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1.  The surgical learning curve and accuracy of minimally invasive lumbar pedicle screw placement using CT based computer-assisted navigation plus continuous electromyography monitoring – a retrospective review of 627 screws in 150 patients 
This study retrospectively assessed the accuracy of placement of lumbar pedicle screws placed by a single surgeon using a minimally-invasive, intra-operative CT-based computer navigated technique in combination with continuous electromyography (EMG) monitoring. The rates of incorrectly positioned screws were reviewed in the context of the surgeon's experience and learning curve.
Data was retrospectively reviewed from all consecutive minimally invasive lumbar fusions performed by the primary author over a period of over 4 years from April 2008 until October 2012. All cases that had utilized computer-assisted intra-operative CT-based image guidance and continuous EMG monitoring to guide percutaneous pedicle screw placement were analysed for the rates of malposition of the pedicle screws. Pedicle screw malposition was defined as having occurred if the screw trajectory was adjusted intraoperatively due to positive EMG responses, or due to breach of the pedicle cortex by more than 2mm on intraoperative CT imaging performed at the end of the instrumentation procedure. Further analysis of the data was undertaken to determine if the rates of malposition changed with the surgeon's experience with the technique.
Six hundred and twenty-seven pedicle screws were placed in one hundred and fifty patients. The overall rate of intraoperative malposition and subsequent adjustment of pedicle screw placement was 3.8% (24 of 627 screws). Screw malposition was detected by intraoperative CT imaging. Warning of potential screw misplacement was provided by use of the EMG monitoring. With increased experience with the technique, rates of intraoperative pedicle screw malposition were found to decrease from 5.1% of screws in the first fifty patients, to 2.0% in the last 50 patients. Only one screw was suboptimally placed at the end of surgery, which did not result in a neurological deficit.
The use of CT-based computer-assisted navigation in combination with continuous EMG monitoring during percutaneous transpedicular screw placement results in very low rates of malposition and neural injury that compare favourably with previously reported rates. Pedicle screw placement accuracy continues to improve as the surgeon becomes more experienced with the technique.
PMCID: PMC4325487
minimally invasive spine surgery; lumbar fusion; Pedicle screw; Spinal navigation; learning curve
2.  Vitamin D Supplementation Suppresses Hypoxia-Stimulated Placental Cytokine Secretion, Hypertension and CD4+ T Cell Stimulation in Response to Placental Ischemia 
To investigate a role of Vitamin D in the pathogenesis of preeclampsia (PE), and to discern any potential benefits of Vitamin D supplementation on hypertension in the RUPP rat model of PE.
Study design
Blood and placentas from normal pregnancies (NP) and PE were collected following elective cesarean delivery without evidence of infection. Circulating Vitamin D was extracted by HPLC and measured via mass spectrometry. Media for placenta explants was supplemented with Vitamin D and exposed to hypoxic (1% O2) or normoxic (6% O2) conditions for 24 hours. ELISAs were performed on media and normalized to total protein to determine cytokine secretion. RUPP rats were supplemented with vitamin D by oral gavage, and blood pressure (MAP) and pup weights were measured in NP and RUPP rats with or without Vitamin D supplementation. Flow cytometry was used to evaluate CD4+ Tcells in control RUPP rats and RUPP rats treated with Vitamin D.
Inflammatory cytokine secretion was higher (p<0.05) while the anti-inflammatory cytokine, IL-10, was significantly lower in the media of PE placentas compared to NP (p=0.005). Vitamin D supplementation decreased hypoxia stimulated pro-inflammatory cytokine secretion (p=0.003) in the media of PE placentas. Vitamin D decreased MAP and circulating CD4+ T cells in the RUPP rat model of PE (p<0.05).
Vitamin D supplementation may be useful in the treatment or prevention of hypertensive disorders in pregnancy.
