Allopregnanolone Levels Are Inversely Associated with Self-Reported Pain Symptoms in U.S. Iraq and Afghanistan-Era Veterans: Implications for Biomarkers and Therapeutics.

BACKGROUND AND OBJECTIVES: Pain symptoms are common among Iraq/Afghanistan-era veterans, many of whom continue to experience persistent pain symptoms despite multiple pharmacological interventions. Preclinical data suggest that neurosteroids such as allopregnanolone demonstrate pronounced analgesic properties, and thus represent logical biomarker candidates and therapeutic targets for pain. Allopregnanolone is also a positive GABAA receptor modulator with anxiolytic, anticonvulsant, and neuroprotective actions in rodent models. We previously reported inverse associations between serum allopregnanolone levels and self-reported pain symptom severity in a pilot study of 82 male veterans. METHODS: The current study investigates allopregnanolone levels in a larger cohort of 485 male Iraq/Afghanistan-era veterans to attempt to replicate these initial findings. Pain symptoms were assessed by items from the Symptom Checklist-90-R (SCL-90-R) querying headache, chest pain, muscle soreness, and low back pain over the past 7 days. Allopregnanolone levels were quantified by gas chromatography/mass spectrometry. RESULTS: Associations between pain ratings and allopregnanolone levels were examined with Poisson regression analyses, controlling for age and smoking. Bivariate nonparametric Mann–Whitney analyses examining allopregnanolone levels across high and low levels of pain were also conducted. Allopregnanolone levels were inversely associated with muscle soreness [P = 0.0028], chest pain [P = 0.032], and aggregate total pain (sum of all four pain items) [P = 0.0001]. In the bivariate analyses, allopregnanolone levels were lower in the group reporting high levels of muscle soreness [P = 0.001]. CONCLUSIONS: These findings are generally consistent with our prior pilot study and suggest that allopregnanolone may function as an endogenous analgesic. Thus, exogenous supplementation with allopregnanolone could have therapeutic potential. The characterization of neurosteroid profiles may also have biomarker utility.

Porcine endothelial cells cocultured with smooth muscle cells became procoagulant in vitro.

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Effect of microRNA modulation on bioartificial muscle function.

Cellular therapies have recently employed the use of small RNA molecules, particularly microRNAs (miRNAs), to regulate various cellular processes that may be altered in disease states. In this study, we examined the effect of transient muscle-specific miRNA inhibition on the function of three-dimensional skeletal muscle cultures, or bioartificial muscles (BAMs). Skeletal myoblast differentiation in vitro is enhanced by inhibiting a proliferation-promoting miRNA (miR-133) expressed in muscle tissues. As assessed by functional force measurements in response to electrical stimulation at frequencies ranging from 0 to 20 Hz, peak forces exhibited by BAMs with miR-133 inhibition (anti-miR-133) were on average 20% higher than the corresponding negative control, although dynamic responses to electrical stimulation in miRNA-transfected BAMs and negative controls were similar to nontransfected controls. Immunostaining for alpha-actinin and myosin also showed more distinct striations and myofiber organization in anti-miR-133 BAMs, and fiber diameters were significantly larger in these BAMs over both the nontransfected and negative controls. Compared to the negative control, anti-miR-133 BAMs exhibited more intense nuclear staining for Mef2, a key myogenic differentiation marker. To our knowledge, this study is the first to demonstrate that miRNA mediation has functional effects on tissue-engineered constructs.

Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.

Musculoskeletal symptoms among female garment factory workers in Sri Lanka.

