Multiplex assays for biomarker research and clinical application: translational science coming of age

Over the last decade, translational science has come into the focus of academic medicine, and significant intellectual and financial efforts have been made to initiate a multitude of bench-to-bedside projects. The quest for suitable biomarkers that will significantly change clinical practice has become one of the biggest challenges in translational medicine. Quantitative measurement of proteins is a critical step in biomarker discovery. Assessing a large number of potential protein biomarkers in a statistically significant number of samples and controls still constitutes a major technical hurdle. Multiplexed analysis offers significant advantages regarding time, reagent cost, sample requirements and the amount of data that can be generated. The two contemporary approaches in multiplexed and quantitative biomarker validation, antibody-based immunoassays and MS-based multiple (or selected) reaction monitoring, are based on different assay principles and instrument requirements. Both approaches have their own advantages and disadvantages and therefore have complementary roles in the multi-staged biomarker verification and validation process. In this review, we discuss quantitative immunoassay and multiple reaction monitoring/selected reaction monitoring assay principles and development. We also discuss choosing an appropriate platform, judging the performance of assays, obtaining reliable, quantitative results for translational research and clinical applications in the biomarker field.

Clinical application and reliability of a post abdominal surgery pain assessment scale (PASPAS) in horses

The aim of this study was to refine a multi-dimensional scale based on physiological and behavioural parameters, known as the post abdominal surgery pain assessment scale (PASPAS), to quantify pain after laparotomy in horses. After a short introduction, eight observers used the scale to assess eight horses at multiple time points after laparotomy. In addition, a single observer was used to test the correlation of each parameter with the total pain index in 34 patients, and the effect of general anaesthesia on PASPAS was investigated in a control group of eight horses. Inter-observer variability was low (coefficient of variation 0.3), which indicated good reliability of PASPAS. The correlation of individual parameters with the total pain index differed between parameters. PASPAS, which was not influenced by general anaesthesia, was a useful tool to evaluate pain in horses after abdominal surgery and may also be useful to investigate analgesic protocols or for teaching purposes.

Free anteromedial thigh flap: clinical application and review of literature

The anteromedial thigh (AMT) flap is reviewed in terms of its vascular anatomy and previous clinical reports in the literature. Our own series of 5 patients treated with this flap for defects in the head and neck region and lower extremity is presented. Although several authors controversially discussed vasculature, we constantly found the pedicle as an emerging septocutaneous perforator at a point where the medial border of the rectus femoris muscle is crossed by the sartorius muscle. In all 5 patients, the AMT flap provided stable coverage with no flap loss. Based on our findings, we conclude that the anteromedial thigh flap offers all the advantages of fasciocutaneous flaps. Therefore, we recommend this flap as an alternative for defects requiring coverages of thin to moderate skin thickness. However, it should be remembered that variations in vascular anatomy are possible.

Anti-eosinophil activity and clinical efficacy of the CRTH2 antagonist OC000459 in eosinophilic esophagitis

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic, Th2-type inflammatory disease. Chemoattractant receptor-homologous molecule on Th2 cells (CRTH2) is a prostaglandin D(2) (PGD(2)) receptor, expressed by Th2 cells and other inflammatory cells, including eosinophils and basophils, that mediates chemotaxis and activation. OC000459 is a selective CRTH2 antagonist and would be expected to suppress eosinophilic tissue inflammation. The purpose of this study was to evaluate the efficacy and safety of an OC000459 monotherapy in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE. METHODS: In this randomized, double-blind, placebo-controlled trial, 26 adult patients (m/f = 22/4; mean age 41 years, range 22-69 years) with active EoE, dependent or resistant to corticosteroids, were treated either with 100 mg OC000459 (n = 14) or placebo (n = 12) twice daily. Pre- and post-treatment disease activity was assessed clinically, endoscopically, histologically, and via biomarkers. The primary end point was the reduction in esophageal eosinophil infiltration. RESULTS: After an 8-week OC000459 treatment, the esophageal eosinophil load decreased significantly, from 114.83 to 73.26 eosinophils per high-power field [(eos/hpf), P = 0.0256], whereas no reduction was observed with placebo (102.80-99.47 eos/hpf, P = 0.870). With OC000459, the physician's global assessment of disease activity improved from 7.13 to 5.18 (P = 0.035). OC000459 likewise reduced extracellular deposits of eosinophil peroxidase and tenascin C, the effects not seen with placebo. No serious adverse events were observed. CONCLUSIONS: An 8-week treatment with the CRTH2-antagonist, OC000459, exerts modest, but significant, anti-eosinophil and beneficial clinical effects in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE and is well tolerated.

