which enabled a ranking between the different formulations. In conclusion, the findings of this study demonstrated that the in vitro and in vivo performance of CCX:PVP amorphous solid dispersions were significantly controlled by the molecular weight of the polymer.
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http://dx.doi.org/10.1016/j.ejpb.2016.02.007DOI Listing
April 2016

Influence of Copolymer Composition on In Vitro and In Vivo Performance of Celecoxib-PVP/VA Amorphous Solid Dispersions.

AAPS J 2016 Mar 14;18(2):416-23. Epub 2016 Jan 14.

Pharmaceutical Science and CMC Biologics, H. Lundbeck A/S, 2500, Valby, Denmark.

Previous studies suggested that an amorphous solid dispersion with a copolymer consisting of both hydrophobic and hydrophilic monomers could improve the dissolution profile of a poorly water-soluble drug compared to the crystalline form. Therefore, this study investigated the influence of the copolymer composition of polyvinylpyrrolidone/vinyl acetate (PVP/VA) on the non-sink in vitro dissolution behavior and in vivo performance of celecoxib (CCX) amorphous solid dispersions. The study showed that the hydrophilic monomer vinylpyrrolidone (VP) was responsible for the generation of CCX supersaturation whereas the hydrophobic monomer vinyl acetate (VA) was responsible for the stabilization of the supersaturated solution. For CCX, there was an optimal copolymer composition around 50-60% VP content where further replacement of VP monomers with VA monomers did not have any biopharmaceutical advantages. A linear relationship was found between the in vitro AUC(0-4h) and in vivo AUC(0-24h) for the CCX:PVP/VA systems, indicating that the non-sink in vitro dissolution method applied in this study was useful in predicting the in vivo performance. These results indicated that when formulating a poorly water-soluble drug as an amorphous solid dispersion using a copolymer, the copolymer composition has a significant influence on the dissolution profile and in vivo performance. Thus, the dissolution profile of a drug can theoretically be tailored by changing the monomer ratio of a copolymer with respect to the required in vivo plasma-concentration profile. As this ratio is likely to be drug dependent, determining the optimal ratio between the hydrophilic (dissolution enhancing) and hydrophobic (crystallization inhibiting) monomers for a given drug is imperative.
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http://dx.doi.org/10.1208/s12248-016-9865-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779114PMC
March 2016

Cannabinoid receptor-specific mechanisms to alleviate pain in sickle cell anemia via inhibition of mast cell activation and neurogenic inflammation.

Haematologica 2016 05 24;101(5):566-77. Epub 2015 Dec 24.

Vascular Biology Center, Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN, USA

Sickle cell anemia is a manifestation of a single point mutation in hemoglobin, but inflammation and pain are the insignia of this disease which can start in infancy and continue throughout life. Earlier studies showed that mast cell activation contributes to neurogenic inflammation and pain in sickle mice. Morphine is the common analgesic treatment but also remains a major challenge due to its side effects and ability to activate mast cells. We, therefore, examined cannabinoid receptor-specific mechanisms to mitigate mast cell activation, neurogenic inflammation and hyperalgesia, using HbSS-BERK sickle and cannabinoid receptor-2-deleted sickle mice. We show that cannabinoids mitigate mast cell activation, inflammation and neurogenic inflammation in sickle mice via both cannabinoid receptors 1 and 2. Thus, cannabinoids influence systemic and neural mechanisms, ameliorating the disease pathobiology and hyperalgesia in sickle mice. This study provides 'proof of principle' for the potential of cannabinoid/cannabinoid receptor-based therapeutics to treat several manifestations of sickle cell anemia.
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http://dx.doi.org/10.3324/haematol.2015.136523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004356PMC
May 2016

Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice.

Haematologica 2015 Dec 20;100(12):1517-25. Epub 2015 Aug 20.

Vascular Biology Center and Division of Hematology-Oncology-Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN, USA

Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia.
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http://dx.doi.org/10.3324/haematol.2015.128736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666327PMC
December 2015

Single-Cell Analysis of [18F]Fluorodeoxyglucose Uptake by Droplet Radiofluidics.

