Discriminating malingered attention Deficit Hyperactivity Disorder from genuine symptom reporting using novel Personality Assessment Inventory validity measures.

It is now widely understood that ADHD can be feigned easily and convincingly. Despite this, almost no methods exist to assist clinicians in identifying when such behavior occurs. Recently, new validity indicators specific to feigned ADHD were reported for the Personality Assessment Inventory (PAI). Derived from a logistic regression, these algorithms are said to have excellent specificity and good sensitivity in identifying feigned ADHD. However, these authors compared those with genuine ADHD only to nonclinical undergraduate students (asked to respond honestly or asked to simulate ADHD); no criterion group of definite malingerers was included. We therefore investigated these new validity indicators with 331 postsecondary students who underwent assessment for possible ADHD and compared scores of those who were eventually diagnosed with ADHD (n = 111) to those who were not [Clinical controls (66), Definite malingerers (36); No diagnosis (117)]. The two proposed PAI algorithms were found to have poor positive predictive value (.19 and .17). Self-report validity measures from the Connors' Adult Attention Rating Scale, and the Negative Impression Management scale on the PAI returned more positive results. Overall, more research is needed to better identify noncredible ADHD presentation, as the PAI-based methods proposed by Aita et al. appear inadequate as symptom validity measures.

Robotic Assessment of Upper Limb Function in a Nonhuman Primate Model of Chronic Stroke.

Stroke is a leading cause of death and disability worldwide and survivors are frequently left with long-term disabilities that diminish their autonomy and result in the need for chronic care. There is an urgent need for the development of therapies that improve stroke recovery, as well as accurate and quantitative tools to measure function. Nonhuman primates closely resemble humans in neuroanatomy and upper limb function and may be crucial in randomized pre-clinical trials for testing the efficacy of stroke therapies. To test the feasibility of robotic assessment of motor function in a NHP model of stroke, two cynomolgus macaques were trained to perform a visually guided reaching task and were also assessed in a passive stretch task using the Kinarm robot. Strokes were then induced in these animals by transiently occluding the middle cerebral artery, and their motor performance on the same tasks was assessed after recovery. Relative to pre-stroke performance, post-stroke hand movements of the affected limb became slower and less accurate. Regression analyses revealed both recovered and compensatory movements to complete movements in different spatial directions. Lastly, we noted decreased range of motion in the elbow joint of the affected limb post-stroke associated with spasticity during passive stretch. Taken together, these studies highlight that sensorimotor deficits in reaching movements following stroke in cynomolgus macaques resemble those in human patients and validate the use of robotic assessment tools in a nonhuman primate model of stroke for identifying and characterizing such deficits.

Poly-Arginine Peptide-18 (R18) Reduces Brain Injury and Improves Functional Outcomes in a Nonhuman Primate Stroke Model.

Poly-arginine peptide-18 (R18) is neuroprotective in different rodent middle cerebral artery occlusion (MCAO) stroke models. In this study, we examined whether R18 treatment could reduce ischemic brain injury and improve functional outcome in a nonhuman primate (NHP) stroke model. A stroke was induced in male cynomolgus macaques by MCAO distal to the orbitofrontal branch of the MCA through a right pterional craniotomy, using a 5-mm titanium aneurysm clip for 90 min. R18 (1000 nmol/kg) or saline vehicle was administered intravenously 60 min after the onset of MCAO. Magnetic resonance imaging (MRI; perfusion-weighted imaging, diffusion-weighted imaging, or T2-weighted imaging) of the brain was performed 15 min, 24 h, and 28 days post-MCAO, and neurological outcome was assessed using the NHP stroke scale (NHPSS). Experimental endpoint was 28 days post-MCAO, treatments were randomized, and all procedures were performed blinded to treatment status. R18 treatment reduced infarct lesion volume by up to 65.2% and 69.7% at 24 h and 28 days poststroke, respectively. Based on NHPSS scores, R18-treated animals displayed reduced functional deficits. This study confirms the effectiveness of R18 in reducing the severity of ischemic brain injury and improving functional outcomes after stroke in a NHP model, and provides further support for its clinical development as a stroke neuroprotective therapeutic.

HPLC-MS/MS Methods for Diacylglycerol and Sphingolipid Molecular Species in Skeletal Muscle.