Clinical outcomes following subgingival application of a novel erythritol powder by means of air polishing in supportive periodontal therapy: a randomized, controlled clinical study

OBJECTIVES The aim of this prospective, randomized, controlled clinical study was to compare the clinical outcomes of the subgingival treatment with erythritol powder by means of an air-polishing (EPAP) device and of scaling and root planing (SRP) during supportive periodontal therapy (SPT). METHOD AND MATERIALS 40 patients enrolled in SPT were randomly assigned to two groups of equal size. Sites had to show signs of inflammation (bleeding on probing [BOP]-positive) and a probing pocket depth (PPD) of ≥ 4 mm, however, without presence of detectable subgingival calculus. During SPT, these sites were treated with EPAP or SRP, respectively. Full mouth and site-specific plaque indices, BOP, PPD, and clinical attachment level (CAL) were recorded at baseline (BL) and at 3 months, whereas the percentage of study sites positive for BOP (BOP+) was considered as primary outcome variable. Additionally, patient comfort using a visual analog scale (VAS) and the time needed to treat per site was evaluated. RESULTS At 3 months, mean BOP level measured 45.1% at test sites and 50.6% at control sites, respectively, without a statistically significant difference between the groups (P > .05). PPD and CAL slightly improved for both groups with comparable mean values at 3 months. Evaluation of patient tolerance showed statistically significantly better values among patients receiving the test treatment (mean VAS [0-10], 1.51) compared to SRP (mean VAS [0-10], 3.66; P = .0012). The treatment of test sites was set to 5 seconds per site. The treatment of control sites, on the other hand, lasted 85 seconds on average. CONCLUSION The new erythritol powder applied with an air-polishing device can be considered a promising modality for repeated instrumentation of residual pockets during SPT. CLINICAL RELEVANCE With regard to clinical outcomes during SPT, similar results can be expected irrespective of the two treatment approaches of hand instrumentation or subgingival application of erythritol powder with an air-polishing device in sites where only biofilm removal is required.

A randomized clinical trial of Behavioral Activation (BA) therapy for improving psychological and physical health in dementia caregivers: results of the Pleasant Events Program (PEP).

Dementia caregiving is associated with elevations in depressive symptoms and increased risk for cardiovascular diseases (CVD). This study evaluated the efficacy of the Pleasant Events Program (PEP), a 6-week Behavioral Activation intervention designed to reduce CVD risk and depressive symptoms in caregivers. One hundred dementia family caregivers were randomized to either the 6-week PEP intervention (N = 49) or a time-equivalent Information-Support (IS) control condition (N = 51). Assessments were completed pre- and post-intervention and at 1-year follow-up. Biological assessments included CVD risk markers Interleukin-6 (IL-6) and D-dimer. Psychosocial outcomes included depressive symptoms, positive affect, and negative affect. Participants receiving the PEP intervention had significantly greater reductions in IL-6 (p = .040), depressive symptoms (p = .039), and negative affect (p = .021) from pre- to post-treatment. For IL-6, clinically significant improvement was observed in 20.0% of PEP participants and 6.5% of IS participants. For depressive symptoms, clinically significant improvement was found for 32.7% of PEP vs 11.8% of IS participants. Group differences in change from baseline to 1-year follow-up were non-significant for all outcomes. The PEP program decreased depression and improved a measure of physiological health in older dementia caregivers. Future research should examine the efficacy of PEP for improving other CVD biomarkers and seek to sustain the intervention's effects.

