A Statistical Framework for Assessing the Relationship between Biomarkers and Clinical Endpoints in Alzheimer's Disease.

Changes in biomarker levels of Alzheimer's disease (AD) reflect underlying pathophysiological changes in the brain and can provide evidence of direct and downstream treatment effects linked to disease modification. Recent results from clinical trials of anti-amyloid ß (Aß) treatments have raised the question of how to best characterize the relationship between AD biomarkers and clinical endpoints. Consensus methodology for assessing such relationships is lacking, leading to inconsistent evaluation and reporting. In this review, we provide a statistical framework for reporting treatment effects on early and late accelerating AD biomarkers and assessing their relationship with clinical endpoints at the subject and group levels. Amyloid positron emission tomography (PET), plasma p-tau, and tau PET follow specific trajectories during AD and are used as exemplar cases to contrast biomarkers with early and late progression. Subject-level correlation was assessed using change from baseline in biomarkers versus change from baseline in clinical endpoints, and interpretation of the correlation is dependent on the biomarker and disease stage. Group-level correlation was assessed using the placebo-adjusted treatment effects on biomarkers versus those on clinical endpoints in each trial. This correlation leverages the fundamental advantages of randomized placebo-controlled trials and assesses the predictivity of a treatment effect on a biomarker or clinical benefit. Harmonization in the assessment of treatment effects on biomarkers and their relationship to clinical endpoints will provide a wealth of comparable data across clinical trials and may yield new insights for the treatment of AD.

Investigating Partially Discordant Results in Phase 3 Studies of Aducanumab.

OBJECTIVES: Efficacy and safety results from the EMERGE (NCT02484547) and ENGAGE (NCT02477800) phase 3 studies of aducanumab in early Alzheimer's disease (AD) have been published. In EMERGE, but not in ENGAGE, high-dose aducanumab demonstrated significant treatment effects across primary and secondary endpoints. Low-dose aducanumab results were consistent across studies with non-significant differences versus placebo that were intermediate to the high-dose arm in EMERGE. The present investigation examined data from EMERGE and ENGAGE through post-hoc analyses to determine factors that contributed to discordant results between the high-dose arms of the two studies. DESIGN: EMERGE and ENGAGE were 2 phase 3, randomized, double-blind, placebo-controlled, parallel-group studies. SETTING: EMERGE and ENGAGE were 2 global multicenter studies involving 348 sites in 20 countries. PARTICIPANTS: Participants in EMERGE and ENGAGE were aged 50 to 85 years and had mild cognitive impairment or mild AD dementia with confirmed amyloid pathology. The randomized and dosed population (all randomized patients who received at least one dose of study treatment) included 1638 patients in EMERGE and 1647 in ENGAGE. INTERVENTION: In EMERGE and ENGAGE, participants were randomized to receive low- or high-dose aducanumab or placebo (1:1:1) once every 4 weeks. MEASUREMENTS: In this paper, 4 areas were investigated through post-hoc analyses to understand the discordance in the high-dose arms of the EMERGE and ENGAGE studies: baseline characteristics, amyloid-related imaging abnormalities, non-normality of the data, and dosing/exposure to aducanumab. RESULTS: Post-hoc analyses showed that outcomes in the ENGAGE high-dose group were affected by an imbalance in a small number of patients with extremely rapid progression and by lower exposure to the target dose of 10 mg/kg. These factors were confounded and present in early enrolled patients but were not present in later-enrolled patients who were randomized to the target dosing regimen of 10 mg/kg after titration. Neither baseline characteristics nor amyloid-related imaging abnormalities contributed to the difference in results between the high-dose arms. CONCLUSIONS: Results were consistent across studies in later enrolled patients in which the incidence of rapidly progressing patients was balanced across treatment arms.

Two Randomized Phase 3 Studies of Aducanumab in Early Alzheimer's Disease.