HPLC-MS/MS has enabled the quantitative analysis of complex mixtures of lipid molecular species. Several separate analyses, using methods that have been optimized for individual lipid classes, provide good lipidomic profiles, but may not be desirable for laboratories constrained by available instrumentation and wanting a higher throughput. Here we describe two methods using binary gradient HiLiC HPLC and triple quadrupole MS that together provide a lipidomic profile for lipids of interest in type 2 diabetes research. Methods for analysis of molecular species of diacylglycerol, ceramide, dihydroceramide, sphingosine, glucosyl- and lactosylceramide, sphingomyelin, and acylcarnitine from skeletal muscle and primary culture cells are described.

Intermuscular adipose tissue directly modulates skeletal muscle insulin sensitivity in humans.

Intermuscular adipose tissue (IMAT) is negatively related to insulin sensitivity, but a causal role of IMAT in the development of insulin resistance is unknown. IMAT was sampled in humans to test for the ability to induce insulin resistance in vitro and characterize gene expression to uncover how IMAT may promote skeletal muscle insulin resistance. Human primary muscle cells were incubated with conditioned media from IMAT, visceral (VAT), or subcutaneous adipose tissue (SAT) to evaluate changes in insulin sensitivity. RNAseq analysis was performed on IMAT with gene expression compared with skeletal muscle and SAT, and relationships to insulin sensitivity were determined in men and women spanning a wide range of insulin sensitivity measured by hyperinsulinemic-euglycemic clamp. Conditioned media from IMAT and VAT decreased insulin sensitivity similarly compared with SAT. Multidimensional scaling analysis revealed distinct gene expression patterns in IMAT compared with SAT and muscle. Pathway analysis revealed that IMAT expression of genes in insulin signaling, oxidative phosphorylation, and peroxisomal metabolism related positively to donor insulin sensitivity, whereas expression of macrophage markers, inflammatory cytokines, and secreted extracellular matrix proteins were negatively related to insulin sensitivity. Perilipin 5 gene expression suggested greater IMAT lipolysis in insulin-resistant individuals. Combined, these data show that factors secreted from IMAT modulate muscle insulin sensitivity, possibly via secretion of inflammatory cytokines and extracellular matrix proteins, and by increasing local FFA concentration in humans. These data suggest IMAT may be an important regulator of skeletal muscle insulin sensitivity and could be a novel therapeutic target for skeletal muscle insulin resistance.

Selection of preclinical models to evaluate intranasal brain cooling for acute ischemic stroke.

Stroke accounts for a large proportion of global mortality and morbidity. Selective hypothermia, via intranasal cooling devices, is a promising intervention in acute ischemic stroke. However, prior to large clinical trials, preclinical studies in large animal models of ischemic stroke are needed to assess the efficacy, safety, and feasibility of intranasal cooling for selective hypothermia as a neuroprotective strategy. Here, we review the available scientific literature for evidence supporting selective hypothermia and make recommendations of a preclinical, large, animal-based, ischemic stroke model that has the greatest potential for evaluating intranasal cooling for selective hypothermia and neuroprotection. We conclude that among large animal models of focal ischemic stroke including pigs, sheep, dogs, and nonhuman primates (NHPs), cynomolgus macaques have nasal anatomy, nasal vasculature, neuroanatomy, and cerebrovasculature that are most similar to those of humans. Moreover, middle cerebral artery stroke in cynomolgus macaques produces functional and behavioral deficits that are quantifiable to a greater degree of precision and detail than those that can be revealed through available assessments for other large animals. These NHPs are also amenable to extensive neuroimaging studies as a means of monitoring stroke evolution and evaluating infarct size. Hence, we suggest that cynomolgus macaques are best suited to assess the safety and efficacy of intranasal selective hypothermia through an evaluation of hyperacute diffusion-weighted imaging and subsequent investigation of chronic functional recovery, prior to randomized clinical trials in humans.

Stroke Longitudinal Volumetric Measures Correlate with the Behavioral Score in Non-Human Primates.

