Publications by authors named "Michele Fiscella"

35 Publications

Versatile live-cell activity analysis platform for characterization of neuronal dynamics at single-cell and network level.

Nat Commun 2020 09 25;11(1):4854. Epub 2020 Sep 25.

Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

Chronic imaging of neuronal networks in vitro has provided fundamental insights into mechanisms underlying neuronal function. Current labeling and optical imaging methods, however, cannot be used for continuous and long-term recordings of the dynamics and evolution of neuronal networks, as fluorescent indicators can cause phototoxicity. Here, we introduce a versatile platform for label-free, comprehensive and detailed electrophysiological live-cell imaging of various neurogenic cells and tissues over extended time scales. We report on a dual-mode high-density microelectrode array, which can simultaneously record in (i) full-frame mode with 19,584 recording sites and (ii) high-signal-to-noise mode with 246 channels. We set out to demonstrate the capabilities of this platform with recordings from primary and iPSC-derived neuronal cultures and tissue preparations over several weeks, providing detailed morpho-electrical phenotypic parameters at subcellular, cellular and network level. Moreover, we develop reliable analysis tools, which drastically increase the throughput to infer axonal morphology and conduction speed.
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http://dx.doi.org/10.1038/s41467-020-18620-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519655PMC
September 2020

Correction to: Recommendations for the Development of Cell-Based Anti-Viral Vector Neutralizing Antibody Assays.

AAPS J 2020 Feb 4;22(2):42. Epub 2020 Feb 4.

Alzheimer's Drug Discovery Foundation, New York, NY, USA.

The first author's name was published incorrectly as "Gorovits Boris". The correct name is "Boris Gorovits".
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http://dx.doi.org/10.1208/s12248-020-0425-8DOI Listing
February 2020

Recommendations for the Development of Cell-Based Anti-Viral Vector Neutralizing Antibody Assays.

AAPS J 2020 01 6;22(2):24. Epub 2020 Jan 6.

Alzheimer's Drug Discovery Foundation, 57 W 57th St #904, New York, USA.

Viral vector-based gene therapies (GTx) have received significant attention in the recent years and the number of ongoing GTx clinical trials is increasing. A platform of choice for many of these studies is adeno-associated virus (AAV). All humans may be exposed to natural AAV infections and could mount an immune response against the virus. Consequently, there can be a high prevalence of pre-existing anti-AAV immunity. This presents a potential limitation for AAV-based GTx due to the potential for AAV-specific antibodies to reduce the efficacy of the GTx. Therefore, appropriate assessment of potential subjects enrolled in these studies should include evaluation for the presence and degree of anti-AAV immunity, including anti-AAV neutralizing antibodies (NAb). Recommendations for the development and validation of cell-based anti-AAV NAb detection methods, including considerations related to selection of appropriate cell line, surrogate vector/reporter gene, assay matrix and controls, and methodologies for calculating assay cut-point are discussed herein. General recommendations for the key assay validation parameters are provided as well as considerations for the development of NAb diagnostic tests. This manuscript is produced by a group of scientists involved in GTx therapeutic development representing various companies. It is our intent to provide recommendations and guidance to industrial and academic laboratories working on viral vector based GTx modalities with the goal of achieving a more consistent approach to anti-AAV NAb assessment.
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http://dx.doi.org/10.1208/s12248-019-0403-1DOI Listing
January 2020

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

Bioanalysis 2019 Dec 10;11(24):2207-2244. Epub 2019 Dec 10.

Janssen R&D, Spring House, PA, USA.

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of , issues 22 and 23 (2019), respectively.
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http://dx.doi.org/10.4155/bio-2019-0271DOI Listing
December 2019

AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression.

J Clin Invest 2019 08 13;129(11):4901-4911. Epub 2019 Aug 13.

Departments of Ophthalmology and Neuroscience, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.
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http://dx.doi.org/10.1172/JCI129085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819121PMC
August 2019

Single-Cell Electrical Stimulation Using CMOS-Based High-Density Microelectrode Arrays.

Front Neurosci 2019 13;13:208. Epub 2019 Mar 13.

Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.

Non-invasive electrical stimulation can be used to study and control neural activity in the brain or to alleviate somatosensory dysfunctions. One intriguing prospect is to precisely stimulate individual targeted neurons. Here, we investigated single-neuron current and voltage stimulation using high-density microelectrode arrays featuring 26,400 bidirectional electrodes at a pitch of 17.5 μm and an electrode area of 5 × 9 μm. We determined optimal waveforms, amplitudes and durations for both stimulation modes. Owing to the high spatial resolution of our arrays and the close proximity of the electrodes to the respective neurons, we were able to stimulate the axon initial segments (AIS) with charges of less than 2 pC. This resulted in minimal artifact production and reliable readout of stimulation efficiency directly at the soma of the stimulated cell. Stimulation signals as low as 70 mV or 100 nA, with pulse durations as short as 18 μs, yielded measurable action potential initiation and propagation. We found that the required stimulation signal amplitudes decreased with cell growth and development and that stimulation efficiency did not improve at higher electric fields generated by simultaneous multi-electrode stimulation.
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http://dx.doi.org/10.3389/fnins.2019.00208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424875PMC
March 2019

Automatic spike sorting for high-density microelectrode arrays.

