a-novel-vivo-gene-transfer-technique-vitro-cell-based-assays-for

A Novel <em>in vivo</em> Gene Transfer Technique and <em>in vitro</em> Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Schizophrenic patients show deficits in working memory (WM) and inhibition of prepotent responses. We examined brain activity while subjects performed tasks that placed demands on WM and overriding prepotent response tendencies, testing predictions that both processes engage overlapping prefrontal cortical (PFC) regions and that schizophrenic patients show reduced PFC activity and performance deficits reflecting both processes.

Prefrontal cortex dysfunction mediates deficits in working memory and prepotent responding in schizophrenia.

Traumatic brain injury (TBI) is often associated with enduring impairments in high-level cognitive functioning, including working memory (WM). We examined WM function in predominantly chronic patients with mild, moderate and severe TBI and healthy comparison subjects behaviorally and, in a small subset of moderate-to-severe TBI patients, with event-related functional magnetic resonance imaging (fMRI), using a visual n-back task that parametrically varied WM load. TBI patients showed severity-dependent and load-related WM deficits in performance accuracy, but not reaction time. Performance of mild TBI patients did not differ from controls; patients with moderate and severe TBI were impaired, relative to controls and mild TBI patients, but only at higher WM-load levels. fMRI results show that TBI patients exhibit altered patterns of activation in a number of WM-related brain regions, including the dorsolateral prefrontal cortex and Broca's area. Examination of the pattern of behavioral responding and the temporal course of activations suggests that WM deficits in moderate-to-severe TBI are due to associative or strategic aspects of WM, and not impairments in active maintenance of stimulus representations. Overall, results demonstrate that individuals with moderate-to-severe TBI exhibit WM deficits that are associated with dysfunction within a distributed network of brain regions that support verbally mediated WM.

Parametric manipulation of working memory load in traumatic brain injury: behavioral and neural correlates.

Psychological distress is both a precipitant and a consequence of ICD shock. Therefore, the assessment of patient anxiety and concerns related to receiving an ICD shock may prompt appropriate psychological referrals and treatment.

Measurement of patient fears about implantable cardioverter defibrillator shock: an initial evaluation of the Florida Shock Anxiety Scale.

The 2001 Institute of Medicine report indicted that the US healthcare system fails to provide high-quality care, and offered 6 aims of improvement that would redesign the delivery of care for the 21st century. This study compared the use of Department of Veterans Affairs (VA) inpatient and outpatient services of cancer patients enrolled in a Cancer Care Coordination/Home-Telehealth (CCHT) program that involved remote management of symptoms (eg, emotional distress, pain) via home-telehealth technologies to a control group of cancer patients receiving standard VA care. Using a matched case-control design, 2 control patients per case were selected, matched by tumor type and cancer stage. There were 43 Cancer CCHT patients and 82 control group patients. Based on a medical record review of each patient, the total number of cancer-related services (defined as visits that were expected given the patients' cancer diagnosis and treatment protocol) and preventable services (defined as visits needed outside of those expected given the cancer diagnosis and planned treatment) were calculated over a 6-month period. Poisson multivariate regression models were used to estimate the adjusted relative risks (RRs) for the effects of the Cancer CCHT program on the service use outcomes. Cancer CCHT patients had significantly fewer preventable services (clinic visits: RR = 0.03, 95% confidence interval [CI] = 0.00-0.24; bed days of care (BDOC) for hospitalization [all-cause]: RR = 0.50, 95% CI = 0.37-0.67; hospitalizations [chemotherapy related]: RR = 0.43, 95% CI = 0.21-0.91; and BDOC for hospitalizations [chemotherapy related]: RR = 0.49, 95% CI = 0.34-0.71) than the control group. This study offered some preliminary evidence that patients enrolled in a Cancer CCHT program can successfully manage multiple complex symptoms without utilizing inpatient and outpatient services.

Healthcare utilization among veterans undergoing chemotherapy: the impact of a cancer care coordination/home-telehealth program.

