Optimising cell-based bioassays via integrated design of experiments (ixDoE) - A practical guide.

For process optimisation Design of Experiments (DoE) has long been established as a more powerful strategy than a One Factor at a Time approach. Nevertheless, DoE is not widely used especially in the field of cell-based bioassay development although it is known that complex interactions often exist. We believe that biopharmaceutical manufacturers are reluctant to move beyond standard practices due to the perceived costs, efforts, and complexity. We therefore introduce the integrated DoE (ixDoE) approach to target a smarter use of DoEs in the bioassay setting, specifically in optimising resources and time. Where in a standard practice 3 to 4 separate DoEs would be performed, our ixDoE approach includes the necessary statistical inference from only a single experimental set. Hence, we advocate for an innovative, ixDoE approach accompanied by a suitable statistical analysis strategy and present this as a practical guide for a typical bioassay development from basic research to biopharmaceutical industry.

Student-Initiated Feedback Using Clinical Encounter Cards during Clinical Rotations in Veterinary Medicine: A Feasibility Study.

Despite its critical role for successful student learning, providing adequate teacher feedback is still a major issue during clinical education. In human medical education, the implementation of clinical encounter cards (CECs) has led to more frequent, timely, and structured teacher feedback. The present study aimed to introduce student-initiated CECs in a veterinary medical setting (clinical rotations). A total of 24 students were randomly assigned to a control group (standard rotations) and an intervention group where they had to ask for teacher feedback using CECs. The feasibility of implementing CECs was evaluated by examining the content of the completed CECs and by using anonymous student and teacher questionnaires. In addition, acceptance of the intervention and changes in feedback behavior were examined from both the teachers' and students' perspectives. Overall, it was shown that using CECs is not only feasible in a veterinary clinical setting but also conducive to more frequent and constructive teacher feedback. However, some teachers postponed completing the CECs due to time pressure, leading to less direct and timely feedback. Moreover, students felt more comfortable asking for feedback from less experienced, younger teachers, and teachers' quantitative ratings and open commentaries seemed to be affected by leniency bias. Finally, a focus group including teachers and students discussed these results in light of their practical experiences. This led to a streamlining of the implementation process and optimizations to facilitate future large-scale implementation. The study has implications for veterinary educators wishing to improve feedback in their institution.

Antimicrobial Formulations of Absorbable Bone Substitute Materials as Drug Carriers Based on Calcium Sulfate.

Substitution of bones is a well-established, necessary procedure to treat bone defects in trauma and orthopedic surgeries. For prevention or treatment of perioperative infection, the implantation of resorbable bone substitute materials carrying antibiotics is a necessary treatment. In this study, we investigated the newly formulated calcium-based resorbable bone substitute materials containing either gentamicin (CaSO4-G [Herafill-G]), vancomycin (CaSO4-V), or tobramycin (Osteoset). We characterized the released antibiotic concentration per unit. Bone substitute materials were implanted in bones of rabbits via a standardized surgical procedure. Clinical parameters and levels of the antibiotic-releasing materials in serum were determined. Local concentrations of antibiotics were measured using antimicrobial tests of bone tissue. Aminoglycoside release kinetics in vitro per square millimeter of bead surface showed the most prolonged release for gentamicin, followed by vancomycin and, with the fastest release, tobramycin. In vivo level in serum detected over 28 days was highest for gentamicin at 0.42 µg/ml, followed by vancomycin at 0.11 µg/ml and tobramycin at 0.04 µg/ml. The clinical parameters indicated high biocompatibility for materials used. None of the rabbits subjected to the procedure showed any adverse reaction. The highest availability of antibiotics at 14.8 µg/g on day 1 in the cortical tibia ex vivo was demonstrated for gentamicin, decreasing within 14 days. In the medulla, vancomycin showed a high level at 444 µg/g on day 1, decreasing continuously over 14 days, whereas gentamicin decreased faster within the initial 3 days. The compared antibiotic formulations varied significantly in release kinetics in serum as well as locally in medulla and cortex.

[Determination of irreversibility of clinical brain death. Electroencephalography and evoked potentials]. / Irreversibilitätsnachweis der klinischen Ausfallssymptome des Gehirns. Elektroenzephalographie und evozierte Potenziale.