Stroke is the second leading cause of death worldwide. Brain imaging data from experimental rodent stroke models suggest that size and location of the ischemic lesion relate to behavioral outcome. However, such a relationship between these two variables has not been established in Non-Human Primate (NHP) models. Thus, we aimed to evaluate whether size, location, and severity of stroke following controlled Middle Cerebral Artery Occlusion (MCAO) in NHP model correlated to neurological outcome. Forty cynomolgus macaques underwent MCAO, after four mortalities, thirty-six subjects were followed up during the longitudinal study. Structural T2 scans were obtained by magnetic resonance imaging (MRI) prior to, 48 h, and 30 days post-MCAO. Neurological function was assessed with the Non-human Primate Stroke Scale (NHPSS). T2 whole lesion volume was calculated per subject. At chronic stages, remaining brain volume was computed, and the affected hemisphere parceled into 50 regions of interest (ROIs). Whole and parceled volumetric measures were analyzed in relation to the NHPSS score. The longitudinal lesion volume evaluation showed a positive correlation with the NHPSS score, whereas the remaining brain volume negatively correlated with the NHPSS. Following ROI parcellation, NHPSS outcome correlated with frontal, temporal, occipital, and middle white matter, as well as the internal capsule, and the superior temporal and middle temporal gyri, and the caudate nucleus. These results represent an important step in stroke translational research by demonstrating close similarities between the NHP stroke model and the clinical characteristics following a human stroke and illustrating significant areas that could represent targets for novel neuroprotective strategies.

Enhanced Platelet-Activating Factor Synthesis Facilitates Acute and Delayed Effects of Ethanol-Intoxicated Thermal Burn Injury.

Thermal burn injuries in patients who are alcohol-intoxicated result in greater morbidity and mortality. Murine models combining ethanol and localized thermal burn injury reproduce the systemic toxicity seen in human subjects, which consists of both acute systemic cytokine production with multiple organ dysfunction, as well as a delayed systemic immunosuppression. However, the exact mechanisms for these acute and delayed effects are unclear. These studies sought to define the role of the lipid mediator platelet-activating factor in the acute and delayed effects of intoxicated burn injury. Combining ethanol and thermal burn injury resulted in increased enzymatic platelet-activating factor generation in a keratinocyte cell line in vitro, human skin explants ex vivo, as well as in murine skin in vivo. Further, the acute increase in inflammatory cytokines, such as IL-6, and the systemic immunosuppressive effects of intoxicated thermal burn injury were suppressed in mice lacking platelet-activating factor receptors. Together, these studies provide a potential mechanism and treatment strategies for the augmented toxicity and immunosuppressive effects of thermal burn injury in the setting of acute ethanol exposure, which involves the pleotropic lipid mediator platelet-activating factor.

Intracellular localization of diacylglycerols and sphingolipids influences insulin sensitivity and mitochondrial function in human skeletal muscle.

BACKGROUND: Accumulation of diacylglycerol (DAG) and sphingolipids is thought to promote skeletal muscle insulin resistance by altering cellular signaling specific to their location. However,the subcellular localization of bioactive lipids in human skeletal muscle is largely unknown. METHODS: We evaluated subcellular localization of skeletal muscle DAGs and sphingolipids in lean individuals (n = 15), endurance-trained athletes (n = 16), and obese men and women with (n = 12) and without type 2 diabetes (n = 15). Muscle biopsies were fractionated into sarcolemmal, cytosolic, mitochondrial/ER, and nuclear compartments. Lipids were measured using liquid chromatography tandem mass spectrometry, and insulin sensitivity was measured using hyperinsulinemic-euglycemic clamp. RESULTS: Sarcolemmal 1,2-DAGs were not significantly related to insulin sensitivity. Sarcolemmal ceramides were inversely related to insulin sensitivity, with a significant relationship found for the C18:0 species. Sarcolemmal sphingomyelins were also inversely related to insulin sensitivity, with the strongest relationships found for the C18:1, C18:0, and C18:2 species. In the mitochondrial/ER and nuclear fractions, 1,2-DAGs were positively related to, while ceramides were inversely related to, insulin sensitivity. Cytosolic lipids as well as 1,3-DAG, dihydroceramides, and glucosylceramides in any compartment were not related to insulin sensitivity. All sphingolipids but only specific DAGs administered to isolated mitochondria decreased mitochondrial state 3 respiration. CONCLUSION: These data reveal previously unknown differences in subcellular localization of skeletal muscle DAGs and sphingolipids that relate to whole-body insulin sensitivity and mitochondrial function in humans. These data suggest that whole-cell concentrations of lipids obscure meaningful differences in compartmentalization and suggest that subcellular localization of lipids should be considered when developing therapeutic interventions to treat insulin resistance. FUNDING: National Institutes of Health General Clinical Research Center (RR-00036), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01DK089170), NIDDK (T32 DK07658), and Colorado Nutrition Obesity Research Center (P30DK048520).