PMCID: PMC4235666  PMID: 25414911
Vitamin D; Hypertension; Preeclampsia; Cytokines
3.  CD4+ T Cells Play a Critical Role in Mediating Hypertension in Response to Placental Ischemia 
Similar to preeclamptic women, hypertension in the chronic Reduced Uterine Perfusion Pressure Rat Model Of Preeclampsia (RUPP) is associated with increased CD4+ T cells, cytokines, sFlt-1 and agonistic autoantibodies to the AngII receptor (AT1-AA). We examined the effect inhibition of T cell co-stimulation in RUPP rats treated with (A) (abatacept, 250 mg/kg, infused i.v. at gestation day 13), on hypertension and sFlt-1, TNF-α and AT1-AA. RUPP surgical procedure was performed on day 14. On day 19 MAP increased from 94+2 mmHg in Normal Pregnant (NP) to 123 ± 3 mmHg in RUPP control rats. This response was attenuated by Abatacept, MAP was 104 ± 2 mmHg in RUPP ± A, and 96 ± 2 mmHg NP ± A. Percent circulating CD4+ T cells were 66 ± 3% in RUPPs compared to 55 ± 3% NP rats (p<0.04) but were normalized in RUPP ± A rats (54 ± 3%). The twofold increase in TNF alpha seen in RUPPs (277 ± 47 pg/ml) was decreased to 80 ± 18 pg/ml in RUPP+A. Placental sFlt-1 was reduced 70 % to 151 ± 28 in RUPP ± A compared 488 ± 61 pg/ml in RUPP (p<0.001). AT1-AA decreased from 20 ± 0.8 bpm in control RUPP to 6 ± 0.7 bpm in RUPP ± A. We next determined the effect of RUPP in causing hypertension in pregnant T cell deficient rats by examining MAP in NP (123 ± 5 mmHg) and RUPP athymic nude rats (123 ± 7 mmHg). In the absence of T cells, hypertension in response to placental ischemia was completely abolished. Collectively these data indicate that CD4+ Tcells in response to placental ischemia play an important role in the pathophysiology of hypertension associated with preeclampsia.
PMCID: PMC4231445  PMID: 25401050
Hypertension; Preeclampsia; Placental ischemic insult
4.  Endothelin-1, oxidative stress and endogenous ANGII: mechanisms of AT1-AA-enhanced Renal and Blood Pressure Response during pregnancy 
Hypertension  2013;62(5):10.1161/HYPERTENSIONAHA.113.01648.
Hypertension during preeclampsia is associated with increased maternal vascular sensitivity to angiotensin II (ANGII). This study was designed to determine mechanisms whereby agonistic autoantibodies to the ANGII type I receptor (AT1-AA) enhance blood pressure (MAP) and renal vascular sensitivity to ANGII during pregnancy. First, we examined MAP and renal artery resistance index (RARI) in response to chronic administration of ANGII or AT1-AA or AT1-AA+ANGII in pregnant rats compared to control pregnant rats. In order to examine mechanisms of heightened sensitivity in response to AT1-AA during pregnancy we examined the role of endogenous ANGII in AT1-AA infused pregnant rats, Endothelin-1 and oxidative stress in AT1-AA+ANGII treated rats. Chronic ANGII increased MAP from 95 +/−2 in NP rats to 115 +/−2 mmHg. Chronic AT1-AA increased MAP to 118+/−1 mmHg in NP rats which further increased to 123+/−2 with AT1-AA+ANGII. Increasing ANGII from (10−11-10−8) decreased Af-Art diameter 15-20% but sharply decreased Af-Art diameter 60% in AT1-AA pretreated vessels. RARI increased from 0.67 in NP rats to 0.70 with AT1-AA infusion, which was exacerbated to 0.74 in AT1-AA + ANGII infused rats. AT1-AA-induced hypertension decreased with Enalapril but was not attenuated. Both tissue ET-1 and ROS increased with AT1-AA+ANGII compared to AT1-AA alone and blockade of either of these pathways had significant effects on MAP or RARI. These data support the hypothesis that AT1-AA, via activation of ET-1 and oxidative stress and interaction with endogenous ANGII, are important mechanisms whereby MAP and renal vascular responses are enhanced during preeclampsia.
PMCID: PMC3845356  PMID: 24041954
Angiotensin II; AT1-AA; Preeclampsia
6.  Integration of L-R Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2 
Developmental cell  2013;26(6):629-644.