Principally, in the fourth update of the rules for the procedure to finally determine the irreversible cessation of function of the cerebrum, the cerebellum and the brainstem, the importance of an electroencephalogram (EEG), somatosensory evoked potentials (SEP) and brainstem auditory evoked potentials (BAEP) are confirmed. This paper presents the reliability and validity of the electrophysiological diagnosis, discusses the amendments in the fourth version of the guidelines and introduces the practical application, problems and sources of error.An EEG is the best established supplementary diagnostic method for determining the irreversibility of clinical brain death syndrome. It should be noted that residual brain activity can often persist for many hours after the onset of brain death syndrome, particularly in patients with primary brainstem lesions. The derivation and analysis of an EEG requires a high level of expertise to be able to safely distinguish artefacts from primary brain activity. The registration of EEGs to demonstrate the irreversibility of clinical brain death syndrome is extremely time consuming.The BAEPs can only be used to confirm the irreversibility of brain death syndrome in serial examinations or in the rare cases of a sustained wave I or sustained waves I and II. Very often, an investigation cannot be reliably performed because of existing sound conduction disturbances or failure of all potentials even before the onset of clinical brain death syndrome. This explains why BAEPs are only used in exceptional cases.The SEPs of the median nerve can be very reliably derived, are technically simple and with few sources of error. A serial investigation is not required and the time needed for examination is short. For these reasons SEPs are given preference over EEGs and BAEPs for establishing the irreversibility of clinical brain death syndrome.

Increased antibiotic release and equivalent biomechanics of a spacer cement without hard radio contrast agents.

We compared a novel calcium carbonate spacer cement (Copal® spacem) to well-established bone cements. Electron microscopic structure and elution properties of the antibiotics ofloxacin, vancomycin, clindamycin, and gentamicin were examined. A knee wear simulator model for articulating cement spacers was established. Mechanical tests for bending strength, flexural modulus, and compressive and fatigue strength were performed. The electron microscopic analysis showed a microporous structure of the spacer cement, and this promoted a significantly higher and longer antibiotic elution. All spacer cement specimens released the antibiotics for a period of up to 50days with the exception of the vancomycin loading. The spacer cement showed significantly less wear scars and fulfilled the ISO 5833 requirements. The newly developed spacer cement is a hydrophilic antibiotic carrier with an increased release. Cement without hard radio contrast agents can improve tribological behaviour of spacers, and this may reduce reactive wear particles and abrasive bone defects.

Clinical use and safety of a novel gentamicin-releasing resorbable bone graft substitute in the treatment of osteomyelitis/osteitis.

OBJECTIVE: A clinical investigation into a new bone void filler is giving first data on systemic and local exposure to the anti-infective substance after implantation. METHOD: A total of 20 patients with post-traumatic/post-operative bone infections were enrolled in this open-label, prospective study. After radical surgical debridement, the bone cavity was filled with this material. The 21-day hospitalisation phase included determination of gentamicin concentrations in plasma, urine and wound exudate, assessment of wound healing, infection parameters, implant resorption, laboratory parameters, and adverse event monitoring. The follow-up period was six months. RESULTS: Systemic exposure to gentamicin after implantation was very low as local gentamicin concentrations were measured in wound exudate after six to ten hours. There were no signs of infectious complication throughout the clinical phase. Four patients had recurrent infections several weeks to months after implantation. The outcome was deemed successful by remission of infection in 16 (80%) of these problematic long-term treated patients. Safety laboratory measurements did not indicate nephrotoxic or hepatotoxic effects. CONCLUSIONS: Local application of calcium sulphate/carbonate bone void filler comprising gentamicin revealed sufficient active local levels of the antibiotic by simultaneous significant low systemic exposure in patients with mostly chronic osteomyelitis/osteitis. The material was safe and well tolerated. Cite this article: Bone Joint Res 2014;3:223-9.

Clinical and pathological nodal staging score for urothelial carcinoma of the bladder: an external validation.

