A high-throughput label-free cell-based biosensor (CBB) system

Cell-based biosensors (CBBs) have important applications in biosecurity and rapid diagnostics. Current CBB technologies have challenges including cell immobilization on the sensors, high throughput fabrication and portability, and rapid detection of responses to environmental changes. We address these challenges by developing an integrated CBB platform that merges cell printing technology, a lensless charge-coupled device (CCD) imaging system, and custom-developed cell image processing software. Cell printing was used to immobilize cells within hydrogel droplets and pattern these droplets on a microfluidic chip. The CCD was used to detect the morphological response of the immobilized cells to external stimuli (e.g., environmental temperature change) using lensless shadow images. The morphological information can be also detected by sensing a small disturbance in cell alignment, i.e., minor alignment changes of smooth muscles cells on the biosensors. The automatic cell alignment quantification software was used to process the cell images (microscopic image was used as an example) and calculate the cell orientation in seconds. The same images were also manually processed as a control to validate and characterize the integrated platform functionality. The results showed software can measure the cell morphology (i.e., orientation) in an automated way without the need for labeling (e.g., florescent staining). Such an integrated CBB system will allow fabrication of CBBs at high throughput as well as rapidly monitor and measure morphological cellular responses

Automated and Adaptable Quantification of Cellular Alignment from Microscopic Images for Tissue Engineering Applications

Cellular alignment plays a critical role in functional, physical, and biological characteristics of many tissue types, such as muscle, tendon, nerve, and cornea. Current efforts toward regeneration of these tissues include replicating the cellular microenvironment by developing biomaterials that facilitate cellular alignment. To assess the functional effectiveness of the engineered microenvironments, one essential criterion is quantification of cellular alignment. Therefore, there is a need for rapid, accurate, and adaptable methodologies to quantify cellular alignment for tissue engineering applications. To address this need, we developed an automated method, binarization-based extraction of alignment score (BEAS), to determine cell orientation distribution in a wide variety of microscopic images. This method combines a sequenced application of median and band-pass filters, locally adaptive thresholding approaches and image processing techniques. Cellular alignment score is obtained by applying a robust scoring algorithm to the orientation distribution. We validated the BEAS method by comparing the results with the existing approaches reported in literature (i.e., manual, radial fast Fourier transform-radial sum, and gradient based approaches). Validation results indicated that the BEAS method resulted in statistically comparable alignment scores with the manual method (coefficient of determination R2=0.92 [R superscript 2 = 0.92]). Therefore, the BEAS method introduced in this study could enable accurate, convenient, and adaptable evaluation of engineered tissue constructs and biomaterials in terms of cellular alignment and organization.National Institutes of Health (U.S.) (NIH R21 (AI087107))National Institutes of Health (U.S.) (NIH R01 (AI081534))Wallace H. Coulter FoundationCenter for Integration of Medicine and Innovative TechnologyUnited States. Army Medical Research and Materiel CommandUnited States. Army. Telemedicine & Advanced Technology Research Cente

Relation of sensory peripheral neuropathy in sjögren syndrome to Anti-Ro/SSA

"Background: Sjögren syndrome is a common, chronic autoimmune disease that typically produces inflammation and poor function of the salivary and lacrimal glands. Other organs can be affected, including the nervous system. Sensory peripheral neuropathy is a common manifestation of the disease. Methods: Eight-eight patients attending a dry eyesYdry mouth clinic were diagnosed to have primary Sjögren syndrome and underwent a neurological examination. Anti-Ro (or SSA) and anti-La (or SSB) were determined using immunodiffusion as well as Inno-Lia and BioPlex ANA screen. Serum vitamin B12 levels were determined using an enzyme-linked microtiter plate assay. Results: Twenty-seven (31%) of the 88 patients had peripheral neuropathy as defined by loss of light touch, proprioception, or vibratory sensation. Anti-Ro and anti-La were found by immunodiffusion in 12 patients, and 8 of these 12 had neuropathy (W2 = 8.46, P = 0.0036, odds ratio = 6.0 compared to those without precipitating anti-Ro and anti- La). Of the 27 patients with only anti-Ro by immunodiffusion, 13 (48.1%) had neuropathy (W2 = 5.587, P = 0.018, compared to those without anti-Ro). There was no relationship of the other, more sensitive measures of anti-Ro and anti-La to neuropathy. In addition, we found no association of serum vitamin B12 levels to neuropathy among these patients with Sjögren syndrome. Conclusions: Sensory peripheral neuropathy is common among patients with Sjögren syndrome and is associated with the presence of anti-Ro and anti-La when determined by immunodiffusion. Copyright © 2012 by Lippincott Williams and amp; Wilkins.

Relation of sensory peripheral neuropathy in sjögren syndrome to Anti-Ro/SSA

Background: Sjögren syndrome is a common, chronic autoimmune disease that typically produces inflammation and poor function of the salivary and lacrimal glands. Other organs can be affected, including the nervous system. Sensory peripheral neuropathy is a common manifestation of the disease. Methods: Eight-eight patients attending a dry eyesYdry mouth clinic were diagnosed to have primary Sjögren syndrome and underwent a neurological examination. Anti-Ro (or SSA) and anti-La (or SSB) were determined using immunodiffusion as well as Inno-Lia and BioPlex ANA screen. Serum vitamin B12 levels were determined using an enzyme-linked microtiter plate assay. Results: Twenty-seven (31%) of the 88 patients had peripheral neuropathy as defined by loss of light touch, proprioception, or vibratory sensation. Anti-Ro and anti-La were found by immunodiffusion in 12 patients, and 8 of these 12 had neuropathy (W2 = 8.46, P = 0.0036, odds ratio = 6.0 compared to those without precipitating anti-Ro and anti- La). Of the 27 patients with only anti-Ro by immunodiffusion, 13 (48.1%) had neuropathy (W2 = 5.587, P = 0.018, compared to those without anti-Ro). There was no relationship of the other, more sensitive measures of anti-Ro and anti-La to neuropathy. In addition, we found no association of serum vitamin B12 levels to neuropathy among these patients with Sjögren syndrome. Conclusions: Sensory peripheral neuropathy is common among patients with Sjögren syndrome and is associated with the presence of anti-Ro and anti-La when determined by immunodiffusion. Copyright © 2012 by Lippincott Williams and amp; Wilkins