BACKGROUND: Alzheimer's disease is a progressive, irreversible, and fatal disease for which accumulation of amyloid beta is thought to play a key role in pathogenesis. Aducanumab is a human monoclonal antibody directed against aggregated soluble and insoluble forms of amyloid beta. OBJECTIVES: We evaluated the efficacy and safety of aducanumab in early Alzheimer's disease. DESIGN: EMERGE and ENGAGE were two randomized, double-blind, placebo-controlled, global, phase 3 studies of aducanumab in patients with early Alzheimer's disease. SETTING: These studies involved 348 sites in 20 countries. PARTICIPANTS: Participants included 1638 (EMERGE) and 1647 (ENGAGE) patients (aged 50-85 years, confirmed amyloid pathology) who met clinical criteria for mild cognitive impairment due to Alzheimer's disease or mild Alzheimer's disease dementia, of which 1812 (55.2%) completed the study. INTERVENTION: Participants were randomly assigned 1:1:1 to receive aducanumab low dose (3 or 6 mg/kg target dose), high dose (10 mg/kg target dose), or placebo via IV infusion once every 4 weeks over 76 weeks. MEASUREMENTS: The primary outcome measure was change from baseline to week 78 on the Clinical Dementia Rating Sum of Boxes (CDR-SB), an integrated scale that assesses both function and cognition. Other measures included safety assessments; secondary and tertiary clinical outcomes that assessed cognition, function, and behavior; and biomarker endpoints. RESULTS: EMERGE and ENGAGE were halted based on futility analysis of data pooled from the first approximately 50% of enrolled patients; subsequent efficacy analyses included data from a larger data set collected up to futility declaration and followed prespecified statistical analyses. The primary endpoint was met in EMERGE (difference of -0.39 for high-dose aducanumab vs placebo [95% CI, -0.69 to -0.09; P=.012; 22% decrease]) but not in ENGAGE (difference of 0.03, [95% CI, -0.26 to 0.33; P=.833; 2% increase]). Results of biomarker substudies confirmed target engagement and dose-dependent reduction in markers of Alzheimer's disease pathophysiology. The most common adverse event was amyloid-related imaging abnormalities-edema. CONCLUSIONS: Data from EMERGE demonstrated a statistically significant change across all four primary and secondary clinical endpoints. ENGAGE did not meet its primary or secondary endpoints. A dose- and time-dependent reduction in pathophysiological markers of Alzheimer's disease was observed in both trials.

Retinal microcirculation imaging in sickle cell disease patients.

OBJECTIVES: To explore the feasibility of a new quantitative method for microvascular function: non-invasive retinal function imaging (RFI). in sickle cell disease (SCD) patients and healthy controls and have it benchmarked against Laser Speckle Contrast Imaging (LSCI) measurements. METHODS: The variability of Microvascular measurements was assessed in 8 SCD patients and 8 healthy matched controls. Measurements were conducted twice on two different study days. RFI was performed for assessment of arterial and venous retinal blood flow. LSCI measurements included post occlusive reactive hyperemia and IBH challenges. Measured variables included basal flow, flow upon occlusion-reperfusion and flow during an IBH. RESULTS: RFI arterial flow and venous flow and LSCI basal flow and peak flow showed excellent intra subject repeatability between days (CVC of 8.5% 9.5%, 7.6% and 7.7% respectively) and between measurements on one day (CVC of 7.0%, 7.7%, 7.6% and 4.7% respectively). RFI arterial flow (p<0.002), and RFI venous flow (p=0.007) differed significantly between SCD patients and controls in as did LSCI basal flow, maximal flow and delta flow during IBH (p<0.0001). CONCLUSIONS: RFI showed low variability for all readout measures, comparable with most microvascular measures from LSCI. The discriminating power of the RFI between SCD patients and controls demonstrate the feasibility of this device for quantitative assessment of the microcirculation in clinical research.

Quantifying Beta-Galactosylceramide Kinetics in Cerebrospinal Fluid of Healthy Subjects Using Deuterium Labeling.

Therapeutics promoting myelin synthesis may enhance recovery in demyelinating diseases, such as multiple sclerosis. However, no suitable method exists to quantify myelination. The turnover of galactosylceramide (myelin component) is indicative of myelination in mice, but its turnover has not been determined in humans. Here, six healthy subjects consumed 120 mL 70% D2 O daily for 70 days to label galactosylceramide. We then used mass spectrometry and compartmental modeling to quantify the turnover rate of galactosylceramide in cerebrospinal fluid. Maximum deuterium enrichment of body water ranged from 1.5-3.9%, whereas that of galactosylceramide was much lower: 0.05-0.14%. This suggests a slow turnover rate, which was confirmed by the model-estimated galactosylceramide turnover rate of 0.00168 day-1 , which corresponds to a half-life of 413 days. Additional studies in patients with multiple sclerosis are needed to investigate whether galactosylceramide turnover could be used as an outcome measure in clinical trials with remyelination therapies.

The Concise Guide to PHARMACOLOGY 2013/14: overview.

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.

Differential dependence of eosinophil chemotactic responses on phosphoinositide 3-kinase (PI3K).