A critical aspect of gut morphogenesis is initiation of a leftward tilt. Failure to do so leads to gut malrotation and volvulus. The direction of tilt is specified by asymmetric cell behaviors within the dorsal mesentery (DM), which suspends the gut tube, and is downstream of Pitx2, the key transcription factor responsible for the transfer of left-right (L-R) information from early gastrulation to morphogenesis. Although Pitx2 is a master regulator of L-R organ development, its cellular targets that drive asymmetric morphogenesis are not known. Using laser microdissection and targeted gene misexpression in the chicken DM, we show that Pitx2-specific effectors mediate Wnt signaling to activate the formin Daam2, a key Wnt effector and itself a Pitx2 target, linking actin dynamics to cadherin-based junctions, to ultimately generate asymmetric cell behaviors. Our work highlights how integration of two conserved cascades may be the ultimate force through which Pitx2 sculpts L-R organs.
PMCID: PMC3965270  PMID: 24091014
7.  Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes 
Developmental biology  2014;393(1):3-9.
Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J × FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.
PMCID: PMC4166609  PMID: 24984260
Gene targeting; Pigmentation; Knockout; Mosaic; Genome editing; Melanocyte
8.  Progesterone supplementation attenuates hypertension and the autoantibody to the angiotensin II type I receptor in response to elevated interleukin-6 during pregnancy 
Preeclampsia is a multisystem disorder recognized as hypertension with proteinuria developing >20 weeks’ gestation. Preeclampsia is associated with chronic immune activation characterized by increased T and B lymphocytes, cytokines, and antibodies activating the angiotensin II type I receptor (AT1-AA). Hypertension in response to elevated interleukin (IL)-6 during pregnancy occurs with increased renin activity and AT1-AA, and reduced kidney function.
We aim to determine whether 17-alpha-hydroxyprogesterone caproate (17-OHPC), progesterone, improved inflammatory pathways during elevated IL-6 in pregnant rats. IL-6 (5 ng/d) was infused via miniosmotic pumps into normal pregnant (NP) rats beginning on day 14 of gestation and 17-OHPC (3.32 mg/kg) was diluted in normal saline and injected on day 18. Blood pressure (mean arterial pressure [MAP]) determination and serum collection were performed on day 19 of gestation.
MAP in NP was 100 ± 3 mm Hg, which increased with IL-6 to 112 ± 4 mm Hg (P < .05). Pregnant rats given 17-OHPC alone had a MAP of 99 ± 3 mm Hg and MAP increased to 103 ± 2 mm Hg in IL-6OHPC. AT1-AA was 1.2 ± 0.5 bpm in NP rats, increased to 17±9 bpm with IL-6 infusion but administration of 17-OHPC significantly blunted AT1-AA to 4 ± 0.8 bpm in NP6OHPC. Total circulating nitrate/nitrite was significantly decreased and placental Ser1177-phosporylated-eNOS/eNOS was lowered with IL-6 infusion. Supplementation of 17-OHPC significantly improved placental Ser1177-phosporylated-eNOS/eNOS however, circulating nitrate/nitrite was unchanged with 17-OHPC supplementation.
This study illustrates that 17-OHPC attenuated hypertension, decreased AT1-AA activity, and improved placental nitric oxide in response to elevated IL-6 during pregnancy and could lend hope to a new potential therapeutic for preeclampsia.
PMCID: PMC4117731  PMID: 24548847
cytokines; hypertension; inflammation; nitric oxide; pregnancy; progesterone; renin angiotensin system
9.  The Roles of Mechanical Stresses in the Pathogenesis of Osteoarthritis 
Cartilage  2013;4(4):286-294.