Radiation therapy generates platelet-activating factor agonists.

Pro-oxidative stressors can suppress host immunity due to their ability to generate oxidized lipid agonists of the platelet-activating factor-receptor (PAF-R). As radiation therapy also induces reactive oxygen species, the present studies were designed to define whether ionizing radiation could generate PAF-R agonists and if these lipids could subvert host immunity. We demonstrate that radiation exposure of multiple tumor cell lines in-vitro, tumors in-vivo, and human subjects undergoing radiation therapy for skin tumors all generate PAF-R agonists. Structural characterization of radiation-induced PAF-R agonistic activity revealed PAF and multiple oxidized glycerophosphocholines that are produced non-enzymatically. In a murine melanoma tumor model, irradiation of one tumor augmented the growth of the other (non-treated) tumor in a PAF-R-dependent process blocked by a cyclooxygenase-2 inhibitor. These results indicate a novel pathway by which PAF-R agonists produced as a byproduct of radiation therapy could result in tumor treatment failure, and offer important insights into potential therapeutic strategies that could improve the overall antitumor effectiveness of radiation therapy regimens.

Chemotherapeutic agents subvert tumor immunity by generating agonists of platelet-activating factor.

Oxidative stress suppresses host immunity by generating oxidized lipid agonists of the platelet-activating factor receptor (PAF-R). Because many classical chemotherapeutic drugs induce reactive oxygen species (ROS), we investigated whether these drugs might subvert host immunity by activating PAF-R. Here, we show that PAF-R agonists are produced in melanoma cells by chemotherapy that is administered in vitro, in vivo, or in human subjects. Structural characterization of the PAF-R agonists induced revealed multiple oxidized glycerophosphocholines that are generated nonenzymatically. In a murine model of melanoma, chemotherapeutic administration could augment tumor growth by a PAF-R-dependent process that could be blocked by treatment with antioxidants or COX-2 inhibitors or by depletion of regulatory T cells. Our findings reveal how PAF-R agonists induced by chemotherapy treatment can promote treatment failure. Furthermore, they offer new insights into how to improve the efficacy of chemotherapy by blocking its heretofore unknown impact on PAF-R activation.

The loss of Gnai2 and Gnai3 in B cells eliminates B lymphocyte compartments and leads to a hyper-IgM like syndrome.

B lymphocytes are compartmentalized within lymphoid organs. The organization of these compartments depends upon signaling initiated by G-protein linked chemoattractant receptors. To address the importance of the G-proteins Gαi2 and Gαi3 in chemoattractant signaling we created mice lacking both proteins in their B lymphocytes. While bone marrow B cell development and egress is grossly intact; mucosal sites, splenic marginal zones, and lymph nodes essentially lack B cells. There is a partial block in splenic follicular B cell development and a 50-60% reduction in splenic B cells, yet normal numbers of splenic T cells. The absence of Gαi2 and Gαi3 in B cells profoundly disturbs the architecture of lymphoid organs with loss of B cell compartments in the spleen, thymus, lymph nodes, and gastrointestinal tract. This results in a severe disruption of B cell function and a hyper-IgM like syndrome. Beyond the pro-B cell stage, B cells are refractory to chemokine stimulation, and splenic B cells are poorly responsive to antigen receptor engagement. Gαi2 and Gαi3 are therefore critical for B cell chemoattractant receptor signaling and for normal B cell function. These mice provide a worst case scenario of the consequences of losing chemoattractant receptor signaling in B cells.

Rgs13 constrains early B cell responses and limits germinal center sizes.