BACKGROUND: Control of eosinophil migration to sites of inflammatory responses is a potentially therapeutic intervention in diseases such as bronchial asthma. Chemoattractants, their receptors and the associated signalling pathways may, therefore, be important targets for novel therapeutics. While several potentially important chemoattractants have been identified, the signalling pathways mediating their actions are incompletely understood. AIMS OF THE STUDY: The role of phosphoinositide 3-kinase (PI3K) in responses of human eosinophils to two important eosinophil chemoattractants -- platelet-activating factor (PAF) and eotaxin (CCL11) -- was studied to determine whether this enzyme activity might be crucial for eosinophil migration. METHODS: Eosinophils were isolated from atopic donor blood by immunomagnetic selection. Chemotaxis was assayed in a 96-well blind-chamber cell fluorescence assay. Respiratory burst and leukotriene C(4) secretion were also assayed. RESULTS: Two PI3K inhibitors, wortmannin and LY294002, caused concentration-dependent inhibition of PAF-induced eosinophil chemotaxis (IC(50) = 0.54 nM and 0.15 microM, respectively) but exhibited at least 100-fold lower potency against eotaxin-induced responses (IC(50) = 48 nM and >100 microM, respectively), indicating that these responses were not dependent upon PI3K. Wortmannin and LY294002 also inhibited PAF induced respiratory burst but not PAF-induced LTC(4) secretion. CONCLUSIONS: We conclude that PI3K-dependence varies with stimulus and response, and that eotaxin-induced eosinophil migration is not controlled by PI3K. This may indicate a limit to the potential of PI3K inhibitors to suppress tissue eosinophilia in diseases such as asthma.

Linking solution chemistry to crystal nucleation: the case of tetrolic acid.

The application of FTIR spectroscopy to concentrated solutions of tetrolic acid shows, for the first time, a direct relationship between molecular self association in solution and H-bonded motifs in the subsequently crystallised solid phases.

Surface-enhanced resonance Raman scattering of black inkjet dyes in solution and in situ printed onto paper.

Understanding the changes that occur when dyes are absorbed onto paper is crucial for the design of new inkjet dyes. This problem is particularly difficult for black dyes that have complex chromophores, and as a result, spectroscopic information on electronic and structural changes can be of importance. Surface-enhanced resonance Raman scattering (SERRS) and electronic structure calculations were used to probe in situ changes in the chromophore in black di-azo dyes printed onto paper. The data indicate that the low-energy chromophore is due mainly to the hydrazone group and the high-energy chromophore to both the azo and hydrazone groups. A comparison of SERRS from the dyes adsorbed onto silver particles in suspension and from the dyes on paper demonstrated a broadening of the chromophore into the red for both dyes and evidence of a structural change in one dye.

Studying human airway pharmacology in microsections: application of videomicrometry.

The influence of endogenously-released mediators and activated eosinophils on the airway lumen and the effect of passive sensitization on anti-immunoglobulin (Ig)-E-induced contractile responses was investigated by videomicrometry. Human bronchial sections of 2-3 mm internal diameter, placed in 250 microL Hank's balanced salt solution on microtitre plates, were monitored and recorded by digitized image analysis. Airway preparations exhibited a spontaneous narrowing (mean+/-SEM -33+/-5% of the luminal area). Removal of the bronchial epithelium almost completely prevented the development of spontaneuous narrowing (-6+/-3%; p<0.001). The addition of platelet-activating factor stimulated human eosinophils to the bronchial sections led to significant narrowing of the airway lumen (-39+/-9%; p<0.05). Passive sensitization induced hyperresponsiveness to polyclonal anti-IgE (-35+/-8%; p<0.01). It is concluded that videomicrometry is suitable for studying interactions between human airways and inflammatory cells, as well as the effect of passive sensitization on smooth muscle reactivity in vitro, without the imposition of preload. Under these conditions, human airways exhibited a spontaneous decrease of the airway lumen over time suggesting a role for epithelium-derived mediators because the development of spontaneous tone was epithelium dependent.

Maternal regulation of the hypothalamic-pituitary-adrenal axis in the 20-day-old rat: consequences of laboratory weaning.