Excessive joint surface loadings, either single (acute impact event) or repetitive (cumulative contact stress), can cause the clinical syndrome of osteoarthritis (OA). Despite advances in treatment of injured joints, the risk of OA following joint injuries has not decreased in the last 50 years. Cumulative excessive articular surface contact stress that leads to OA results from post-traumatic joint incongruity and instability, and joint dysplasia, but also may cause OA in patients without known joint abnormalities. In vitro investigations show that excessive articular cartilage loading triggers release of reactive oxygen species (ROS) from mitochondria, and that these ROS cause chondrocyte death and matrix degradation. Preventing release of ROS or inhibiting their effects preserves chondrocytes and their matrix. Fibronectin fragments released from articular cartilage subjected to excessive loads also stimulate matrix degradation; inhibition of molecular pathways initiated by these fragments prevents this effect. Additionally, injured chondrocytes release alarmins that activate chondroprogentior cells in vitro that propogate and migrate to regions of damaged cartilage. These cells also release chemokines and cytokines that may contribute to inflammation that causes progressive cartilage loss. Distraction and motion of osteoarthritic human ankles can promote joint remodeling, decrease pain and improve joint function in patients with end-stage post-traumatic OA. These advances in understanding of how altering mechanical stresses can lead to remodeling of osteoarthritic joints and how excessive stress causes loss of articular cartilage, including identification of mechanically induced mediators of cartilage loss, provide the basis for new biologic and mechanical approaches to the prevention and treatment of OA.
PMCID: PMC4109888  PMID: 25067995
post-traumatic osteoarthritis; joint injury; mechanical loading of joints; joint instability; alarmins
10.  Inhaled birch pollen extract induces airway hyperresponsiveness via oxidative stress but independently of pollen-intrinsic NADPH oxidase activity, or the Toll-like receptor 4-TRIF pathway 
Oxidative stress in allergic asthma may result from oxidase activity or pro-inflammatory molecules in pollens. Signaling via TLR4 and its adaptor TRIF has been implicated in reactive oxygen species (ROS)-mediated acute lung injury and in T helper 2 immune responses. We investigated the contributions of oxidative stress and TLR4/TRIF signaling to experimental asthma induced by birch pollen exposure exclusively via the airways. Mice were exposed to native or heat-inactivated white birch pollen extract (BPEx) intratracheally and injected with the antioxidants, N-acetyl-L-cysteine (NAC) or dimethylthiourea (DMTU) prior to sensitization, challenge, or all allergen exposures, to assess the role of oxidative stress and pollen-intrinsic NADPH oxidase activity in allergic sensitization, inflammation and airway hyperresponsiveness (AHR). Additionally, TLR4 signaling was antagonized concomitantly with allergen exposure, or the development of allergic airway disease was evaluated in TLR4 or TRIF knockout mice. NAC inhibited BPEx-induced eosinophilic airway inflammation and AHR except when given exclusively during sensitization, whereas DMTU was inhibitory even when administered with the sensitization alone. Heat-inactivation of BPEx had no effect on the development of allergic airway disease. Oxidative stress-mediated AHR was also TLR4- and TRIF-independent, however, TLR4 deficiency decreased, while TRIF deficiency increased BPEx-induced airway inflammation. In conclusion, oxidative stress plays a significant role in allergic sensitization to pollen via the airway mucosa, but the pollen-intrinsic NADPH oxidase activity and TLR4 or TRIF signaling are unnecessary for the induction of allergic airway disease and AHR. Pollen extract does, however, activate TLR4, thereby enhancing airway inflammation which is restrained by the TRIF-dependent pathway.
PMCID: PMC3983962  PMID: 23776177
12.  Pyrethroid susceptibility of malaria vectors in four Districts of western Kenya 
Parasites & Vectors  2014;7:310.
Increasing pyrethroid resistance in malaria vectors has been reported in western Kenya where long lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the mainstays of vector control. To ensure the sustainability of insecticide-based malaria vector control, monitoring programs need to be implemented. This study was designed to investigate the extent and distribution of pyrethroid resistance in 4 Districts of western Kenya (Nyando, Rachuonyo, Bondo and Teso). All four Districts have received LLINs while Nyando and Rachuonyo Districts have had IRS campaigns for 3–5 years using pyrethroids. This study is part of a programme aimed at determining the impact of insecticide resistance on malaria epidemiology.
Three day old adult mosquitoes from larval samples collected in the field, were used for bioassays using the WHO tube bioassay, and mortality recorded 24 hours post exposure. Resistance level was assigned based on the 2013 WHO guidelines where populations with <90% mortality were considered resistant. Once exposed, samples were identified to species using PCR.
