Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening.

A metabolic biomarker-based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery-phase detection of potential human developmental toxicants. In this study, metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure-based biomarker assay using these metabolites, along with a cytotoxicity endpoint, was then developed using a 9-point dose-response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity, an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy, but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity, 100% specificity). The assay had a high concordance (≥75%) with existing in vivo models, demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests.

Metabolic biomarkers of prenatal alcohol exposure in human embryonic stem cell-derived neural lineages.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. The mechanisms underlying FASD are incompletely understood, and biomarkers to identify those at risk are lacking. Here, we perform metabolomic analysis of embryoid bodies and neural lineages derived from human embryonic stem (hES) cells to identify the neural secretome produced in response to ethanol (EtOH) exposure. METHODS: WA01 and WA09 hES cells were differentiated into embryoid bodies, neural progenitors, or neurons. Cells along this progression were cultured for 4 days with 0, 0.1, or 0.3% EtOH. Supernatants were subjected to C18 chromatography followed by ESI-QTOF-MS. Features were annotated using public databases, and the identities of 4 putative biomarkers were confirmed with purified standards and comparative MS/MS. RESULTS: EtOH treatment induced statistically significant changes to metabolite abundance in human embryoid bodies (180 features), neural progenitors (76 features), and neurons (42 features). There were no shared significant features between different cell types. Fifteen features showed a dose-response to EtOH. Four chemical identities were confirmed: L-thyroxine, 5'-methylthioadenosine, and the tryptophan metabolites, L-kynurenine and indoleacetaldehyde. One feature with a putative annotation of succinyladenosine was significantly increased in both EtOH treatments. Additional features were selective to EtOH treatment but were not annotated in public databases. CONCLUSIONS: EtOH exposure induces statistically significant changes to the metabolome profile of human embryoid bodies, neural progenitors, and neurons. Several of these metabolites are normally present in human serum, suggesting their usefulness as potential serum FASD biomarkers. These findings suggest the biochemical pathways that are affected by EtOH in the developing nervous system and delineate mechanisms of alcohol injury during human development.

A Targeted Metabolomics-Based Assay Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Identifies Structural and Functional Cardiotoxicity Potential.

Implementing screening assays that identify functional and structural cardiotoxicity earlier in the drug development pipeline has the potential to improve safety and decrease the cost and time required to bring new drugs to market. In this study, a metabolic biomarker-based assay was developed that predicts the cardiotoxicity potential of a drug based on changes in the metabolism and viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Assay development and testing was conducted in 2 phases: (1) biomarker identification and (2) targeted assay development. In the first phase, metabolomic data from hiPSC-CM spent media following exposure to 66 drugs were used to identify biomarkers that identified both functional and structural cardiotoxicants. Four metabolites that represent different metabolic pathways (arachidonic acid, lactic acid, 2'-deoxycytidine, and thymidine) were identified as indicators of cardiotoxicity. In phase 2, a targeted, exposure-based biomarker assay was developed that measured these metabolites and hiPSC-CM viability across an 8-point concentration curve. Metabolite-specific predictive thresholds for identifying the cardiotoxicity potential of a drug were established and optimized for balanced accuracy or sensitivity. When predictive thresholds were optimized for balanced accuracy, the assay predicted the cardiotoxicity potential of 81 drugs with 86% balanced accuracy, 83% sensitivity, and 90% specificity. Alternatively, optimizing the thresholds for sensitivity yields a balanced accuracy of 85%, 90% sensitivity, and 79% specificity. This new hiPSC-CM-based assay provides a paradigm that can identify structural and functional cardiotoxic drugs that could be used in conjunction with other endpoints to provide a more comprehensive evaluation of a drug's cardiotoxicity potential.

Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity.