Anal Chem 2015 Jul 15;87(13):6667-73. Epub 2015 Jun 15.

‡Department of Chemistry and Biochemistry, Santa Clara University, Santa Clara, California 95053, United States.

Radiolabels can be used to detect small biomolecules with high sensitivity and specificity without interfering with the biochemical activity of the labeled molecule. For instance, the radiolabeled glucose analogue, [18F]fluorodeoxyglucose (FDG), is routinely used in positron emission tomography (PET) scans for cancer diagnosis, staging, and monitoring. However, despite their widespread usage, conventional radionuclide techniques are unable to measure the variability and modulation of FDG uptake in single cells. We present here a novel microfluidic technique, dubbed droplet radiofluidics, that can measure radiotracer uptake for single cells encapsulated into an array of microdroplets. The advantages of this approach are multiple. First, droplets can be quickly and easily positioned in a predetermined pattern for optimal imaging throughput. Second, droplet encapsulation reduces cell efflux as a confounding factor, because any effluxed radionuclide is trapped in the droplet. Last, multiplexed measurements can be performed using fluorescent labels. In this new approach, intracellular radiotracers are imaged on a conventional fluorescence microscope by capturing individual flashes of visible light that are produced as individual positrons, emitted during radioactive decay, traverse a scintillator plate placed below the cells. This method is used to measure the cell-to-cell heterogeneity in the uptake of tracers such as FDG in cell lines and cultured primary cells. The capacity of the platform to perform multiplexed measurements was demonstrated by measuring differential FDG uptake in single cells subjected to different incubation conditions and expressing different types of glucose transporters. This method opens many new avenues of research in basic cell biology and human disease by capturing the full range of stochastic variations in highly heterogeneous cell populations in a repeatable and high-throughput manner.
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http://dx.doi.org/10.1021/acs.analchem.5b00792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669076PMC
July 2015

The fucosylation inhibitor, 2-fluorofucose, inhibits vaso-occlusion, leukocyte-endothelium interactions and NF-ĸB activation in transgenic sickle mice.

PLoS One 2015 23;10(2):e0117772. Epub 2015 Feb 23.

Department of Medicine, Vascular Biology Center, Division of Hematology, Oncology and Transplantation, Minneapolis, Minnesota, United States of America.

2-Fluorofucose (2FF) blocks the fucosylation and the tethering of sialyl-Lewisx tetrasaccharide and structural variants on leukocytes and red blood cells to P- and E-selectins on activated endothelial cell surfaces. Because P- and E-selectin are required for vaso-occlusion in murine sickle cell disease (SCD), we investigated whether 2FF would inhibit vaso-occlusion in SCD mice. Microvascular stasis was measured in subcutaneous venules in NY1DD and HbSS-Townes SCD mice with dorsal skin-fold chambers after infusion of hemoglobin or exposure to hypoxia/reoxygenation. 2FF in drinking water or administered by gavage inhibited stasis in sickle mice in a dose-responsive manner. Significant inhibitory effects on stasis were seen 1 day post-treatment. 2FF treatment of SCD mice also significantly reduced leukocyte rolling and adhesion along the vessel walls of SCD mice and the static adhesion of neutrophils and sickle red blood cells isolated from 2FF-treated SCD mice to resting and activated endothelial cells. Total white blood cell counts increased in response to 2FF. NF-ĸB activation and VCAM-1 and E-selectin expression were inhibited in the livers of SCD mice consistent with an overall decrease in vascular inflammation and ischemia-reperfusion physiology. Pretreatment with 2FF completely eliminated heme-induced lethality in HbSS-Townes mice, consistent with the observed anti-inflammatory and anti-adhesive properties of 2FF in SCD mice. These data suggest that 2FF may be beneficial for preventing or treating vaso-occlusive crises in SCD patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117772PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338063PMC
January 2016