An. arabiensis comprised at least 94% of all An. gambiae s.l. in Bondo, Rachuonyo and Nyando. Teso was a marked contrast case with 77% of all samples being An. gambiae s.s. Mortality to insecticides varied widely between clusters even in one District with mortality to deltamethrin ranging from 45-100%, while to permethrin the range was 30-100%. Mortality to deltamethrin in Teso District was < 90% in 4 of 6 clusters tested in An arabiensis and <90% in An. gambiae s.s in 5 of 6 clusters tested. To permethrin, mortality ranged between 5.9-95%, with <90% mortality in 9 of 13 and 8 of 13 in An. arabiensis and An. gambiae s.s. respectively. Cluster specific mortality of An. arabiensis between permethin and deltamethrin were not correlated (Z = 2.9505, P = 0.2483).
High levels of pyrethroid resistance were observed in western Kenya. This resistance does not seem to be associated with either species or location. Insecticide resistance can vary within small geographical areas and such heterogeneity may make it possible to evaluate the impact of resistance on malaria and mosquito parameters within similar eco-epidemiological zones.
PMCID: PMC4094666  PMID: 24996418
Mortality; Insecticide resistance; Pyrethroids; IRS
13.  Concomitant Exposure to Ovalbumin and Endotoxin Augments Airway Inflammation but Not Airway Hyperresponsiveness in a Murine Model of Asthma 
PLoS ONE  2014;9(6):e98648.
Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1β, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells.We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA.
PMCID: PMC4072597  PMID: 24968337
14.  Progesterone blunts vascular endothelial cell secretion of endothelin-1 in response to placental ischemia 
Preeclampsia (PE) is associated with hypertension and elevated endothelin (ET-1), an indicator of endothelial cell activation and dysfunction. Reduction of uteroplacental perfusion (RUPP) in the pregnant rat model of PE is characterized by elevated mean arterial pressure, inflammatory cytokines, and activation of the ET-1 system. We aim to determine whether 17-alpha-hydroxyprogesterone caproate (17-OHPC) or progesterone suppresses these pathways.
Plasma progesterone was purified from normal pregnant (NP) and PE patients and measured via enzyme-linked immunosorbent assay. Human umbilical vein endothelial cells were exposed to the sera with or without progesterone added and ET-1 was measured. Pregnant rats underwent the RUPP procedure with or without intraperitoneal 17-OHPC. Mean arterial pressure was compared in RUPP vs NP rats. Human umbilical vein endothelial cells were exposed to NP or RUPP sera, with and without progesterone and ET-1 measured.
Progesterone was significantly decreased in PE women compared with NP women. In response to human sera, ET-1 was elevated in PE women compared to NP women, and decreased with addition of progesterone. Mean arterial pressure was significantly elevated in RUPP vs NP rats but was attenuated by 17-OHPC. ET-1 secretion was stimulated significantly by RUPP compared to NP rat sera, but attenuated by progesterone.
Circulating progesterone is significantly lower in PE women compared to controls. 17-OHPC attenuates hypertension in response to placental ischemia in RUPP rats. Progesterone blunts vascular ET-1 stimulated at cellular level by sera from PE women or RUPP rats. Decreased circulating progesterone is associated with stimulation of ET-1. 17-OHPC supplementation blunts hypertension and progesterone blunts endothelial cell ET-1 secretion in response to placental ischemia.
PMCID: PMC4052216  PMID: 23545163
endothelin; placental ischemia; preeclampsia; progesterone
A common in vitro model for studying acute mechanical damage in cartilage is to impact an isolated osteochondral or cartilage specimen with a metallic impactor. The mechanics of a cartilage-on-cartilage (COC) impact, as encountered in vivo, are likely different than those of a metal-on-cartilage (MOC) impact. The hypothesis of this study was that impacted in vitro COC and MOC specimens would differ in their impact behavior, mechanical properties, chondrocyte viability, cell metabolism, and histologic structural damage. Osteochondral specimens were impacted with either an osteochondral plug or a metallic cylinder at the same delivered impact energy per unit area, and processed after 14 days in culture. The COC impacts resulted in about half of the impact maximum stress and a quarter of the impact maximum stress rate of change, as compared to the MOC impacts. The impacted COC specimens had smaller changes in mechanical properties, smaller decreases in chondrocyte viability, higher total proteoglycan content, and less histologic structural damage, as compared to the impacted MOC specimens. If metal-on-cartilage impact conditions are to be used for modeling of articular injuries and post-traumatic osteoarthritis, the differences between COC and MOC impacts must be kept in mind.