The missing link between clinical states and biomarkers in mental disorders

Current diagnostic definitions of psychiatric disorders based on collections of symptoms encompass very heterogeneous populations and are thus likely to yield spurious results when exploring biological correlates of mental disturbances. It has been suggested that large studies of biomarkers across diagnostic entities may yield improved clinical information. Such a view is based on the concept of assessment as a collection of symptoms devoid of any clinical judgment and interpretation. Yet, important advances have been made in recent years in clinimetrics, the science of clinical judgment. The current clinical taxonomy in psychiatry, which emphasizes reliability at the cost of clinical validity, does not include effects of comorbid conditions, timing of phenomena, rate of progression of an illness, responses to previous treatments, and other clinical distinctions that demarcate major prognostic and therapeutic differences among patients who otherwise seem to be deceptively similar since they share the same psychiatric diagnosis. Clinimetrics may provide the missing link between clinical states and biomarkers in psychiatry, building pathophysiological bridges from clinical manifestations to their neurobiological counterparts.

Combinations of dexmedetomidine and alfaxalone with butorphanol in cats: application of an innovative stepwise optimisation method to identify optimal clinical doses for intramuscular anaesthesia

OBJECTIVES The aim of this study was to optimise dexmedetomidine and alfaxalone dosing, for intramuscular administration with butorphanol, to perform minor surgeries in cats. METHODS Initially, cats were assigned to one of five groups, each composed of six animals and receiving, in addition to 0.3 mg/kg butorphanol intramuscularly, one of the following: (A) 0.005 mg/kg dexmedetomidine, 2 mg/kg alfaxalone; (B) 0.008 mg/kg dexmedetomidine, 1.5 mg/kg alfaxalone; (C) 0.012 mg/kg dexmedetomidine, 1 mg/kg alfaxalone; (D) 0.005 mg/kg dexmedetomidine, 1 mg/kg alfaxalone; and (E) 0.012 mg/kg dexmedetomidine, 2 mg/kg alfaxalone. Thereafter, a modified 'direct search' method, conducted in a stepwise manner, was used to optimise drug dosing. The quality of anaesthesia was evaluated on the basis of composite scores (one for anaesthesia and one for recovery), visual analogue scales and the propofol requirement to suppress spontaneous movements. The medians or means of these variables were used to rank the treatments; 'unsatisfactory' and 'promising' combinations were identified to calculate, through the equation first described by Berenbaum in 1990, new dexmedetomidine and alfaxalone doses to be tested in the next step. At each step, five combinations (one new plus the best previous four) were tested. RESULTS None of the tested combinations resulted in adverse effects. Four steps and 120 animals were necessary to identify the optimal drug combination (0.014 mg/kg dexmedetomidine, 2.5 mg/kg alfaxalone and 0.3 mg/kg butorphanol). CONCLUSIONS AND RELEVANCE The investigated drug mixture, at the doses found with the optimisation method, is suitable for cats undergoing minor clinical procedures.

Clinical activity of pemphigus vulgaris relates to IgE autoantibodies against desmoglein 3

Pemphigus vulgaris (PV) is a severe autoimmune bullous skin disease and is primarily associated with IgG against desmoglein 3 (dsg3), a desmosomal adhesion protein. In light of the recent association of autoreactive T helper (Th) 2 cells with active PV, the present study sought to relate the occurrence of Th2-regulated dsg3-specific autoantibody subtypes, i.e. IgE and IgG4, in 93 well-characterized PV patients. Patients with acute onset PV (n=37) showed the highest concentrations of serum IgE and IgG4 autoantibodies, which were significantly lower in PV patients in remission (n=14). Furthermore, there was a strong correlation between dsg3-reactive IgE and IgG4 in acute onset, but not in chronic active (n=42) or remittent patients. Additionally, intercellular IgE deposits were detected in the epidermis of acute onset PV. Thus, dsg3-specific IgE and IgG4 autoantibodies are related to acute onset disease which provides additional support to the concept that PV is a Th2-driven autoimmune disorder.

Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

BACKGROUND: Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases. METHODS: sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression. RESULTS: sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition. CONCLUSIONS: This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.