Leukocyte capture on inflamed endothelium is facilitated by a shift in LFA-1 from low to high affinity that supports binding to ICAM-1. LFA-1 bonds help anchor polymorphonuclear leukocytes (PMN) to inflamed endothelium in shear flow, and their redistribution to the leading edge guides pseudopod formation, migration, and extravasation. These events can be disrupted at the plasma membrane by stabilizing LFA-1 in a low- or intermediate-affinity state with allosteric small molecules. We hypothesized that a minimum dimeric bond formation between high-affinity LFA-1 and ICAM-1 under shear stress is necessary to catalyze transmembrane signaling of directed cell migration. Microspheres and substrates were derivatized with monomeric or dimeric ICAM-1 to simulate the surface of inflamed endothelium under defined ligand valence. Binding to dimeric ICAM-1, and not monomeric ICAM-1, was sufficient to elicit assembly of F-actin and phosphorylation of Src family kinases that colocalized with LFA-1 on adherent PMN. Genetic deletion or small molecule inhibition of Src family kinases disrupted their association with LFA-1 that correlated with diminished polarization of arrested PMN and abrogation of transmigration on inflamed endothelium. We conclude that dimeric bond clusters of LFA-1/ICAM-1 provide a key outside-in signal for orienting cytoskeletal dynamics that direct PMN extravasation at sites of inflammation.

Transmigration of neutrophils across inflamed endothelium is signaled through LFA-1 and Src family kinase.

Orai1 was reported to function as a calcium channel subunit that facilitates store operated calcium entry (SOCE) in T cells and is necessary for formation of the immune synapse. We reasoned that SOCE via Orai1 might regulate PMNs activation during recruitment to inflamed endothelium. Orai1 function was assessed by real-time imaging of calcium transients as PMNs were stimulated to roll, arrest, and migrate on E-selectin and ICAM-1 in shear flow. Calcium entry was significantly reduced when Orai1 function was impaired by heterozygous knockout in a mouse model or by siRNA knockdown in HL-60 cells. Reduced Orai-1 expression correlated with the delayed onset of arrest and reduced ability to transition to a polarized migratory phenotype. Inhibition of SOCE by treatment with 2-APB, or blocking phospholipase C (PLC) mediated calcium store release with U73122, abrogated formyl peptide induced calcium elevation, and delayed subsequent cell arrest and polarization. These results suggest that calcium entry via Orai1 is the predominant SOCE that cooperates with cytoplasmic calcium store release in coordinating integrin-dependent PMN arrest and migration in the acute response to inflammation.

Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow.

Cardiovascular patients with reduced cardiovascular output and capacity such as those with congestive heart failure (CHF) have demonstrated cognitive-related dysfunction. The use of cardiac resynchronization therapy (CRT) is considered standard care for CHF patients who do not improve despite optimal medical therapy. Cardiac resynchronization therapy may improve neurocognitive and psychosocial functioning in patients by increasing cardiac output and cerebral perfusion.

Cardiac resynchronization therapy: a pilot study examining cognitive change in patients before and after treatment.

The primary purposes of this study were to determine if controls, and mild and moderate/severe traumatic brain injury (TBI) patients performed differently on a battery of executive functioning (EF) tests, and to identify the operating characteristics of EF tests in this population. Participants consisted of 46 brain-injured individuals and 24 healthy controls. All participants completed an extensive battery of EF tests. Results showed that mild TBI participants performed worse than controls on the Trail Making Test Part B, and that moderate/severe TBI participants consistently performed worse than either group on a variety of EF measures. Tests of EF exhibited a wide range of operating characteristics, suggesting that some EF tests are better than others in identifying TBI-related neurocognitive impairment. Predictive values were better for individuals with moderate/severe TBI than mild TBI. Overall, the Digit Span Backward Test showed the best positive predictive power in differentiating TBI. Our results provide useful data that may guide test selection in evaluating EF in patients with traumatic brain injury.

Operating characteristics of executive functioning tests following traumatic brain injury.

Acute inflammation triggers the innate immune response of neutrophils that efficiently traffic from the bloodstream to concentrate at high numbers at the site of tissue infection or wounding. A gatekeeper in this process is activation of β(2) integrins, which form bond clusters with ICAM-1 on the endothelial surface. These bond clusters serve dual functions of providing adhesive strength to anchor neutrophils under the shear forces of blood flow and directional guidance for cell polarization and subsequent transmigration on inflamed endothelium. We hypothesized that shear forces transmitted through high-affinity LFA-1 facilitates the cooperation with the calcium release-activated channel Orai1 in directing localized cytoskeletal activation and directed migration. By using vascular mimetic microfluidic channels, we observed neutrophil arrest on a substrate of either ICAM-1 or allosteric Abs that stabilize a high- or low-affinity conformation of LFA-1. Neutrophils captured via low-affinity LFA-1 did not exhibit intracellular calcium flux, F-actin polymerization, cell polarization, or directional migration under shear flow. In contrast, high-affinity LFA-1 provided orientation along a uropod-pseudopod axis that required calcium flux through Orai1. We demonstrate how the shear stress of blood flow can transduce distinct outside-in signals at focal sites of high-affinity LFA-1 that provide contact-mediated guidance for neutrophil emigration.