Germinal centers (GCs) are microanatomic structures that develop in secondary lymphoid organs in response to antigenic stimulation. Within GCs B cells clonally expand and their immunoglobulin genes undergo class switch recombination and somatic hypermutation. Transcriptional profiling has identified a number of genes that are prominently expressed in GC B cells. Among them is Rgs13, which encodes an RGS protein with a dual function. Its canonical function is to accelerate the intrinsic GTPase activity of heterotrimeric G-protein α subunits at the plasma membrane, thereby limiting heterotrimeric G-protein signaling. A unique, non-canonical function of RGS13 occurs following translocation to the nucleus, where it represses CREB transcriptional activity. The functional role of RGS13 in GC B cells is unknown. To create a surrogate marker for Rgs13 expression and a loss of function mutation, we inserted a GFP coding region into the Rgs13 genomic locus. Following immunization GFP expression rapidly increased in activated B cells, persisted in GC B cells, but declined in newly generated memory B and plasma cells. Intravital microscopy of the inguinal lymph node (LN) of immunized mice revealed the rapid appearance of GFP(+) cells at LN interfollicular regions and along the T/B cell borders, and eventually within GCs. Analysis of WT, knock-in, and mixed chimeric mice indicated that RGS13 constrains extra-follicular plasma cell generation, GC size, and GC B cell numbers. Analysis of select cell cycle and GC specific genes disclosed an aberrant gene expression profile in the Rgs13 deficient GC B cells. These results indicate that RGS13, likely acting at cell membranes and in nuclei, helps coordinate key decision points during the expansion and differentiation of naive B cells.

Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

FcepsilonRI and Thy-1 domains have unique protein and lipid compositions.

Receptor activation leads to the dynamic remodeling of the plasma membrane. Previous work using immunoelectron microscopy showed that aggregated high-affinity receptor for immunoglobulin E (FcRI) and aggregated Thy-1, a glycerophosphoinositol (GPI)-anchored protein, have distinct membrane distributions. We now report lipidomics analysis of FcRI- and Thy-1-enriched vesicles obtained by magnetic bead isolation in the absence of detergent. Protein analyses show that FcRI domains are enriched in receptors and associated signaling molecules, whereas Thy-1 domains are devoid of FcRI subunits. Positive and negative ion electrospray mass spectrometry demonstrated that both domains retained a complex mixture of phospholipid classes and molecular species, predominantly glycerophosphocholine, glycerophosphoethanolamine (GPE), and sphingomyelin as well as glycerophosphoserine and GPI lipids. Analysis of total acyl groups showed that < 50% of fatty acids in these domains are fully saturated, inconsistent with the recruitment of aggregated receptors or GPI-anchored proteins to liquid ordered domains. However, further analysis showed that FcRI domains contain two times more sphingomyelin and a high ratio of cholesterol to total fatty acid content compared with Thy 1-enriched domains. Remarkably, plasmenyl glycerophosphoethanolamine phospholipids (plasmalogen GPE) were also 2.5-3 times more abundant in FcRI domains than in the Thy-1 microdomains, whereas most diacyl GPE molecular species were equally abundant in the two domains.

Endotoxins stimulate neutrophil adhesion followed by synthesis and release of platelet-activating factor in microparticles.

Lipopolysaccharides and triacyl-cysteine-modified proteins of Gram-negative and positive organisms are potent endotoxins. Animal models show that the receptor for platelet-activating factor (PAF) is responsible for many of the deleterious effects of endotoxin, where regulated, localized PAF production localizes the inflammatory response. In contrast, biologically active analogs of PAF (PAF-like lipids) are generated by oxidative attack on phospholipids by chemical reactions that are unregulated and unlocalized. The identity and distribution of the PAF receptor ligand in endotoxemia is unknown. We found human polymorphonuclear leukocytes (PMNs) were a significant source of PAF receptor agonists after stimulation by either class of endotoxin. Production of PAF receptor agonists required that the PMN adhere to a surface, and adhesion (and therefore accumulation of PAF-like bioactivity) in response to endotoxic stimulation was delayed for several minutes. PAF-like oxidized phospholipids were found by mass spectroscopy, but biosynthetic PAF accounted for most of the phospholipid agonists arising from endotoxic stimulation. A significant portion of the PAF made by PMNs was secreted, in contrast to its near complete retention by other inflammatory cells. Endotoxic stimulation induced a respiratory burst with the production of superoxide and the formation and shedding of microparticles. Free and microparticle-bound PAF appeared in the media, and blocking microvesiculation with calpeptin blocked PAF release. The released material activated platelets, and platelets co-aggregated with endotoxin-stimulated PMNs. Adherent PMNs therefore behave differently than suspended cells and are a significant source of free PAF after endotoxin exposure. Leukocytes can couple endotoxic challenge to the widespread circulatory and inflammatory effects of endotoxin.