PURPOSE: Radical cystectomy (RC) and pelvic lymph node dissection (LND) are standard treatments for muscle-invasive urothelial carcinoma of the bladder. Lymph node staging is a prerequisite for clinical decision-making regarding adjuvant chemotherapy and follow-up regimens. Recently, the clinical and pathological nodal staging scores (cNSS and pNSS) were developed. Prior to RC, cNSS determines the minimum number of lymph nodes required to be dissected; pNSS quantifies the accuracy of negative nodal staging based on pT stage and dissected LNs. cNSS and pNSS have not been externally validated, and their relevance for prediction of cancer-specific mortality (CSM) has not been assessed. METHODS: In this retrospective study of 2,483 RC patients from eight German centers, we externally validated cNSS and pNSS and determined their prediction of CSM. All patients underwent RC and LND. Median follow-up was 44 months. cNSS and pNSS sensitivities were evaluated using the original beta-binominal models. Adjusted proportional hazards models were calculated for pN0 patients to assess the predictive value of cNSS and pNSS for CSM. RESULTS: cNSS and pNSS both pass external validation. Adjusted for other clinical parameters, cNSS can predict outcome after RC. pNSS has no independent impact on prediction of CSM. The retrospective design is the major limitation of the study. CONCLUSIONS: In the present external validation, we confirm the validity of both cNSS and pNSS. cNSS is an independent predictor of CSM, thus rendering it useful as a tool for planning the extent of LND.

The effect of airflow on thermographically determined temperature of the distal forelimb of the horse.

REASONS FOR PERFORMING STUDY: Current literature suggests that thermographic imaging of horses should be performed in a draught-free room. However, studies on the effect of airflow on determined temperature have not been published. OBJECTIVES: To investigate effects of airflow on thermographically determined temperature of horses' forelimbs; to assess the relationship of wind velocity, rectal temperature, ambient temperature and humidity. METHODS: Thermographic images were obtained for the forelimbs of 6 horses in a draught-free room. Three replicates (R) with defined wind velocities (R1, 0.5-1.0 m/s; R2, 1.3-2.6 m/s; and R3, 3.0-4.0 m/s) were conducted. Each replicate consisted of a baseline image, a 15 min phase with the wind on and a 15 min phase with the wind off. We exposed only the right leg to airflow and determined the temperature by thermography with the wind on and wind off. Temperature differences between baseline and wind on, between wind on and wind off and between different wind velocities were analysed by a general linear model, Student's paired t test and ANOVA. RESULTS: After the onset of wind, the temperature on the right forelimb decreased within 1-3 min (by approximately 0.6°C at R1, 1.5°C at R2 and 2.1°C at R3). With the wind off, the temperature increased within 3 min (by approximately 1.2°C at R1, 1.7°C at R2 and 2.1°C at R3). With increasing wind velocity, the temperature differences between baseline and wind on and between wind on and wind off increased significantly. CONCLUSIONS: Barely noticeable wind velocities caused a decrease in thermographically determined temperatures of the forelimbs of the horse. Further research is required to assess the influence of airflow on other parts of the body and at different ambient temperatures, as well as the effect on horses with inflammatory lesions, especially of the distal limbs. POTENTIAL RELEVANCE: It is essential for practitioners to perform thermography on horses in a draught-free environment in order to avoid false-positive or -negative diagnoses.

Clinical outcome of neurointerventional emergency treatment of extra- or intracranial tandem occlusions in acute major stroke: antegrade approach with wallstent and solitaire stent retriever.

BACKGROUND: Acute large cerebral artery occlusions respond poorly to systemic thrombolysis with recombinant tissue plasminogen activator (rTPA) alone. The value of stent retriever-based mechanical thrombectomy in patients with additional extracranial occlusion of the internal carotid artery (ICA), who require acute a priori extracranial stenting in order to reach the intracranial obstruction site, is not well known. We determined the outcome after emergency revascularization in acute stroke with tandem occlusions of the anterior circulation. METHODS: According to specific inclusion/exclusion criteria, eligible stroke patients with large artery occlusions underwent mechanical recanalization with the Solitaire stent retriever. In case of a tandem occlusion, we performed an acute stenting with the Wallstent before thrombectomy. From October 2009 to March 2011, 50 patients were treated according to this protocol; time frames, clinical data, recanalization rates, and midterm outcome were recorded. RESULTS: Forty-one patients had a large artery occlusion in the anterior circulation and nine in the posterior circulation. Mechanical recanalization was successful in 35/41 cases (85 %). Six of 41 patients (15 %) died in the acute phase. In 17/41 patients (42 %), thrombectomy was preceded by an emergency stenting in the extracranial portion of the internal carotid artery (ICA). National Institutes of Health Stroke Scale (NIHSS)/modified Rankin Scale (mRS) scores showed significant improvement in both the stenting group and the nonstenting group; there were no significant differences between the groups. At 90 days, 54 % of patients with emergency stenting had a good outcome. CONCLUSIONS: Acute extracranial stenting with the Wallstent combined with intracranial Solitaire-based thrombectomy is safe and may lead to an improvement in neurological outcome in patients with an otherwise poor prognosis under i.v. thrombolysis alone.