The Stemina devTOX quickPredict platform is a human pluripotent stem cell-based assay that predicts the developmental toxicity potential based on changes in cellular metabolism following chemical exposure [Palmer, J. A., Smith, A. M., Egnash, L. A., Conard, K. R., West, P. R., Burrier, R. E., Donley, E. L. R., and Kirchner, F. R. (2013). Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening. Birth Defects Res. B Dev. Reprod. Toxicol. 98, 343-363]. Using this assay, we screened 1065 ToxCast phase I and II chemicals in single-concentration or concentration-response for the targeted biomarker (ratio of ornithine to cystine secreted or consumed from the media). The dataset from the Stemina (STM) assay is annotated in the ToxCast portfolio as STM. Major findings from the analysis of ToxCast_STM dataset include (1) 19% of 1065 chemicals yielded a prediction of developmental toxicity, (2) assay performance reached 79%-82% accuracy with high specificity (> 84%) but modest sensitivity (< 67%) when compared with in vivo animal models of human prenatal developmental toxicity, (3) sensitivity improved as more stringent weights of evidence requirements were applied to the animal studies, and (4) statistical analysis of the most potent chemical hits on specific biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain. The results of this study will be useful to improving our ability to predict in vivo developmental toxicants based on in vitro data and in silico models.

A human induced pluripotent stem cell-based in vitro assay predicts developmental toxicity through a retinoic acid receptor-mediated pathway for a series of related retinoid analogues.

The relative developmental toxicity potency of a series of retinoid analogues was evaluated using a human induced pluripotent stem (iPS) cell assay that measures changes in the biomarkers ornithine and cystine. Analogue potency was predicted, based on the assay endpoint of the ornithine/cystine (o/c) ratio, to be all-trans-retinoic acid>TTNPB>13-cis-retinoic acid≈9-cis-retinoic acid>acitretin>etretinate>retinol. These rankings correlate with in vivo data and demonstrate successful application of the assay to rank a series of related toxic and non-toxic compounds. The retinoic acid receptor α (RARα)-selective antagonist Ro 41-5253 inhibited the cystine perturbation caused by all-trans-retinoic acid, TTNPB, 13-cis-retinoic acid, 9-cis-retinoic acid, and acitretin. Ornithine was altered independent of RARα in all retinoids except acitretin. These results suggest a role for an RARα-mediated mechanism in retinoid-induced developmental toxicity through altered cystine metabolism.

Slump stretching in the management of non-radicular low back pain: a pilot clinical trial.

The purpose of this study was to determine if slump stretching results in improvements in pain, centralization of symptoms, and disability in patients with non-radicular low back pain (LBP) with likely mild to moderate neural mechanosensitivity. Thirty consecutive patients referred to physical therapy by their primary care physician for LBP who met all eligibility criteria including a positive slump test but who had a negative straight-leg-raise test (SLR) agreed to participate in the study. All patients completed several self-report measures including a body diagram, numeric pain rating scale (NPRS), and the modified Oswestry Disability Index (ODI). Patients were randomized to receive lumbar spine mobilization and exercise (n = 14) or lumbar spine mobilization, exercise, and slump stretching (n = 16). All patients were treated in physical therapy twice weekly for 3 weeks for a total of 6 visits. Upon discharge, outcome measures were re-assessed. Independent t-tests were used to assess differences between groups at baseline and discharge. No baseline differences existed between the groups (P > .05). At discharge, patients who received slump stretching demonstrated significantly greater improvements in disability (9.7 points on the ODI, P < .001), pain (.93 points on the NPRS, P = .001), and centralization of symptoms (P < .01) than patients who did not. The results suggest that slump stretching is beneficial for improving short-term disability, pain, and centralization of symptoms. Future studies should examine whether these benefits are maintained at a longer-term follow-up.

The use of a lumbar spine manipulation technique by physical therapists in patients who satisfy a clinical prediction rule: a case series.