J Neurophysiol 2018 12 12;120(6):3155-3171. Epub 2018 Sep 12.

Department of Biosystems Science and Engineering, ETH Zurich, Basel , Switzerland.

High-density microelectrode arrays can be used to record extracellular action potentials from hundreds to thousands of neurons simultaneously. Efficient spike sorters must be developed to cope with such large data volumes. Most existing spike sorting methods for single electrodes or small multielectrodes, however, suffer from the "curse of dimensionality" and cannot be directly applied to recordings with hundreds of electrodes. This holds particularly true for the standard reference spike sorting algorithm, principal component analysis-based feature extraction, followed by k-means or expectation maximization clustering, against which most spike sorters are evaluated. We present a spike sorting algorithm that circumvents the dimensionality problem by sorting local groups of electrodes independently with classical spike sorting approaches. It is scalable to any number of recording electrodes and well suited for parallel computing. The combination of data prewhitening before the principal component analysis-based extraction and a parameter-free clustering algorithm obviated the need for parameter adjustments. We evaluated its performance using surrogate data in which we systematically varied spike amplitudes and spike rates and that were generated by inserting template spikes into the voltage traces of real recordings. In a direct comparison, our algorithm could compete with existing state-of-the-art spike sorters in terms of sensitivity and precision, while parameter adjustment or manual cluster curation was not required. NEW & NOTEWORTHY We present an automatic spike sorting algorithm that combines three strategies to scale classical spike sorting techniques for high-density microelectrode arrays: 1) splitting the recording electrodes into small groups and sorting them independently; 2) clustering a subset of spikes and classifying the rest to limit computation time; and 3) prewhitening the spike waveforms to enable the use of parameter-free clustering. Finally, we combined these strategies into an automatic spike sorter that is competitive with state-of-the-art spike sorters.
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http://dx.doi.org/10.1152/jn.00803.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314465PMC
December 2018

Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex.

Nat Neurosci 2017 Jul 22;20(7):960-968. Epub 2017 May 22.

Neural Circuits Laboratory, Friedrich Miescher Institute, Basel, Switzerland.

How neuronal computations in the sensory periphery contribute to computations in the cortex is not well understood. We examined this question in the context of visual-motion processing in the retina and primary visual cortex (V1) of mice. We disrupted retinal direction selectivity, either exclusively along the horizontal axis using FRMD7 mutants or along all directions by ablating starburst amacrine cells, and monitored neuronal activity in layer 2/3 of V1 during stimulation with visual motion. In control mice, we found an over-representation of cortical cells preferring posterior visual motion, the dominant motion direction an animal experiences when it moves forward. In mice with disrupted retinal direction selectivity, the over-representation of posterior-motion-preferring cortical cells disappeared, and their responses at higher stimulus speeds were reduced. This work reveals the existence of two functionally distinct, sensory-periphery-dependent and -independent computations of visual motion in the cortex.
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http://dx.doi.org/10.1038/nn.4566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490790PMC
July 2017

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud.

Bioanalysis 2017 Apr 24;9(7):505-516. Epub 2017 Mar 24.

WuXi Apptec, Plainsboro, NJ, USA.

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.
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http://dx.doi.org/10.4155/bio-2017-5000DOI Listing
April 2017

9th GCC closed forum: CAPA in regulated bioanalysis; method robustness, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, regulatory audit experiences and electronic laboratory notebooks.

Bioanalysis 2016 Mar 26;8(6):487-95. Epub 2016 Feb 26.

WuXi/XBL, 107 Morgan Lane, Plainsboro, NJ, USA.

The 9th GCCClosed Forum was held just prior to the 2015 Workshop on Recent Issues in Bioanalysis (WRIB) in Miami, FL, USA on 13 April 2015. In attendance were 58 senior-level participants, from eight countries, representing 38 CRO companies offering bioanalytical services. The objective of this meeting was for CRO bioanalytical representatives to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues selected at this year's closed forum include CAPA, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, and ELNs. A summary of the industry's best practices and the conclusions from the discussion of these topics is included in this meeting report.
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http://dx.doi.org/10.4155/bio.16.16DOI Listing
March 2016

Structures of Neural Correlation and How They Favor Coding.