PMCID: PMC3740544  PMID: 23335281
Articular cartilage; biochemical analysis; histology; impact testing; post-traumatic osteoarthritis
16.  Noncontact quantitative biomechanical characterization of cardiac muscle using shear wave imaging optical coherence tomography 
Biomedical Optics Express  2014;5(7):1980-1992.
We report on a quantitative optical elastographic method based on shear wave imaging optical coherence tomography (SWI-OCT) for biomechanical characterization of cardiac muscle through noncontact elasticity measurement. The SWI-OCT system employs a focused air-puff device for localized loading of the cardiac muscle and utilizes phase-sensitive OCT to monitor the induced tissue deformation. Phase information from the optical interferometry is used to reconstruct 2-D depth-resolved shear wave propagation inside the muscle tissue. Cross-correlation of the displacement profiles at various spatial locations in the propagation direction is applied to measure the group velocity of the shear waves, based on which the Young’s modulus of tissue is quantified. The quantitative feature and measurement accuracy of this method is demonstrated from the experiments on tissue-mimicking phantoms with the verification using uniaxial compression test. The experiments are performed on ex vivo cardiac muscle tissue from mice with normal and genetically altered myocardium. Our results indicate this optical elastographic technique is useful as a noncontact tool to assist the cardiac muscle studies.
PMCID: PMC4102343  PMID: 25071943
(170.6935) Tissue characterization; (170.4500) Optical coherence tomography
17.  Imaging biopsy composition at ACL reconstruction 
Early-stage osteoarthritis (OA) includes glycosaminoglycan (GAG) loss and collagen disruption that cannot be seen on morphological magnetic resonance imaging (MRI). T1ρ MRI is a measurement that probes the low-frequency rate of exchange between protons of free water and those from water associated with macromolecules in the cartilage’s extracellular matrix. While it has been hypothesized that increased water mobility resulting from early osteoarthritic changes cause elevated T1ρ MRI values, there remain several unknown mechanisms influencing T1ρ measurements in cartilage. The purpose of this work was to relate histological and biochemical metrics directly measured from osteochondral biopsies and fluid specimens with quantitative MRI-detected changes of in vivo cartilage composition.
Patients and methods
Six young patients were enrolled an average of 41 days after acute anterior cruciate ligament (ACL) rupture. Femoral trochlear groove osteochondral biopsies, serum, and synovial fluid were harvested during ACL reconstruction to complement a presurgery quantitative MRI study (T1ρ, T2, delayed gadolinium-enhanced MRI of cartilage [dGEMRIC] relaxation times). A high-resolution MRI scan of the excised osteochondral biopsy was also collected. Analyses of in vivo T1ρ images were compared with ex vivo T1ρ imaging, GAG assays and histological GAG distribution in the osteochondral biopsies, and direct measures of bone and cartilage turnover markers and “OA marker” 3B3 in serum and synovial fluid samples.
T1ρ relaxation times in patients with a torn ACL were elevated from normal, indicating changes consistent with general fluid effusion after blunt joint trauma. Increased chondrogenic progenitor cell (CPC) production of chondroprotective lubricin may relate to cartilage surface disruption by blunt trauma and CPC amplification of joint inflammation. Disparity between ex vivo and matched in vivo MRI of trochlear cartilage suggests MRI signal differences that may be related to the synovial fluid environment. T1ρ is emerging as a promising MRI biomarker to relate noninvasive measures of whole-joint condition and cartilage composition to direct measures of cartilage changes in the acute phase of joint injuries.
PMCID: PMC4028072  PMID: 24855396
proteoglycan; osteochondral biopsy; T1ρ; biomarker
18.  AEOL10150: A Novel Therapeutic for Rescue Treatment Following Toxic Gas Lung Injury1 
Free radical biology & medicine  2010;50(5):602-608.