STUDY DESIGN: A case series of patients with low back pain (LBP) who satisfy a clinical prediction rule (CPR). BACKGROUND: A CPR that identifies patients with LBP who are likely to respond with rapid and prolonged reductions in pain and disability following spinal manipulation was developed and recently validated. The CPR developed to predict favorable response to manipulation investigated the effects of only 1 manipulation technique. The accuracy of the CPR for predicting outcomes using other manipulation techniques is not known. The purpose of the case series was to describe the outcomes of patients presenting to physical therapy with LBP who met the CPR and were treated with an alternative lumbar manipulation technique. CASE DESCRIPTION: Consecutive patients referred to physical therapy who satisfied the eligibility criteria, including the presence of at least 4 of the 5 criteria on the CPR, were invited to participate in the case series. Patients were treated for 2 visits with a side-lying lumbar manipulation technique, followed by a basic range of motion exercise. Patients who exhibited a 50% reduction or greater in disability, as measured by the Oswestry Disability Index (ODI), were considered to have experienced a successful outcome. OUTCOMES: A total of 12 patients participated in the case series. The mean age of the group was 39 years (SD, 8.9 years) and the median duration of symptoms was 19 days (range, 8-148 days). Of the 12 patients who participated in this case series, the mean reduction in disability as measured with the ODI was 57% (SD, 9%). Only 1 patient did not surpass the 50% reduction in ODI scores. DISCUSSION: Eleven of the 12 patients (92%) in this case series who satisfied the CPR and were treated with an alternative lumbar manipulation technique demonstrated a successful outcome in 2 visits. It is plausible that patients with LBP who satisfy the CPR may obtain a successful outcome with either manipulation technique directed at the lumbopelvic region.

Immediate effects of thoracic manipulation in patients with neck pain: a randomized clinical trial.

Mechanical neck pain is a common occurrence in the general population resulting in a considerable economic burden. Often physical therapists will incorporate manual therapies directed at the cervical spine including joint mobilization and manipulation into the management of patients with cervical pain. Although the effectiveness of mobilization and manipulation of the cervical spine has been well documented, the small inherent risks associated with these techniques has led clinicians to frequently utilize manipulation directed at the thoracic spine in this patient population. It is hypothesized that thoracic spine manipulation may elicit similar therapeutic benefits as cervical spine manipulation while minimizing the magnitude of risk associated with the cervical technique. The purpose of this randomized clinical trial was to investigate the immediate effects of thoracic spine manipulation on perceived pain levels in patients presenting with neck pain. The results suggest that thoracic spine manipulation results in immediate analgesic effects in patients with mechanical neck pain. Further studies are needed to determine the effects of thoracic spine manipulation in patients with neck pain on long-term outcomes including function and disability.

Manual physical therapy, cervical traction, and strengthening exercises in patients with cervical radiculopathy: a case series.

STUDY DESIGN: A case series of consecutive patients with cervical radiculopathy. BACKGROUND: A multitude of physical therapy interventions have been proposed to be effective in the management of cervical radiculopathy. However, outcome studies using consistent treatment approaches on a well-defined sample of patients are lacking. The purpose of this case series is to describe the outcomes of a consecutive series of patients presenting to physical therapy with cervical radiculopathy and managed with the use of manual physical therapy, cervical traction, and strengthening exercises. CASE DESCRIPTION: Eleven consecutive patients (mean age, 51.7 years; SD, 8.2) who presented with cervical radiculopathy on the initial examination were treated with a standardized approach, including manual physical therapy, cervical traction, and strengthening exercises of the deep neck flexors and scapulothoracic muscles. At the initial evaluation all patients completed self-report measures of pain and function, including a numeric pain rating scale (NPRS), the Neck Disability Index (NDI), and the Patient-Specific Functional Scale (PSFS). All patients again completed the outcome measures, in addition to the global rating of change (GROC), at the time of discharge from therapy and at a 6-month follow-up session OUTCOMES: Ten of the 11 patients (91%) demonstrated a clinically meaningful improvement in pain and function following a mean of 7.1 (SD, 1.5) physical therapy visits and at the 6-month follow-up. DISCUSSION: Ninety-one percent (10 of 11) of patients with cervical radiculopathy in this case series improved, as defined by the patients classifying their level of improvement as at least "quite a bit better" on the GROC. However, because a cause-and-effect relationship cannot be inferred from a case series, follow-up randomized clinical trials should be performed to further investigate the effectiveness of manual physical therapy, cervical traction, and strengthening exercises in a homogeneous group of patients with cervical radiculopathy.