Neuron 2016 Jan;89(2):409-22

Department of Physics, Ecole Normale Supérieure, 75005 Paris, France; Laboratoire de Physique Statistique, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Université Denis Diderot, 75005 Paris, France; Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA. Electronic address:

The neural representation of information suffers from "noise"-the trial-to-trial variability in the response of neurons. The impact of correlated noise upon population coding has been debated, but a direct connection between theory and experiment remains tenuous. Here, we substantiate this connection and propose a refined theoretical picture. Using simultaneous recordings from a population of direction-selective retinal ganglion cells, we demonstrate that coding benefits from noise correlations. The effect is appreciable already in small populations, yet it is a collective phenomenon. Furthermore, the stimulus-dependent structure of correlation is key. We develop simple functional models that capture the stimulus-dependent statistics. We then use them to quantify the performance of population coding, which depends upon interplays of feature sensitivities and noise correlations in the population. Because favorable structures of correlation emerge robustly in circuits with noisy, nonlinear elements, they will arise and benefit coding beyond the confines of retina.
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http://dx.doi.org/10.1016/j.neuron.2015.12.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424879PMC
January 2016

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity.

Neuron 2016 Jan 17;89(1):177-93. Epub 2015 Dec 17.

Neural Circuit Laboratories, Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; Department of Ophthalmology, University of Basel, Mittlere Strasse 91, 4031 Basel, Switzerland. Electronic address:

Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease.
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http://dx.doi.org/10.1016/j.neuron.2015.11.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712192PMC
January 2016

A method for electrophysiological characterization of hamster retinal ganglion cells using a high-density CMOS microelectrode array.

Front Neurosci 2015 13;9:360. Epub 2015 Oct 13.

Bio Engineering Laboratory, Department of Biosystems Science and Engineering, ETH Zurich Basel, Switzerland.

Knowledge of neuronal cell types in the mammalian retina is important for the understanding of human retinal disease and the advancement of sight-restoring technology, such as retinal prosthetic devices. A somewhat less utilized animal model for retinal research is the hamster, which has a visual system that is characterized by an area centralis and a wide visual field with a broad binocular component. The hamster retina is optimally suited for recording on the microelectrode array (MEA), because it intrinsically lies flat on the MEA surface and yields robust, large-amplitude signals. However, information in the literature about hamster retinal ganglion cell functional types is scarce. The goal of our work is to develop a method featuring a high-density (HD) complementary metal-oxide-semiconductor (CMOS) MEA technology along with a sequence of standardized visual stimuli in order to categorize ganglion cells in isolated Syrian Hamster (Mesocricetus auratus) retina. Since the HD-MEA is capable of recording at a higher spatial resolution than most MEA systems (17.5 μm electrode pitch), we were able to record from a large proportion of RGCs within a selected region. Secondly, we chose our stimuli so that they could be run during the experiment without intervention or computation steps. The visual stimulus set was designed to activate the receptive fields of most ganglion cells in parallel and to incorporate various visual features to which different cell types respond uniquely. Based on the ganglion cell responses, basic cell properties were determined: direction selectivity, speed tuning, width tuning, transience, and latency. These properties were clustered to identify ganglion cell types in the hamster retina. Ultimately, we recorded up to a cell density of 2780 cells/mm(2) at 2 mm (42°) from the optic nerve head. Using five parameters extracted from the responses to visual stimuli, we obtained seven ganglion cell types.
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http://dx.doi.org/10.3389/fnins.2015.00360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602149PMC
November 2015

Visual coding with a population of direction-selective neurons.

J Neurophysiol 2015 Oct 19;114(4):2485-99. Epub 2015 Aug 19.

Bio Engineering Laboratory, ETH Zurich, Basel, Switzerland;

The brain decodes the visual scene from the action potentials of ∼20 retinal ganglion cell types. Among the retinal ganglion cells, direction-selective ganglion cells (DSGCs) encode motion direction. Several studies have focused on the encoding or decoding of motion direction by recording multiunit activity, mainly in the visual cortex. In this study, we simultaneously recorded from all four types of ON-OFF DSGCs of the rabbit retina using a microelectronics-based high-density microelectrode array (HDMEA) and decoded their concerted activity using probabilistic and linear decoders. Furthermore, we investigated how the modification of stimulus parameters (velocity, size, angle of moving object) and the use of different tuning curve fits influenced decoding precision. Finally, we simulated ON-OFF DSGC activity, based on real data, in order to understand how tuning curve widths and the angular distribution of the cells' preferred directions influence decoding performance. We found that probabilistic decoding strategies outperformed, on average, linear methods and that decoding precision was robust to changes in stimulus parameters such as velocity. The removal of noise correlations among cells, by random shuffling trials, caused a drop in decoding precision. Moreover, we found that tuning curves are broad in order to minimize large errors at the expense of a higher average error, and that the retinal direction-selective system would not substantially benefit, on average, from having more than four types of ON-OFF DSGCs or from a perfect alignment of the cells' preferred directions.
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http://dx.doi.org/10.1152/jn.00919.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620130PMC
October 2015

Recommendations for the development and validation of confirmatory anti-drug antibody assays.