New therapeutics designed as rescue treatments following toxic gas injury such as chlorine (Cl2) are an emerging area of interest. We tested the effects of the metalloporphyrin catalytic antioxidant AEOL10150, a compound that scavenges peroxynitrite, inhibits lipid peroxidation, and has SOD and catalase-like activities, on Cl2-induced airway injury.
Balb/C mice received 100 ppm Cl2 gas for five minutes. Four groups were studied; Cl2 only, Cl2 followed by AEOL10150 one and nine hours after exposure, AEOL10150 only, and control. Twenty-four hours following Cl2 gas exposure airway responsiveness to aerosolized methacholine (6.25–50 mg/mL) was measured using a small animal ventilator. Bronchoalveolar lavage (BAL) was performed to assess airway inflammation and protein. Whole lung tissue was assayed for 4-hydroxynonenal. In separate groups, lungs were collected at 72 hours following Cl2 injury to evaluate epithelial cell proliferation.
Mice exposed to Cl2 showed a significantly higher airway resistance than control, Cl2/AEOL10150, or AEOL10150-only treated animals in response to methacholine challenge. Eosinophils, neutrophils and macrophages were elevated in BAL of Cl2-exposed mice. AEOL10150 attenuated the increases in neutrophils and macrophages. AEOL10150 prevented Cl2-induced increase in BAL fluid protein. Chlorine induced an increase in number of proliferating airway epithelial cells, an effect AEOL10150 attenuated. 4-hydroxynonenal levels in the lung were increased following Cl2 and this effect was prevented with AEOL10150.
AEOL10150 is an effective rescue treatment for Cl2-induced airway hyperresponsiveness, airway inflammation, injury-induced airway epithelial cell regeneration and oxidative stress.
PMCID: PMC4026011  PMID: 21156205
19.  Prejudice Events and Traumatic Stress among Heterosexuals and Lesbians, Gay Men and Bisexuals 
Journal of aggression, maltreatment & trauma  2013;22(5):10.1080/10926771.2013.785455.
This mixed-methods study examined associations between prejudice events and posttraumatic stress disorder (PTSD) among 382 lesbians, gays, and bisexuals (LGB) and 126 heterosexuals. Using the Composite International Diagnostic Interview, we assessed PTSD with a relaxed Criterion A1; that is, we allowed events that did not involve threat to life or physical integrity to also qualify as traumatic. We first assessed whether exposure to prejudice-related qualifying events differed with respect to participants’ sexual orientation and race. We found that White LGBs were more likely than White heterosexuals to encounter a prejudice-related qualifying event, and among LGBs, Black and Latino LGBs were no more likely than White LGBs to experience this type of event. We then used qualitative analysis of participants’ brief narratives to examine prejudice events that precipitated Relaxed Criterion A1 PTSD among 8 participants. Two themes emerged: (a) the need to make major changes and (b) compromised sense of safety and security following exposure to the prejudice event.
PMCID: PMC3885323  PMID: 24415898
lesbian; gay; and bisexual; PTSD; Criterion A1; prejudice; discrimination
20.  A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3′ untranslated region-mediated gene regulation 
Nucleic Acids Research  2014;42(10):e86.
Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3′ untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3′ UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3′ UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome—a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3′ UTR of interest. The pBUTR (piggyBac-based 3′ UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.
PMCID: PMC4041432  PMID: 24753411
21.  Sphingosine 1-Phosphate (S1P) Induced Interleukin-8 (IL-8) Release Is Mediated by S1P Receptor 2 and Nuclear Factor κB in BEAS-2B Cells 
PLoS ONE  2014;9(4):e95566.
The airway epithelium may release pro-inflammatory cytokines and chemokines in the asthmatic airway. Sphingosine 1-phosphate (S1P) is a bioactive lipid, increased in the airways of asthmatics, that may trigger the release of the potent neutrophil chemoattractant Interleukin-8 (IL-8) by epithelial cells. S1P is a ligand for 5 G protein-coupled receptors, S1PR1-5. We wished to explore the mechanisms of S1P induced IL-8 secretion with regard to the receptor(s) and downstream signaling events involved. Our results indicate that S1P induced IL-8 release is mediated by S1PR2 and the transcription factor NF-κB. Since the Epidermal Growth Factor Receptor (EGFR) and reactive oxygen species (ROS) have been implicated in IL-8 release in response to activation of other G protein-coupled receptors, we examined their importance in S1P induced IL-8 release and established that they are not involved. This study reveals S1PR2 and NF-κB as potential therapeutic targets in neutrophilic airway diseases such as severe asthma.