Semantic event-related potential components reflect severity of comprehension deficits in aphasia.

BACKGROUND AND OBJECTIVES: Several cognitive event-related potential (ERP) components such as mismatch negativity, P300, N400, and the late positive component (LPC) have been studied in aphasia. The aim of this study was to determine whether a modified semantic incongruity paradigm can serve as a more graded differentiation of ERP changes in patients with mild versus severe comprehension deficits. METHODS: A total of 20 aphasic patients with minor and severe comprehension deficits and 20 young and elderly healthy controls were examined while reading 4-word sentences ending in a semantically congruent or noncongruent word. RESULTS: In contrast to young controls and to patients with mild comprehension deficits, aphasic patients with severe comprehension deficits exhibit an early positivity in the time window from 200 to 400 milliseconds and no N400 after the presentation of nonrecurrent semantically incongruent words. Patients with mild comprehension deficits were found to have an N400 with prolonged latency in comparison with the controls. An age effect in the control groups was detected as well. DISCUSSION: Semantic access and integration are performed differently in aphasic subjects with severe comprehension deficits. These differences in lexical-semantic processing must be taken into account in rehabilitation approaches that aim to improve comprehension deficits. Moreover, the findings may contribute to the design of therapy studies by employing a physiological measure that can discriminate among patients at baseline and at the end of an intervention.

New anti-infective coatings of surgical sutures based on a combination of antiseptics and fatty acids.

Wound infection is a complication feared in surgery. The aim of this study was to develop new anti-infective coatings of surgical sutures and to compare the anti-microbial effectiveness and biocompatibility to the well-established Vicryl Plus. Synthetic absorbable PGA surgical sutures were coated with three different chlorhexidine concentrations and two different octenidine concentrations in combination with palmitic acid and lauric acid. Drug-release kinetics lasting 96 h were studied in phosphate-buffered saline at 37 degrees C. Anti-infective characteristics were determined by measuring the change in optical density of Staphylococcus aureus suspensions charged with coated sutures over time. Microorganisms adsorbed at the surface of coated sutures were assessed on blood agar plates and coated sutures eluted for 24 h were placed on bacterial lawns cultured on Mueller-Hinton plates to prove retained anti-microbial potency. A cell proliferation assay was performed to assess the degree of cytotoxicity. Anti-infective characteristics and biocompatibility were compared to Vicryl Plus. A coating technology for slow-release drug-delivery systems on surgical sutures could be developed. All coatings showed a continuous drug release within 96 h. Individual chlorhexidine and octenidine coated sutures showed superior anti-infective characteristics but inferior biocompatibility in comparison to Vicryl Plus. We conclude that the developed anti-infective suture coatings consisting of lipid-based drug-delivery systems in combination with antiseptics are highly effective against bacterial colonization in vitro; however, drug doses have to be adjusted to improve biocompatibility.

Short-term modulation of the ipsilateral primary sensory cortex by nociceptive interference revealed by SEPs.

We studied the modulation of the topographic arrangement of the human ipsilateral primary somatosensory cortex following interference of nociceptive stimuli by means of dipole source analysis. Multichannel somatosensory evoked potentials were obtained by electrical stimulation of digits 1 and 5 of the left hand before, during and after the application of pain to digits 2-4 of the right hand. The primary cortical response of the SEP (N20) was obtained for dipole localization of the representation of the primary sensory cortex receiving input from digits 1 to 5. The 3D-distance between these sides was calculated for further analysis. To account for possible attentional effects recordings were performed while simultaneously to this intervention subjects were asked to turn their attention to the right or left hand in a pseudorandom order. The application of pain induced an expansion of the 3D-distance between digits 1 and 5. Focusing attention to the stimulated limb or the site of the intervention did not yield to an additional effect. Our results provide further evidence for the presence of a quickly adapting interaction between primary somatosensory areas of both hemispheres following an interference of nociceptive stimulation in SEPs. This modifying process is probably mediated by interhemispheric and intercortical connections leading to hyperexcitability of the primary sensory cortex contralateral to that receiving nociceptive input. Spatial attention does not seem to have an impact on this kind of short-term intercortical plasticity.