Bioanalysis 2015 ;7(13):1619-31

Covance, Inc., 3635 Concorde Parkway, Suite 100 Chantilly, VA 20151, USA.

Identification and characterization of anti-drug antibodies is a critical component of biopharmaceutical drug development. The tiered approach for immunogenicity testing consists of screening, confirmatory, and characterization assays. Herein, we provide recommendations for confirmatory assays by expanding upon published guidance and present common practices across the industry. The authors recommend scientific approaches for development and validation of confirmatory assays using competition methods in ligand-binding assays, along with statistical formulae for routine use and validation. The paper will assist in understanding the confirmatory assay, and carefully implementing validation criteria a priori, as well as during sample analysis. These approaches represent the authors' current knowledge and practices, with the aim that more uniform practices will be applied across the industry.
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http://dx.doi.org/10.4155/bio.15.96DOI Listing
April 2016

A network comprising short and long noncoding RNAs and RNA helicase controls mouse retina architecture.

Nat Commun 2015 Jun 4;6:7305. Epub 2015 Jun 4.

1] Neural Circuit Laboratories, Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland [2] Biozentrum, University of Basel, 4056 Basel, Switzerland.

Brain regions, such as the cortex and retina, are composed of layers of uniform thickness. The molecular mechanism that controls this uniformity is not well understood. Here we show that during mouse postnatal development the timed expression of Rncr4, a retina-specific long noncoding RNA, regulates the similarly timed processing of pri-miR-183/96/182, which is repressed at an earlier developmental stage by RNA helicase Ddx3x. Shifting the timing of mature miR-183/96/182 accumulation or interfering with Ddx3x expression leads to the disorganization of retinal architecture, with the photoreceptor layer being most affected. We identify Crb1, a component of the adhesion belt between glial and photoreceptor cells, as a link between Rncr4-regulated miRNA metabolism and uniform retina layering. Our results suggest that the precise timing of glia-neuron interaction controlled by noncoding RNAs and Ddx3x is important for the even distribution of cells across layers.
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http://dx.doi.org/10.1038/ncomms8305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468907PMC
June 2015

High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels.

Lab Chip 2015 Jul 14;15(13):2767-80. Epub 2015 May 14.

ETH Zurich, Bio Engineering Laboratory, Department of Biosystems Science and Engineering, Mattenstrasse 26, CH-4058 Basel, Switzerland.

Studies on information processing and learning properties of neuronal networks would benefit from simultaneous and parallel access to the activity of a large fraction of all neurons in such networks. Here, we present a CMOS-based device, capable of simultaneously recording the electrical activity of over a thousand cells in in vitro neuronal networks. The device provides sufficiently high spatiotemporal resolution to enable, at the same time, access to neuronal preparations on subcellular, cellular, and network level. The key feature is a rapidly reconfigurable array of 26 400 microelectrodes arranged at low pitch (17.5 μm) within a large overall sensing area (3.85 × 2.10 mm(2)). An arbitrary subset of the electrodes can be simultaneously connected to 1024 low-noise readout channels as well as 32 stimulation units. Each electrode or electrode subset can be used to electrically stimulate or record the signals of virtually any neuron on the array. We demonstrate the applicability and potential of this device for various different experimental paradigms: large-scale recordings from whole networks of neurons as well as investigations of axonal properties of individual neurons.
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http://dx.doi.org/10.1039/c5lc00133aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421573PMC
July 2015

Recommendations on incurred sample stability (ISS) by GCC.

Bioanalysis 2014 Sep;6(18):2385-90

Quintiles Bioanalytical & ADME Labs, Ithaca, NY, USA.

The topic of incurred sample stability (ISS) has generated considerable discussion within the bioanalytical community in recent years. The subject was an integral part of the seventh annual Workshop on Recent Issues in Bioanalysis (WRIB) held in Long Beach, CA, USA, in April 2013, and at the Global CRO Council for Bioanalysis (GCC) meeting preceding it. Discussion at both events focused on the use of incurred samples for ISS purposes in light of results from a recent GCC survey completed by member companies. This paper reports the consensus resulting from these discussions and serves as a useful reference for depicting ISS issues and concerns, summarizing the GCC survey results and providing helpful recommendations on ISS in the context of bioanalytical method development and application.
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http://dx.doi.org/10.4155/bio.14.155DOI Listing
September 2014

A 1024-Channel CMOS Microelectrode Array With 26,400 Electrodes for Recording and Stimulation of Electrogenic Cells In Vitro.