PMCID: PMC3990666  PMID: 24743449
22.  Yin-Yang 1, a new player in early heart development 
Circulation research  2013;112(6):876-877.
PMCID: PMC3955736  PMID: 23493301
23.  Odontoid pannus formation in a patient with ankylosing spondylitis causing atlanto-axial instability 
BMJ Case Reports  2012;2012:bcr1120115178.
Ankylosing spondylitis is one of the commonest inflammatory diseases of the axial skeleton and can be complicated by atlanto-axial instability. This serious and likely underestimated complication can be easily overlooked. However, there are clear features which can help alert suspicion to initiate the appropriate investigations with imaging that is very effective at diagnosing and assessing this complication. The authors report an unusual case where odontoid pannus formation, akin to that seen in rheumatoid arthritis, was the underlying cause.
PMCID: PMC3291001  PMID: 22665557
24.  Fatigue is Specific to Working Muscles: No Cross-over with Single-leg Cycling in Trained Cyclists 
Fatigue induced via a maximal isometric contraction of a single-limb muscle group can evoke a “cross-over” of fatigue that reduces voluntary muscle activation and maximum isometric force in the rested contralateral homologous muscle group. We asked whether a cross-over of fatigue also occurs when fatigue is induced via high-intensity endurance exercise involving a substantial muscle mass. Specifically, we used high-intensity single-leg cycling to induce fatigue and evaluated associated effects on maximum cycling power (Pmax) in the fatigued ipsilateral leg (FATleg) as well as the rested contralateral leg (RESTleg). On separate days, 12 trained cyclists performed right leg Pmax trials before and again 30s, 3, 5, and 10min after a cycling time trial (TT, 10min) performed either with their right or left leg. Fatigue was estimated by comparing exercise-induced changes in Pmax and maximum handgrip isometric force (Fmax). Mean power produced during the right and left leg TT’s did not differ (203±8 vs. 199±8W). Compared to pre-TT, FATleg Pmax was reduced by 22±3% at 30s post-TT and remained reduced by 9±2% at 5min post-TT (both P<0.05). Despite considerable power loss in the FATleg, post-TT RESTleg Pmax (596–603W) did not differ from pre-TT values (596±35W). There were no alterations in handgrip Fmax (529–547N). Our data suggest that any potential cross-over of fatigue, if present at all, was not sufficient to measurably compromise RESTleg Pmax in trained cyclists. These results along with the lack of changes in handgrip Fmax indicate that impairments in maximal voluntary neuromuscular function were specific to working muscles.
PMCID: PMC3934423  PMID: 22806085
Muscle fatigue; central fatigue; neuromuscular function; contralateral limb
25.  Mechanical Stress and ATP Synthesis are coupled by Mitochondrial Oxidants in Articular Cartilage 
Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. It is believed that physiological strains cause deformation of the mitochondria, potentially releasing ROS for energy production. We hypothesized that mechanical loading stimulates ATP synthesis via mitochondrial release of ROS. Bovine osteochondral explants were dynamically loaded at 0.5Hz with amplitude of 0.25MPa for 1 Hour. Cartilage response to mechanical loading was assessed by imaging with dihydroethidium (ROS indicator) and a Luciferase based ATP assay. Electron transport inhibitor rotenone and mitochondrial ROS scavenger MitoQ significantly suppressed mechanically induced ROS production and ATP synthesis. Our findings indicate that mitochondrial ROS are produced as a result of physiological mechanical strains. Taken together with our previous findings of ROS involvement in blunt impact injuries, mitochondrial ROS are important contributors to cartilage metabolic adaptation and their precise role in the pathogenesis of osteoarthritis warrants further investigation.
PMCID: PMC3678272  PMID: 22930474
Articular Cartilage; Oxidants; Reactive Oxygen Species; Mechanical stress; Glycolysis

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