Interference of tactile and pain stimuli on thalamocortical signal processing in humans revealed by median nerve SEPs.

OBJECTIVE: We investigated the interference of tactile and painful stimuli on human early somatosensory evoked potentials (SEPs) including high frequency oscillations (HFOs) to further study thalamocortical processing of somatosensory information. METHODS: Multi-channel median nerve SEPs were recorded during (1) no interference, (2) sensory interference by tactile stimulation to digits 2 and 3, and (3) application of pain to the same digits. Spatio-temporal source analysis separated brain stem (S1), thalamic (S2) and two cortical sources (S3, S4), which were evaluated for the low (20-450 Hz) and high (450-750 Hz) frequency portion of the signal. RESULTS: Low frequency SEPs showed a decrease of activity at cortical source S3 during both conditions, while thalamic source S2 was significantly increased during pain interference. HFOs showed an increase of cortical source S3 and in trend of thalamic source S2 and cortical source S4 during both kinds of interference. CONCLUSIONS: Although the painful stimulus might not be specific for the nociceptive afferents, the present data affirm that at this early stage of sensory information processing within the primary sensory cortex (area 3b, area 1) pain is handled similar to sensory interference. SIGNIFICANCE: HFOs might represent an intrinsic "somatosensory alerting" system which reacts to both interference stimuli in a similar way, therefore indicating an interference without a qualitative evaluation.

[Severe encephalitis without correlate in MRI]. / Schwerste Enzephalitiden ohne Korrelat im MRT.

Encephalitis could be a life-threatening disease depending on localisation and infectious agent. Neuroimaging, especially MRI, is an important component in the diagnosis. The recent investigations demonstrate that diffusion abnormalities are the first and sensitive signs of viral encephalitis. We describe five patients with severe encephalitis with normal MR imaging. Three of five patients were intermittently mechanically ventilated, two of these longer than 4 weeks. The other two patients suffered from a severe psychosyndrome with seriously limited ability to communicate throughout 6 weeks. At the time of first MRI examination all patients were noticeably ill. Four of five patients had at least one follow-up MRI. The MRI examinations included FLAIR sequences and DWI in four of five patients. Contrast-enhancement was detectable in none of our patients. Follow-up examinations revealed that symptoms of encephalitis were noticeably or completely regressive.

[Pseudoangiitis in bilateral ocular ischemia]. / Pseudoangiitis bei beidseitiger okulärer Ischämie.

BACKGROUND: The term 'frosted branch angiitis' was initially used to describe an idiopathic form of vasculitis in association with panuveitis. It has since also been used to describe the angiographic phenomenon of diffuse leakage along retinal vessels against the background of other ocular and systemic diseases. METHODS: We describe a 57-year-old male patient with acute bilateral reduction of visual acuity, headaches and absence of any pulse at the temporal arteries. Fluorescence angiography showed bilateral diffuse leakage along all the retinal vessels, which resembled frosted branches. Laboratory parameters and histology were not indicative of vasculitis. Imaging showed complete occlusion of both common carotid arteries and a hypoplastic vertebral artery on the left. CONCLUSIONS: Ocular ischemia may imitate primarily inflammatory conditions in its angiographic appearance.

A case of cerebral Whipple's disease initially presenting with isolated focal myoclonus.

Neurological manifestations in Whipple's disease are highly variable and tend to occur at later stages of the disease. However, isolated, focal neurological symptoms are reported to be rare. Here we describe the successful treatment of a case of cerebral Whipple's disease initially presenting solely with isolated myoclonic jerks of the left hand and forearm evolving to a segmental myoclonus at a later stage. Additionally, we present - to our knowledge - a novel treatment by administration of immunomodulatory therapy (IVIg) in addition to established antibiotics.