IEEE J Solid-State Circuits 2014 Nov;49(11):2705-2719

Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, 4058 Basel, Switzerland.

To advance our understanding of the functioning of neuronal ensembles, systems are needed to enable simultaneous recording from a large number of individual neurons at high spatiotemporal resolution and good signal-to-noise ratio. Moreover, stimulation capability is highly desirable for investigating, for example, plasticity and learning processes. Here, we present a microelectrode array (MEA) system on a single CMOS die for recording and stimulation. The system incorporates 26,400 platinum electrodes, fabricated by in-house post-processing, over a large sensing area (3.85 × 2.10 mm) with sub-cellular spatial resolution (pitch of 17.5 μm). Owing to an area and power efficient implementation, we were able to integrate 1024 readout channels on chip to record extracellular signals from a user-specified selection of electrodes. These channels feature noise values of 2.4 μV in the action-potential band (300 Hz-10 kHz) and 5.4 μV in the local-field-potential band (1 Hz-300 Hz), and provide programmable gain (up to 78 dB) to accommodate various biological preparations. Amplified and filtered signals are digitized by 10 bit parallel single-slope ADCs at 20 kSamples/s. The system also includes 32 stimulation units, which can elicit neural spikes through either current or voltage pulses. The chip consumes only 75 mW in total, which obviates the need of active cooling even for sensitive cell cultures.
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http://dx.doi.org/10.1109/JSSC.2014.2359219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424881PMC
November 2014

Bioavailability, Pharmacokinetics, and Safety of Belimumab Administered Subcutaneously in Healthy Subjects.

Clin Pharmacol Drug Dev 2013 Oct 26;2(4):349-57. Epub 2013 Aug 26.

Covance Clinical Research Unit, Inc., Daytona Beach, FL, USA.

This Phase 1 study evaluated the absolute bioavailability, pharmacokinetics (PK), tolerability, and safety of belimumab 200 mg/mL administered subcutaneously (SC) to healthy subjects as a single dose and as multiple doses up to 240 mg. In all, 118 subjects (age range 18-55 years; body weight 51-115 kg) were enrolled. Seventy-eight subjects received a single dose of belimumab 240 mg intravenously, or 2 × 120, 1 × 240, or 1 × 200 mg SC. Forty subjects received 4 weekly injections of belimumab 2 × 120 or 1 × 200 mg SC. Randomization was stratified by weight (<75 kg vs. ≥75 kg) and injection site (abdomen vs. thigh). Following single belimumab SC doses, bioavailability was 74-82%, indicating that belimumab SC was well absorbed, and bioavailability was similar among the three SC groups. Following 4 weekly belimumab SC doses, bioavailability was similar to that following single SC administration. Four subjects had persistent positive immune responses; neutralizing antibodies in these subjects were not detected and there was no apparent impact on PK. Belimumab was generally well tolerated after single and multiple SC dosing, and 200 mg SC weekly dosing is expected to provide an exposure similar to 10 mg/kg intravenously every 28 days.
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http://dx.doi.org/10.1002/cpdd.54DOI Listing
October 2013

Tracking axonal action potential propagation on a high-density microelectrode array across hundreds of sites.

Nat Commun 2013 ;4:2181

Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

Axons are traditionally considered stable transmission cables, but evidence of the regulation of action potential propagation demonstrates that axons may have more important roles. However, their small diameters render intracellular recordings challenging, and low-magnitude extracellular signals are difficult to detect and assign. Better experimental access to axonal function would help to advance this field. Here we report methods to electrically visualize action potential propagation and network topology in cortical neurons grown over custom arrays, which contain 11,011 microelectrodes and are fabricated using complementary metal oxide semiconductor technology. Any neuron lying on the array can be recorded at high spatio-temporal resolution, and simultaneously precisely stimulated with little artifact. We find substantial velocity differences occurring locally within single axons, suggesting that the temporal control of a neuron's output may contribute to neuronal information processing.
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http://dx.doi.org/10.1038/ncomms3181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5419423PMC
January 2014

Pre-existing biotherapeutic-reactive antibodies: survey results within the American Association of Pharmaceutical Scientists.

AAPS J 2013 Jul 26;15(3):852-5. Epub 2013 Apr 26.

Pharmacokinetics, Dynamics & Metabolism (PDM-NBE), Pfizer Inc., Andover, MA, USA.