Metabolomics as a tool for discovery of biomarkers of autism spectrum disorder in the blood plasma of children.

BACKGROUND: The diagnosis of autism spectrum disorder (ASD) at the earliest age possible is important for initiating optimally effective intervention. In the United States the average age of diagnosis is 4 years. Identifying metabolic biomarker signatures of ASD from blood samples offers an opportunity for development of diagnostic tests for detection of ASD at an early age. OBJECTIVES: To discover metabolic features present in plasma samples that can discriminate children with ASD from typically developing (TD) children. The ultimate goal is to identify and develop blood-based ASD biomarkers that can be validated in larger clinical trials and deployed to guide individualized therapy and treatment. METHODS: Blood plasma was obtained from children aged 4 to 6, 52 with ASD and 30 age-matched TD children. Samples were analyzed using 5 mass spectrometry-based methods designed to orthogonally measure a broad range of metabolites. Univariate, multivariate and machine learning methods were used to develop models to rank the importance of features that could distinguish ASD from TD. RESULTS: A set of 179 statistically significant features resulting from univariate analysis were used for multivariate modeling. Subsets of these features properly classified the ASD and TD samples in the 61-sample training set with average accuracies of 84% and 86%, and with a maximum accuracy of 81% in an independent 21-sample validation set. CONCLUSIONS: This analysis of blood plasma metabolites resulted in the discovery of biomarkers that may be valuable in the diagnosis of young children with ASD. The results will form the basis for additional discovery and validation research for 1) determining biomarkers to develop diagnostic tests to detect ASD earlier and improve patient outcomes, 2) gaining new insight into the biochemical mechanisms of various subtypes of ASD 3) identifying biomolecular targets for new modes of therapy, and 4) providing the basis for individualized treatment recommendations.

The reliability and construct validity of the Neck Disability Index and patient specific functional scale in patients with cervical radiculopathy.

STUDY DESIGN: Cohort study of patients with cervical radiculopathy undergoing physical therapy. OBJECTIVES: Examine the test-retest reliability, construct validity, and minimum levels of detectable and clinically important change for the Neck Disability Index (NDI) and Patient Specific Functional Scale (PSFS) in cohort of patients with cervical radiculopathy. SUMMARY OF BACKGROUND DATA: To date, no studies have investigated the psychometric properties of the NDI or PSFS in a cohort of patients with cervical radiculopathy. METHODS: Thirty-eight patients with cervical radiculopathy undergoing physical therapy completed the NDI and PSFS, and Numerical Pain Rating Scale (NPRS) at the baseline examination and at a follow-up. In addition, at follow-up, patients completed a 15-point global rating of change (GROC), which was used to dichotomize patients as improved or stable. Changes in the NDI and PSFS were then used to assess test-retest reliability, construct validity, and minimal levels of detectable and clinically important change. RESULTS: Test-retest reliability was moderate for the NDI (intraclass correlation coefficient [ICC] = 0.68; 95% confidence interval [CI], 0.30-0.90) and high for the PSFS (ICC = 0.82; 95% CI, 0.54-0.93). The PSFS was more responsive to change than the NDI. The minimal detectable change for the NDI was 10.2 and for the PSFS 2.1. The minimally clinically important change for the NDI was 7.0 and PSFS 2.0. CONCLUSIONS: Our results suggest that the PSFS exhibits superior reliability, construct validity, and responsiveness in this cohort of patients with cervical radiculopathy compared with the NDI. Further research is needed to examine the ability of these measures to accurately reflect changes in individuals, as well as large samples of patients.