Migrational guidance of neutrophils is mechanotransduced via high-affinity LFA-1 and calcium flux.

This study examines whether serum levels of glial fibrillary acidic protein breakdown products (GFAP-BDP) are elevated in patients with mild and moderate traumatic brain injury compared with controls and whether they are associated with traumatic intracranial lesions on computed tomography (CT) scan (positive CT result) and with having a neurosurgical intervention.

Elevated levels of serum glial fibrillary acidic protein breakdown products in mild and moderate traumatic brain injury are associated with intracranial lesions and neurosurgical intervention.

Leukocyte trafficking to acute sites of injury or infection requires spatial and temporal cues that fine tune precise sites of firm adhesion and guide migration to endothelial junctions where they undergo diapedesis to sites of insult. Many detailed studies on the location and gradient of chemokines such as IL-8 and other CXCR ligands reveal that their recognition shortly after selectin-mediated capture and rolling exerts acute effects on integrin activation and subsequent binding to their ligands on the endothelium, which directs firm adhesion, adhesion strengthening, and downstream migration. In this process, G-protein coupled receptor (GPCR) signaling has been found to play an integral role in activating and mobilizing intracellular stores of calcium, GTPases such as Rap-1 and Rho and cytokeletal proteins such as Talin and F-actin to facilitate cell polarity and directional pseudopod formation. A critical question remaining is how intracellular Ca(2+) flux from CRAC channels such as Orai1 synergizes with cytosolic stores to mediate a rapid flux which is critical to the onset of PMN arrest and polarization. Our review will highlight a specific role for calcium as a signaling messenger in activating focal clusters of integrins bound to the cytoskeleton which allows the cell to attain a migratory phenotype. The precise interplay between chemokines, selectins, and integrins binding under the ubiquitous presence of shear stress from blood flow provides an essential cooperative signaling mechanism for effective leukocyte recruitment.

Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest.

Neutrophil arrest and migration on inflamed endothelium involves a conformational shift in CD11a/CD18 (leukocyte function antigen-1; LFA-1) to a high-affinity and clustered state that determines the strength and lifetime of bond formation with ICAM-1. Cytoskeletal adapter proteins Kindlin-3 and Talin-1 anchor clustered LFA-1 to the cytoskeleton and facilitate the transition from neutrophil rolling to arrest. We recently reported that tensile force acts on LFA-1 bonds inducing their colocalization with Orai1, the predominant membrane store operated Ca(2+) channel that cooperates with the endoplasmic reticulum to elicit cytosolic flux. Because Kindlin-3 was recently reported to initiate LFA-1 clustering in lymphocytes, we hypothesized that it cooperates with Orai1 and LFA-1 in signaling local Ca(2+) flux necessary for shear-resistant neutrophil arrest. Using microfluidic flow channels combined with total internal reflection fluorescence microscopy, we applied defined shear stress to low- or high-affinity LFA-1 and imaged the spatiotemporal regulation of bond formation with Kindlin-3 recruitment and Ca(2+) influx. Orai1 and Kindlin-3 genes were silenced in neutrophil-like HL-60 cells to assess their respective roles in this process. Kindlin-3 was enriched within focal clusters of high-affinity LFA-1, which promoted physical linkage with Orai1. This macromolecular complex functioned to amplify inside-out Ca(2+) signaling in response to IL-8 stimulation by catalyzing an increased density of Talin-1 and consolidating LFA-1 clusters within sites of contact with ICAM-1. In this manner, neutrophils use focal adhesions as mechanosensors that convert shear stress-mediated tensile force into local bursts of Ca(2+) influx that catalyze cytoskeletal engagement and an adhesion-strengthened migratory phenotype.

Leukocyte function antigen-1, kindlin-3, and calcium flux orchestrate neutrophil recruitment during inflammation.