Identification of a Sjögren's syndrome susceptibility locus at OAS1 that influences isoform switching, protein expression, and responsiveness to type I interferons

Sjögren’s syndrome (SS) is a common, autoimmune exocrinopathy distinguished by keratoconjunctivitis sicca and xerostomia. Patients frequently develop serious complications including lymphoma, pulmonary dysfunction, neuropathy, vasculitis, and debilitating fatigue. Dysregulation of type I interferon (IFN) pathway is a prominent feature of SS and is correlated with increased autoantibody titers and disease severity. To identify genetic determinants of IFN pathway dysregulation in SS, we performed cis-expression quantitative trait locus (eQTL) analyses focusing on differentially expressed type I IFN-inducible transcripts identified through a transcriptome profiling study. Multiple cis-eQTLs were associated with transcript levels of 2'-5'-oligoadenylate synthetase 1 (OAS1) peaking at rs10774671 (PeQTL= 6.05 × 10−14). Association of rs10774671 with SS susceptibility was identified and confirmed through meta-analysis of two independent cohorts (Pmeta= 2.59 × 10−9; odds ratio = 0.75; 95% confidence interval = 0.66–0.86). The risk allele of rs10774671 shifts splicing of OAS1 from production of the p46 isoform to multiple alternative transcripts, including p42, p48, and p44. We found that the isoforms were differentially expressed within each genotype in controls and patients with and without autoantibodies. Furthermore, our results showed that the three alternatively spliced isoforms lacked translational response to type I IFN stimulation. The p48 and p44 isoforms also had impaired protein expression governed by the 3' end of the transcripts. The SS risk allele of rs10774671 has been shown by others to be associated with reduced OAS1 enzymatic activity and ability to clear viral infections, as well as reduced responsiveness to IFN treatment. Our results establish OAS1 as a risk locus for SS and support a potential role for defective viral clearance due to altered IFN response as a genetic pathophysiological basis of this complex autoimmune disease. © 2017 Public Library of Science. All Rights Reserved

Differentially expressed transcripts between 115 anti-Ro/SSA positive SS cases and 56 controls identified through transcriptome profiling.

<p>(A) We identified 73 genes (represented by 83 probes on the heatmap) differentially expressed between anti-Ro/SSA positive SS cases and healthy controls (absolute FC >2 and <i>q</i><0.05). Among the differentially expressed genes, 57 were type I IFN-regulated genes (black bar on right) and formed an IFN signature where most genes were overexpressed in SS patients (yellow indicates overexpressed genes compared to controls). (B) The 57 differentially expressed type I IFN-regulated genes were re-clustered in anti-Ro/SSA positive SS cases using <i>k</i>-means (<i>k</i> = 3) algorithm and heterogeneity of the IFN signature levels in anti-Ro/SSA positive SS cases was observed.</p

Composition of independent cohorts used in the genetic association analyses.

<p>Composition of independent cohorts used in the genetic association analyses.</p

Identification of a Sjögren's syndrome susceptibility locus at OAS1 that influences isoform switching, protein expression, and responsiveness to type I interferons

Sjogren's syndrome (SS) is a common, autoimmune exocrinopathy distinguished by keratoconjunctivitis sicca and xerostomia. Patients frequently develop serious complications including lymphoma, pulmonary dysfunction, neuropathy, vasculitis, and debilitating fatigue. Dysregulation of type I interferon (IFN) pathway is a prominent feature of SS and is correlated with increased autoantibody titers and disease severity. To identify genetic determinants of IFN pathway dysregulation in SS, we performed cis-expression quantitative trait locus (eQTL) analyses focusing on differentially expressed type I IFN-inducible transcripts identified through a transcriptome profiling study. Multiple cis-eQTLs were associated with transcript levels of 2'-5'-oligoadenylate synthetase 1 (OAS1) peaking at rs10774671 (PeQTL = 6.05 x 10(-14)). Association of rs10774671 with SS susceptibility was identified and confirmed through meta-analysis of two independent cohorts (P-meta = 2.59 x 10(-9); odds ratio = 0.75; 95% confidence interval = 0.66-0.86). The risk allele of rs10774671 shifts splicing of OAS1 from production of the p46 isoform to multiple alternative transcripts, including p42, p48, and p44. We found that the isoforms were differentially expressed within each genotype in controls and patients with and without autoantibodies. Furthermore, our results showed that the three alternatively spliced isoforms lacked translational response to type I IFN stimulation. The p48 and p44 isoforms also had impaired protein expression governed by the 3' end of the transcripts. The SS risk allele of rs10774671 has been shown by others to be associated with reduced OAS1 enzymatic activity and ability to clear viral infections, as well as reduced responsiveness to IFN treatment. Our results establish OAS1 as a risk locus for SS and support a potential role for defective viral clearance due to altered IFN response as a genetic pathophysiological basis of this complex autoimmune disease