Evaluation using hoof wall strain gauges of a therapeutic shoe and a hoof cast with a heel wedge as potential supportive therapy for horses with laminitis.

OBJECTIVE: To evaluate using strain gauges, a hoof cast with heel wedge, and a therapeutic shoe with unsupported toe for their effectiveness in redistribution of load from the dorsal hoof wall. STUDY DESIGN: In vitro biomechanical study. SAMPLE POPULATION: Twenty forelimb specimens. METHODS: Rosette strain gauges were placed on the dorsal and lateral hoof wall of 20 normal shaped hooves. Limbs were loaded vertically using a tensile testing machine with a 1 Hz sinusoidally cycling load up to 3000 N during 15 seconds. Mean values of principal strain and direction at 2500 N load were calculated for 3 experimental conditions (unshod, therapeutic shoe with unsupported toe, and hoof cast with heel elevation) and tested by ANOVA (P<.05). RESULTS: Vertical limb loading in an unshod hoof leads to a biaxial compression of the dorsal wall with high longitudinal compression (epsilon2 = -1515 microm/m). Principal strain at the dorsal wall (epsilon2) was decreased by 23% with the therapeutic shoe and by 59% with the hoof cast. On the lateral hoof wall principal strain was unchanged with the shoe, but increased by 34% with the cast. CONCLUSIONS: Strain measurements indicate unloading of the dorsal hoof wall by both methods with the cast being more effective than the shoe. CLINICAL RELEVANCE: The hoof cast with wedge offers substantial unloading of the dorsal wall, but increases load on the quarter. Therefore a hoof cast would likely be most helpful in acute laminitis when palmar structures can still bear load. The therapeutic shoe offers rehabilitation and regrowth of the dorsal wall without increased load on the quarter wall.

Different origins of low- and high-frequency components (600 Hz) of human somatosensory evoked potentials.

OBJECTIVE: Human median nerve somatosensory evoked potentials (SEPs) contain a low-amplitude (<500 nV) high-frequency (approximately 600 Hz) burst of repetitive wavelets (HFOs) which are superimposed onto the primary cortical response 'N20.' This study aimed to further clarify the cortical and subcortical structures involved in the generation of the HFOs. METHODS: 128-Channel recordings were obtained to right median nerve stimulation of 10 right-handed healthy human subjects and in 7 of them additional to right ulnar nerve. Data were evaluated by applying principal component analysis and dipole source analysis. RESULTS: Different source evaluation strategies provided converging evidence for a cortical HFO origin, with two different almost orthogonally oriented generators being active in parallel, but with a phase shift of a quarter of their oscillatory period, while the low-frequency 'N20' is adequately modeled by one tangential dipole source. Median and ulnar derived low-frequency and HFO cortical sources show a somatotopic order. Additionally, generation of the HFOs was localized in subcortical, near-thalamic and subthalamic source sites. The near-thalamic dipole was located at significantly different sites in HFO and low-frequency data. CONCLUSIONS: The cortical HFO source constellation points to a 'precortical' source in terminals of thalamocortical fibers and a second intracortical HFO origin. Furthermore, HFOs are also generated at subcortical and even subthalamic sites. Near-thalamic, the HFO and low-frequency signals are generated or modulated by different neuron populations involved in the thalamocortical outflow.

Activation of the human posterior parietal and temporoparietal cortices during audiotactile interaction.

We recorded cortical-evoked responses with a whole-scalp neuromagnetometer to study human brain dynamics associated with audiotactile interaction. The subjects received unilateral auditory (A) or tactile (T) stimuli, or both stimuli simultaneously (AT), alternating to the left and right side. Responses to AT stimuli differed significantly from the algebraic sum of responses to A and T stimuli (A + T) at 75-85 and 105-130 ms and indicated suppressive audiotactile interaction. Source modeling revealed that the earlier interaction occurred in the contralateral posterior parietal cortex and the later interaction in the contralateral parietal opercula between the SII cortex and the auditory cortex. The interaction was significantly stronger in the left than the right hemisphere. In most subjects, AT responses were far more similar to T than to A responses, suggesting suppression of auditory processing during the spatially and temporally concordant audiotactile stimuli in which the tactile component was subjectively more salient.