The immunogenicity profile of a biotherapeutic is determined by a multitude of product and patient-related risk factors that can influence the observed incidence and clinical consequences of immunogenicity. Pre-existing antibodies, i.e., biotherapeutic-reactive antibodies present in samples from treatment-naïve subjects, have been commonly observed during immunogenicity assessments; however their relevance in terms of the safety and efficacy of a biotherapeutic is poorly understood. An American Association of Pharmaceutical Scientists-sponsored survey was conducted to gather information about the prevalence, nature, and consequences of pre-existing antibodies in clinical and nonclinical studies. The survey results indicate that pre-existing antibodies against a variety of biotherapeutics (e.g., mAbs, fusion proteins) are frequently encountered, especially in the context of autoimmune diseases, but that the methods and approaches used to detect, characterize, and report these antibodies vary. In most cases, pre-existing antibodies did not appear to have clinical consequences; however, a few of the respondents reported having observed an effect on pharmacokinetic, pharmacodynamic, safety, and/or efficacy parameters. The findings from this survey are an important first step in evaluating the potential risks associated with the presence of pre-existing antibodies and highlight the importance of standardizing the approaches for detection and characterization of these antibodies. Cross-industry sharing of case studies and relevant data collection will help better inform biotherapeutic risk/benefit profiles and provide deeper understanding of the biological consequences of pre-existing antibodies.
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http://dx.doi.org/10.1208/s12248-013-9492-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691423PMC
July 2013

Bacillus anthracis protective antigen kinetics in inhalation spore-challenged untreated or levofloxacin/ raxibacumab-treated New Zealand white rabbits.

Toxins (Basel) 2013 Jan 14;5(1):120-38. Epub 2013 Jan 14.

Human Genome Sciences, Inc., 14200 Shady Grove Road, Rockville, MD 20850, USA.

Inhaled Bacillus anthracis spores germinate and the subsequent vegetative growth results in bacteremia and toxin production. Anthrax toxin is tripartite: the lethal factor and edema factor are enzymatic moieties, while the protective antigen (PA) binds to cell receptors and the enzymatic moieties. Antibiotics can control B. anthracis bacteremia, whereas raxibacumab binds PA and blocks lethal toxin effects. This study assessed plasma PA kinetics in rabbits following an inhaled B. anthracis spore challenge. Additionally, at 84 h post-challenge, 42% of challenged rabbits that had survived were treated with either levofloxacin/placebo or levofloxacin/raxibacumab. The profiles were modeled using a modified Gompertz/second exponential growth phase model in untreated rabbits, with added monoexponential PA elimination in treated rabbits. Shorter survival times were related to a higher plateau and a faster increase in PA levels. PA elimination half-lives were 10 and 19 h for the levofloxacin/placebo and levofloxacin/raxibacumab groups, respectively, with the difference attributable to persistent circulating PA-raxibacumab complex. PA kinetics were similar between untreated and treated rabbits, with one exception: treated rabbits had a plateau phase nearly twice as long as that for untreated rabbits. Treated rabbits that succumbed to disease had higher plateau PA levels and shorter plateau duration than surviving treated rabbits.
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http://dx.doi.org/10.3390/toxins5010120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564073PMC
January 2013

Recording from defined populations of retinal ganglion cells using a high-density CMOS-integrated microelectrode array with real-time switchable electrode selection.

J Neurosci Methods 2012 Oct 23;211(1):103-13. Epub 2012 Aug 23.

Bio Engineering Laboratory, ETH Zurich, Basel, Switzerland.

In order to understand how retinal circuits encode visual scenes, the neural activity of defined populations of retinal ganglion cells (RGCs) has to be investigated. Here we report on a method for stimulating, detecting, and subsequently targeting defined populations of RGCs. The possibility to select a distinct population of RGCs for extracellular recording enables the design of experiments that can increase our understanding of how these neurons extract precise spatio-temporal features from the visual scene, and how the brain interprets retinal signals. We used light stimulation to elicit a response from physiologically distinct types of RGCs and then utilized the dynamic-configurability capabilities of a microelectronics-based high-density microelectrode array (MEA) to record their synchronous action potentials. The layout characteristics of the MEA made it possible to stimulate and record from multiple, highly overlapping RGCs simultaneously without light-induced artifacts. The high-density of electrodes and the high signal-to-noise ratio of the MEA circuitry allowed for recording of the activity of each RGC on 14±7 electrodes. The spatial features of the electrical activity of each RGC greatly facilitated spike sorting. We were thus able to localize, identify and record from defined RGCs within a region of mouse retina. In addition, we stimulated and recorded from genetically modified RGCs to demonstrate the applicability of optogenetic methods, which introduces an additional feature to target a defined cell type. The developed methodologies can likewise be applied to other neuronal preparations including brain slices or cultured neurons.
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http://dx.doi.org/10.1016/j.jneumeth.2012.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5419501PMC
October 2012

Applicability of independent component analysis on high-density microelectrode array recordings.

J Neurophysiol 2012 Jul 4;108(1):334-48. Epub 2012 Apr 4.

Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

Emerging complementary metal oxide semiconductor (CMOS)-based, high-density microelectrode array (HD-MEA) devices provide high spatial resolution at subcellular level and a large number of readout channels. These devices allow for simultaneous recording of extracellular activity of a large number of neurons with every neuron being detected by multiple electrodes. To analyze the recorded signals, spiking events have to be assigned to individual neurons, a process referred to as "spike sorting." For a set of observed signals, which constitute a linear mixture of a set of source signals, independent component (IC) analysis (ICA) can be used to demix blindly the data and extract the individual source signals. This technique offers great potential to alleviate the problem of spike sorting in HD-MEA recordings, as it represents an unsupervised method to separate the neuronal sources. The separated sources or ICs then constitute estimates of single-neuron signals, and threshold detection on the ICs yields the sorted spike times. However, it is unknown to what extent extracellular neuronal recordings meet the requirements of ICA. In this paper, we evaluate the applicability of ICA to spike sorting of HD-MEA recordings. The analysis of extracellular neuronal signals, recorded at high spatiotemporal resolution, reveals that the recorded data cannot be modeled as a purely linear mixture. As a consequence, ICA fails to separate completely the neuronal signals and cannot be used as a stand-alone method for spike sorting in HD-MEA recordings. We assessed the demixing performance of ICA using simulated data sets and found that the performance strongly depends on neuronal density and spike amplitude. Furthermore, we show how postprocessing techniques can be used to overcome the most severe limitations of ICA. In combination with these postprocessing techniques, ICA represents a viable method to facilitate rapid spike sorting of multidimensional neuronal recordings.
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http://dx.doi.org/10.1152/jn.01106.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421571PMC
July 2012

The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics.

Anal Bioanal Chem 2011 Mar 31;399(7):2313-29. Epub 2010 Jul 31.

Department Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich, Basel, Switzerland.

Planar microelectrode arrays (MEAs) are devices that can be used in biomedical and basic in vitro research to provide extracellular electrophysiological information about biological systems at high spatial and temporal resolution. Complementary metal oxide semiconductor (CMOS) is a technology with which MEAs can be produced on a microscale featuring high spatial resolution and excellent signal-to-noise characteristics. CMOS MEAs are specialized for the analysis of complete electrogenic cellular networks at the cellular or subcellular level in dissociated cultures, organotypic cultures, and acute tissue slices; they can also function as biosensors to detect biochemical events. Models of disease or the response of cellular networks to pharmacological compounds can be studied in vitro, allowing one to investigate pathologies, such as cardiac arrhythmias, memory impairment due to Alzheimer's disease, or vision impairment caused by ganglion cell degeneration in the retina.
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http://dx.doi.org/10.1007/s00216-010-3968-1DOI Listing
March 2011

Changes in B-lymphocyte stimulator protein levels during treatment with albinterferon alfa-2b in patients with chronic hepatitis C who have failed previous interferon therapy.

Hepatol Res 2009 May 30;39(5):455-62. Epub 2008 Dec 30.

Metropolitan Research, Arlington, Virginia, VA, USA.

Aim:   The pharmacodynamics of albinterferon alfa-2b (alb-IFN), a novel recombinant protein consisting of interferon-α-2b genetically fused to human albumin, was evaluated in patients with chronic hepatitis C with a previous non-response to interferon-α-based therapy. B-lymphocyte stimulator (BLyS) is an essential in vivo regulator of B-lymphocyte homeostasis. This analysis examined the relationship between serum BLyS level and virologic response across a range of alb-IFN doses.

Methods:   In all, 115 patients were randomized initially to three alb-IFN treatment arms (900 and 1200 µg every two weeks [q2wk], and 1200 µg every four weeks) with weight-based ribavirin, followed by sequential enrollment in two higher dose arms (1500 and 1800 µg q2wk). Serum BLyS level was assessed by enzyme-linked immunosorbent assay.

Results:   Serum BLyS levels at baseline were lower in African-Americans (P < 0.001). Significant BLyS inductions were observed at weeks 12 and 24 versus pretreatment; in general, serum BLyS levels returned to pretreatment levels following treatment completion. Induction of BLyS was greater in the highest dose group; a significant dose-response trend was observed at weeks 12 (P = 0.002) and 24 (P < 0.001), as well as a significant time trend, with further BLyS induction increases at week 24 versus 12 (P < 0.001). Week 24 BLyS level change correlated with hepatitis C virus RNA reduction (r = -0.28; P = 0.006), driven primarily by patients with BLyS increases > 400%, but did not correlate with sustained virologic response.

Conclusion:   Higher alb-IFN doses demonstrated dose-related BLyS increases, although the correlation with virologic response was modest.
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http://dx.doi.org/10.1111/j.1872-034X.2008.00475.xDOI Listing
May 2009

Safety and antiviral activity of albinterferon alfa-2b in prior interferon nonresponders with chronic hepatitis C.