Neutrophil arrest and migration on inflamed endothelium is dependent upon a conformational shift in CD11a/CD18 (LFA-1) from a low to high affinity and clustered state which determines the strength and lifetime of bond formation with intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal adaptor proteins kindlin-3 and talin-1 anchor clustered LFA-1 to the cytoskeleton and support the transition from neutrophil rolling to arrest. We employ microfluidic flow channels and total internal reflection fluorescence microscopy to evaluate the spatiotemporal regulation of LFA-1 affinity and bond formation that facilitate the transition from neutrophil rolling to arrest. Methodology is presented to correlate the relationship between integrin conformation, bond formation with ICAM-1, and cytoskeletal engagement and adhesion strengthening necessary to achieve a migratory phenotype.

Detection of bidirectional signaling during integrin activation and neutrophil adhesion.

Both Glial Fibrillary Acidic Protein (GFAP) and S100β are found in glial cells and are released into serum following a traumatic brain injury (TBI) but the clinical utility of S100β has been questioned because of its release from bone. This study examined the ability of GFAP and S100β to detect intracranial lesions on CT in trauma patients and also assessed biomarker performance in patients with fractures and extracranial injuries on head CT. This prospective cohort study enrolled a convenience sample of adult trauma patients at a Level I trauma center with and without mild and moderate TBI (MMTBI). Serum samples were obtained within 4 hours of injury. The primary outcome was the presence of traumatic intracranial lesions on CT scan. There were 397 general trauma patients enrolled, 209 (53%) had a MMTBI and 188 (47%) had trauma without MMTBI. Out of the 262 patients with a head CT, 20 (8%) had intracranial lesions. There were 137 (35%) trauma patients who sustained extracranial fractures below the head to the torso and extremities. Levels of S100β were significantly higher in patients with fractures compared to those without fractures (p<0.001) whether MMTBI was present or not. However, GFAP levels were not significantly affected by the presence of fractures (p>0.05). The area under the ROC curve (AUC) for predicting intracranial lesions on CT for GFAP was 0.84 (0.73-0.95) and for S100β was 0.78 (0.67-0.89). However, in the presence of extracranial fractures, the AUC for GFAP increased to 0.93 (0.86-1.00) and for S100β it decreased to 0.75 (0.61-0.88). In a general trauma population, GFAP out-performed S100β in detecting intracranial CT lesions, particularly in the setting of extracranial fractures.

GFAP Out-performs S100B In Detecting Traumatic Intracranial Lesions On CT In Trauma Patients With Mild Traumatic Brain Injury And Those With Extracranial Lesions.

Article	Year
Prefrontal cortex dysfunction mediates deficits in working memory and prepotent responding in schizophrenia. Biological psychiatry\| PubMed ID: 12513942	2003
Parametric manipulation of working memory load in traumatic brain injury: behavioral and neural correlates. Journal of the International Neuropsychological Society : JINS\| PubMed ID: 15327720	2004
Measurement of patient fears about implantable cardioverter defibrillator shock: an initial evaluation of the Florida Shock Anxiety Scale. Pacing and clinical electrophysiology : PACE\| PubMed ID: 16784427	2006
Healthcare utilization among veterans undergoing chemotherapy: the impact of a cancer care coordination/home-telehealth program. The Journal of ambulatory care management\| PubMed ID: 17873662	2007
Transmigration of neutrophils across inflamed endothelium is signaled through LFA-1 and Src family kinase. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 19050286	2008
Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow. Blood\| PubMed ID: 19965684	2010
Cardiac resynchronization therapy: a pilot study examining cognitive change in patients before and after treatment. Clinical cardiology\| PubMed ID: 20186985	2010
Operating characteristics of executive functioning tests following traumatic brain injury. The Clinical neuropsychologist\| PubMed ID: 21069617	2010
Migrational guidance of neutrophils is mechanotransduced via high-affinity LFA-1 and calcium flux. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 21632714	2011
Elevated levels of serum glial fibrillary acidic protein breakdown products in mild and moderate traumatic brain injury are associated with intracranial lesions and neurosurgical interventio... Annals of emergency medicine\| PubMed ID: 22071014	2012
Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest. Frontiers in immunology\| PubMed ID: 22787461	2012
Leukocyte function antigen-1, kindlin-3, and calcium flux orchestrate neutrophil recruitment during inflammation. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 23144497	2012
Detection of bidirectional signaling during integrin activation and neutrophil adhesion. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 24504956	2014
GFAP Out-performs S100B In Detecting Traumatic Intracranial Lesions On CT In Trauma Patients With Mild Traumatic Brain Injury And Those With Extracranial Lesions. Journal of neurotrauma\| PubMed ID: 24903744	2014

Neha Dixit

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