There is a large body of evidence that the development of the hypothalamic-pituitary-adrenal (HPA) system in the rat is under maternal regulation. One method used to study the influence of the dam-pup interaction in neonates and weanlings is the separation of mother and litter for 24 h. Previous studies showed that, even at the time of weaning, maternal deprivation results in a dysregulation of the HPA axis at multiple levels. However, the maternal deprivation paradigm usually includes deprivation of food and water, and it was not clear to which extent the observed effects are due to either maternal cues or dehydration and fasting. The primary purpose of the present study was to determine the role of fasting and/or maternal separation on the HPA axis at the time of weaning. Pups at 20 days after parturition are capable of self-feeding and no longer require tactile stimulation to induce eliminative functions. The results indicated that 24 h of fasting led to increased basal levels and further increases in stress induced corticosterone secretion. Fasting also appeared to contribute to the down regulation of basal glucocorticoid receptor mRNA in the hippocampus. In contrast, abrupt weaning irrespective of fasting or dehydration resulted in a suppressed adrenocorticotropin hormone response to an injection of isotonic saline. Although there was an effect of maternal separation on corticotropin-releasing factor mRNA in the paraventricular nucleus, this effect was further exacerbated by the absence of food. Finally, all rats that were separated from their dams showed more efficient negative-feedback. Thus, different aspects of the HPA system appear to respond differentially to either the absence of food or the absence of the mother or both.

A case-control study of tobacco use and other non-occupational risk factors for t(14;18) subtypes of non-Hodgkin's lymphoma (United States).

OBJECTIVE: Non-Hodgkin's lymphoma (NHL) encompasses diverse subtypes, and analyzing NHL as a single outcome may mask associations. In a new approach we evaluated associations with subtypes defined by the t(14;18) translocation, reasoning that cases within these subtypes would have more common risk factors than all NHL combined. METHODS: Archival biopsies from cases in a population-based NHL study were assayed for t(14;18) using polymerase chain reaction amplification. Exposures in 68 t(14;18)-positive and 114-negative cases were compared with 1245 controls. The expectation-maximization algorithm was used to fit polytomous regression models based on all available information, including data from 440 unclassified cases. RESULTS: Family history of hemolymphatic cancer was associated with t(14;18)-negative NHL (odds ratio (OR) 2.4, 95% confidence interval (CI) 1.4 3.9). but not t(14;18)-positive NHL. Cigarette smoking was weakly associated with t(14;18)-positive NHL (OR 1.7, CI 0.9-3.3), but ORs decreased as smoking increased. Chewing tobacco was associated with t(14;18)-positive NHL, particularly when used before age 18 (OR 2.5. CI 1.0-6.0, 13 exposed cases). Odds ratios for both case-subtypes were doubled among hair-dye users. CONCLUSIONS: Cigarette smoking was not clearly associated with t(14;18)-positive NHL. Family history may be a marker for factors that act specifically through t(14;18)-negative pathogenic mechanisms.

The role of CD28-B7 costimulation in allergen-induced cytokine release by bronchial mucosa from patients with moderately severe asthma.

BACKGROUND: T cells play an important role in airway inflammation in asthma through the release of T(H)2 cytokines. Optimal T-cell activation by antigen-presenting cells requires co-stimulatory signaling, such as the interaction of CD80, CD86, or both with CD28. In patients with mild allergic asthma, the fusion protein cytotoxic T-lymphocyte antigen 4Ig (CTLA-4Ig), which inhibits CD28-mediated signaling, blocks the release of IL-5 and IL-13 from bronchial explant cultures exposed to the allergen Dermatophagoides pteronyssinus. OBJECTIVES: To assess costimulation in more severe forms of atopic asthma, we have compared the ability of CTLA-4Ig to block allergen-induced cytokine responses of bronchial explants and PBMCs from patients with moderately severe asthma. METHODS: Bronchial explants and PBMCs were cultured in vitro, and cytokine expression was measured by means of quantitative RT-PCR and ELISA. RESULTS: Constitutive mRNA transcripts for IL-5, IL-13, and GM-CSF were detected in the tissue explants, but only IL-5 mRNA increased significantly with allergen stimulation. Consistent with increased transcription, allergen-stimulated IL-5 protein release into explant supernatants, but this was not blocked by CTLA-4Ig. Allergen did not induce GM-CSF release, and IL-13 protein could not be detected in the explant supernatants under any condition. In contrast, allergen enhanced production of IL-5 and IL-13 by PBMC cultures from the same subjects, and this was inhibited effectively by CTLA-4Ig. CONCLUSIONS: These data suggest that IL-5 production in the airways of subjects with moderately severe asthma is largely independent of CD28-mediated costimulation. The different requirements for CD28-mediated costimulation in PBMC cultures and bronchial tissue cultures emphasizes the importance of the tissue microenvironment in pulmonary inflammatory responses in severe asthma.

Agricultural risk factors for t(14;18) subtypes of non-Hodgkin's lymphoma.