OBJECTIVES: To assess the prevalence of musculoskeletal symptoms and their association with sociodemographic risk factors among female garment factory workers in Sri Lanka. METHODS: 1058 randomly selected female garment factory workers employed in the free trade zone of Kogalla, Sri Lanka were recruited to complete two interviewer-administered questionnaires assessing musculoskeletal symptoms and health behaviors. DISCUSSION: Musculoskeletal complaints among female garment workers in the FTZ of Kogalla are less common than expected. Sociocultural factors may have resulted in underreporting and similarly contribute to the low rates of healthcare utilization by these women. RESULTS: 164 (15.5%) of workers reported musculoskeletal symptoms occurring more than 3 times or lasting a week or more during the previous 12-month period. Back (57.3%) and knee (31.7%) were the most common sites of pain. Although most symptomatic women reported that their problems interfered with work and leisure activities, very few missed work as a result of their pain. Prevalence correlated positively with increased age and industry tenure of less than 12 months. Job type, body mass index, and education were not significant predictors of musculoskeletal symptoms.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Association between attention-deficit/hyperactivity disorder symptoms and obesity and hypertension in early adulthood: a population-based study.

OBJECTIVE: To examine the associations between attention-deficit/hyperactivity disorder (ADHD) symptoms, obesity and hypertension in young adults in a large population-based cohort. DESIGN, SETTING AND PARTICIPANTS: The study population consisted of 15,197 respondents from the National Longitudinal Study of Adolescent Health, a nationally representative sample of adolescents followed from 1995 to 2009 in the United States. Multinomial logistic and logistic models examined the odds of overweight, obesity and hypertension in adulthood in relation to retrospectively reported ADHD symptoms. Latent curve modeling was used to assess the association between symptoms and naturally occurring changes in body mass index (BMI) from adolescence to adulthood. RESULTS: Linear association was identified between the number of inattentive (IN) and hyperactive/impulsive (HI) symptoms and waist circumference, BMI, diastolic blood pressure and systolic blood pressure (all P-values for trend <0.05). Controlling for demographic variables, physical activity, alcohol use, smoking and depressive symptoms, those with three or more HI or IN symptoms had the highest odds of obesity (HI 3+, odds ratio (OR)=1.50, 95% confidence interval (CI) = 1.22-2.83; IN 3+, OR = 1.21, 95% CI = 1.02-1.44) compared with those with no HI or IN symptoms. HI symptoms at the 3+ level were significantly associated with a higher OR of hypertension (HI 3+, OR = 1.24, 95% CI = 1.01-1.51; HI continuous, OR = 1.04, 95% CI = 1.00-1.09), but associations were nonsignificant when models were adjusted for BMI. Latent growth modeling results indicated that compared with those reporting no HI or IN symptoms, those reporting 3 or more symptoms had higher initial levels of BMI during adolescence. Only HI symptoms were associated with change in BMI. CONCLUSION: Self-reported ADHD symptoms were associated with adult BMI and change in BMI from adolescence to adulthood, providing further evidence of a link between ADHD symptoms and obesity.

Targeting Gbeta gamma signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis.

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. betagamma subunits of heterotrimeric G proteins (Gbetagamma) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gbetagamma signaling (betaARKct), we evaluated the role of Gbetagamma in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gbetagamma. Furthermore, we studied the effects of in vivo adenoviral-mediated betaARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the betaARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gbetagamma plays a critical role in physiological VSM proliferation, and targeted Gbetagamma inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo.

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.

Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7(+) cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

Peace and war: trajectories of posttraumatic stress disorder symptoms before, during, and after military deployment in Afghanistan.

In the study reported here, we examined posttraumatic stress disorder (PTSD) symptoms in 746 Danish soldiers measured on five occasions before, during, and after deployment to Afghanistan. Using latent class growth analysis, we identified six trajectories of change in PTSD symptoms. Two resilient trajectories had low levels across all five times, and a new-onset trajectory started low and showed a marked increase of PTSD symptoms. Three temporary-benefit trajectories, not previously described in the literature, showed decreases in PTSD symptoms during (or immediately after) deployment, followed by increases after return from deployment. Predeployment emotional problems and predeployment traumas, especially childhood adversities, were predictors for inclusion in the nonresilient trajectories, whereas deployment-related stress was not. These findings challenge standard views of PTSD in two ways. First, they show that factors other than immediately preceding stressors are critical for PTSD development, with childhood adversities being central. Second, they demonstrate that the development of PTSD symptoms shows heterogeneity, which indicates the need for multiple measurements to understand PTSD and identify people in need of treatment.