Clin Gastroenterol Hepatol 2009 Feb 7;7(2):212-8. Epub 2008 Nov 7.

University of Florida College of Medicine, Box 100214, Room M-440, Gainesville, Florida 32610-0214, USA.

Background & Aims: Pegylated interferon alfa-2a/2b is used in combination with ribavirin to treat patients with chronic hepatitis C (CHC), although many do not achieve a sustained virologic response (SVR). Albinterferon alfa-2b, a recombinant protein consisting of interferon alfa-2b fused to human albumin, may increase drug exposure. This phase 2 study evaluated the safety/efficacy of albinterferon in CHC patients who had not responded to interferon-based regimens.

Methods: A total of 115 patients were assigned to 5 groups given 1200 microg albinterferon every 4 weeks or 900, 1200, 1500, or 1800 microg every 2 weeks, plus oral ribavirin, for 48 weeks. The primary efficacy end point was achievement of an SVR after 24 weeks. Treatment was extended to 72 weeks for 6 slow responders who were negative for hepatitis C virus RNA after 24 weeks.

Results: The types of adverse events were similar across groups; the overall discontinuation rate as a result of adverse events was 10.4%. Reductions in absolute neutrophil counts were less frequent in the every 4 weeks group and comparable among the every 2 weeks groups. The overall SVR rate was 17% (11% for previous nonresponders to pegylated interferon-alfa/ribavirin with genotype 1 infection). An SVR occurred in 3 of 6 slow responders by 72 weeks. The greatest reductions in hepatitis C virus RNA in nonresponders to pegylated interferon-alfa/ribavirin with genotype 1 infection were observed in the 1800-microg group.

Conclusions: In patients with CHC who did not respond to interferon-based regimens, higher doses of albinterferon had significant early antiviral activity and a low incidence of adverse events, with the types of adverse events similar to those observed with interferon.
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http://dx.doi.org/10.1016/j.cgh.2008.10.035DOI Listing
February 2009

Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products.

J Pharm Biomed Anal 2008 Dec 19;48(5):1267-81. Epub 2008 Sep 19.

Clinical Pharmacology Sciences, Centocor Research & Development Inc., Radnor, PA 19087, USA.

Most biological drug products elicit some level of anti-drug antibody (ADA) response. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. In humans, ADA often causes no detectable clinical effects, but in the instances of some therapeutic proteins these antibodies have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In nonclinical (preclinical) studies, ADA can affect drug exposure, complicating the interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess the immunogenic potential of biological drug molecules, and be able to correlate laboratory results with clinical events, it is important to develop reliable laboratory test methods that provide valid assessments of antibody responses in both nonclinical and clinical studies. For this, method validation is considered important, and is a necessary bioanalytical component of drug marketing authorization applications. Existing regulatory guidance documents dealing with the validation of methods address immunoassays in a limited manner, and in particular lack information on the validation of immunogenicity methods. Hence this article provides scientific recommendations for the validation of ADA immunoassays. Unique validation performance characteristics are addressed in addition to those provided in existing regulatory documents pertaining to bioanalyses. The authors recommend experimental and statistical approaches for the validation of immunoassay performance characteristics; these recommendations should be considered as examples of best practice and are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry.
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http://dx.doi.org/10.1016/j.jpba.2008.09.020DOI Listing
December 2008

Albinterferon alpha-2b: a genetic fusion protein for the treatment of chronic hepatitis C.

Nat Biotechnol 2007 Dec;25(12):1411-9

Human Genome Sciences, Inc., 14200 Shady Grove Road, Rockville, Maryland 21042, USA.

Treatment regimens based on the use of interferon-alpha (IFN-alpha) remain the cornerstone of therapy for chronic hepatitis C virus infection, which affects nearly 170 million people worldwide. Treatment options include unmodified IFN-alpha given three times weekly or pegylated IFNs given once weekly. The albumin-fusion platform takes advantage of the long half-life of human albumin to provide a new treatment approach that allows the dosing frequency of IFN-alpha to be reduced in individuals with chronic hepatitis C. Albinterferon alpha-2b (alb-IFN), a recombinant polypeptide composed of IFN-alpha2b genetically fused to human albumin, has an extended half-life and early evidence indicates that it is efficacious and well tolerated. Pharmacodynamic modeling supports treatment with alb-IFN at 2- or 4-week intervals. Phase 3 registration trials are in progress. The albumin-fusion platform is currently being applied to other important bioactive peptides with short half-lives. These fusion proteins, which are at present in different phases of clinical development, might lead to improved therapies across a broad range of diseases.
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http://dx.doi.org/10.1038/nbt1364DOI Listing
December 2007