The t(14;18) translocation is a common somatic mutation in non-Hodgkin's lymphoma (NHL) that is associated with bcl-2 activation and inhibition of apoptosis. We hypothesized that some risk factors might act specifically along t(14;18)-dependent pathways, leading to stronger associations with t(14;18)-positive than t(14;18)-negative non-Hodgkin's lymphoma. Archival biopsies from 182 non-Hodgkin's lymphoma cases included in a case-control study of men in Iowa and Minnesota (the Factors Affecting Rural Men, or FARM study) were assayed for t(14;18) using polymerase chain reaction amplification; 68 (37%) were t(14;18)-positive. We estimated adjusted odds ratios (OR) and 95% confidence intervals (CI) for various agricultural risk factors and t(14;18)-positive and -negative cases of non-Hodgkin's lymphoma, based on polytomous logistic regression models fit using the expectation-maximization (EM) algorithm. T(14;18)-positive non-Hodgkin's lymphoma was associated with farming (OR 1.4, 95% CI = 0.9-2.3), dieldrin (OR 3.7, 95% CI = 1.9-7.0), toxaphene (OR 3.0, 95% CI = 1.5-6.1), lindane (OR 2.3, 95% CI = 1.3-3.9), atrazine (OR 1.7, 95% CI = 1.0-2.8), and fungicides (OR 1.8, 95% CI = 0.9-3.6), in marked contrast to null or negative associations for the same self-reported exposures and t(14;18)-negative non-Hodgkin's lymphoma. Causal relations between agricultural exposures and t(14;18)-positive non-Hodgkin's lymphoma are plausible, but associations should be confirmed in a larger study. Results suggest that non-Hodgkin's lymphoma classification based on the t(14;18) translocation is of value in etiologic research.

The contribution of interleukin (IL)-4 and IL-13 to the epithelial-mesenchymal trophic unit in asthma.

Interleukin (IL)-4 and IL-13 are key proinflammatory cytokines in asthma. Studies in transgenic mice show that both cytokines cause inflammation, but only IL-13 causes subepithelial fibrosis, a characteristic feature of asthma. We compared the in vitro profibrogenic effects of IL-4 and IL-13 using bronchial fibroblasts from asthmatic subjects. In the presence of transforming growth factor (TGF)-beta the cells transformed into contractile myofibroblasts and expressed alpha-smooth muscle actin and procollagen I. IL-4 and IL-13 also stimulated proliferation, but were relatively ineffective in promoting myofibroblast transformation. TGF-beta was more potent than the cytokines in stimulating release of endothelin-1 and vascular endothelial growth factor, whereas IL-4 and IL-13 were more potent stimuli for eotaxin release. Although neither IL-4 nor IL-13 induced profibrotic responses, both cytokines caused a corticosteroid-insensitive stimulation of TGF-beta2 release from primary bronchial epithelial cells. These data indicate that epithelial activation by IL-13 or IL-4 plays a critical role in initiating remodeling through release of TGF-beta2. TGF-beta2 then activates the underlying myofibroblasts to secrete matrix proteins and smooth muscle and vascular mitogens to propagate remodeling changes into the submucosa. In contrast, direct activation of submucosal fibroblasts by IL-4 and IL-13 has a proinflammatory effect via eotaxin release and recruitment of eosinophils into the airways.

Gene expression profiling in the amygdala: an approach to examine the molecular substrates of mammalian behavior.

The molecular substrates of behavior have been difficult to assess because of the large number of messenger RNAs (mRNAs) expressed in a given brain region, the heterogeneous composition of the CNS, and the complexity of mammalian behavior. To gain insight into the molecular components of behavior requires an understanding of the anatomy associated with a specific behavior and the ability to examine multiple gene expression in discrete brain regions. Neuroanatomical and behavioral studies have demonstrated that the amygdaloid complex is an essential component of the neural pathways mediating behaviors, such as fear, anxiety, learning, and memory. The amygdala is composed of several interconnected subnuclei and it is the modulation of information, as it flows through these subnuclei, that underlies amygdala function. To examine the molecular components of the amygdala, we have combined the antisense RNA (aRNA) amplification procedure with microarray technology. This experimental approach permits the simultaneous detection and quantification of numerous mRNAs in fixed tissue sections. Our initial experiment examines region-specific gene expression in naïve mice in order to map the molecular relationship between the subregions of the amygdala. This report provides a general overview of the techniques used to examine regional gene expression, suggests future experiments, and describes a theoretical framework for examining the molecular analysis of behavior.