The impact of the developmental timing of trauma exposure on PTSD symptoms and psychosocial functioning among older adults.

The present study examined the impact of the developmental timing of trauma exposure on posttraumatic stress disorder (PTSD) symptoms and psychosocial functioning in a large sample of community-dwelling older adults (N = 1,995). Specifically, we investigated whether the negative consequences of exposure to traumatic events were greater for traumas experienced during childhood, adolescence, young adulthood, midlife, or older adulthood. Each of these developmental periods is characterized by age-related changes in cognitive and social processes that may influence psychological adjustment following trauma exposure. Results revealed that older adults who experienced their currently most distressing traumatic event during childhood exhibited more severe symptoms of PTSD and lower subjective happiness compared with older adults who experienced their most distressing trauma after the transition to adulthood. Similar findings emerged for measures of social support and coping ability. The differential effects of childhood compared with later life traumas were not fully explained by differences in cumulative trauma exposure or by differences in the objective and subjective characteristics of the events. Our findings demonstrate the enduring nature of traumatic events encountered early in the life course and underscore the importance of examining the developmental context of trauma exposure in investigations of the long-term consequences of traumatic experiences.

Memory in posttraumatic stress disorder: properties of voluntary and involuntary, traumatic and nontraumatic autobiographical memories in people with and without posttraumatic stress disorder symptoms.

One hundred fifteen undergraduates rated 15 word-cued memories and their 3 most negatively stressful, 3 most positive, and 7 most important events and completed tests of personality and depression. Eighty-nine also recorded involuntary memories online for 1 week. In the first 3-way comparisons needed to test existing theories, comparisons were made of memories of stressful events versus control events and involuntary versus voluntary memories in people high versus low in posttraumatic stress disorder (PTSD) symptom severity. For all participants, stressful memories had more emotional intensity, more frequent voluntary and involuntary retrieval, but not more fragmentation. For all memories, participants with greater PTSD symptom severity showed the same differences. Involuntary memories had more emotional intensity and less centrality to the life story than voluntary memories. Meeting the diagnostic criteria for traumatic events had no effect, but the emotional responses to events did. In 533 undergraduates, correlations among measures were replicated and the Negative Intensity factor of the Affect Intensity Measure correlated with PTSD symptom severity. No special trauma mechanisms were needed to account for the results, which are summarized by the autobiographical memory theory of PTSD.

When a trauma becomes a key to identity: Enhanced integration of trauma memories predicts posttraumatic stress disorder symptoms

The Centrality of Event Scale (CES) measures the extent to which a traumatic memory forms a central component of personnal identity, a turning point in the life story and a reference point for everyday inferences. In two studies, we show that the CES is positively correlated with severity of PTSD symptoms, even when controlling for measures of anxiety, depression, dissociation and self-consciousness. The findings contradict the widespread view that poor integration of the traumatic memory into one's life story is a main cause of PTSD symptoms. Instead, enhanced integration appears to be a key issue. Copyright © 2006 John Wiley & Sons, Ltd.

The centrality of event scale: a measure of integrating a trauma into one's identity and its relation to post-traumatic stress disorder symptoms.

We introduce a new scale that measures how central an event is to a person's identity and life story. For the most stressful or traumatic event in a person's life, the full 20-item Centrality of Event Scale (CES) and the short 7-item scale are reliable (alpha's of .94 and .88, respectively) in a sample of 707 undergraduates. The scale correlates .38 with PTSD symptom severity and .23 with depression. The present findings are discussed in relation to previous work on individual differences related to PTSD symptoms. Possible connections between the CES and measures of maladaptive attributions and rumination are considered along with suggestions for future research.