Assessment of Rapid Optimized 96-well Tray Flow Cytometric Crossmatch (Halifax-FCXM) with Luminex Single Antigen Test

IntroductionFlow cytometric crossmatch assay (FCXM) is a sensitive cell-based method for evaluating the presence of donor-specific antibodies (DSA) before transplantation. Recently, 96-well tray FCXM protocol (Halifax FCXM) with improved test efficiency has been introduced. The objective of the present study was to assess the performance of Halifax FCXM by correlating with DSA results based on single antigen bead (SAB) assays (virtual crossmatch, VXM).MethodsA total of 341 FCXMs were evaluated for the detection of HLA-DSA. A positive VXM was defined as having at least one HLA - DSA (HLA-A, B, Cw, DR, DQB1) with ≥ 1000 MFI (mean fluorescence intensity) identified by SAB assay.ResultsOf a total 341 cases, 113 showed class I VXM (+) with class I DSA MFI ≥ 1000 exclusively against one or more donor HLA class I antigens (HLA-A, B, Cw), 72 had class I-/II + DSA, and 156 had VXM(-). Halifax T-FCXM showed a sensitivity of 87.6% (99/113) and a specificity of 98.2% (224/228) for detecting class I VXM (+). The concordance between T-FCXM and class I VXM was 94.7% (323/341). Halifax B-FCXM showed a sensitivity of 58.3% (42/72) and a specificity of 98.7% (154/156) for detecting class I-/II + DSAs. The concordance between B-FCXM and class I-/II + VXM was 86.0% (196/228). When we separately analyzed data, B-FCXM detected HLA-DR (+) (68.8%) and HLA-DQ (+) DSAs (71.0%) similarly (P > 0.05). T-FCXM detected 87.6%, 97.2%, and 98.2% of class I DSA-positive cases with MFI values (sumDSA) ≥ 1000, ≥ 3000, and ≥ 5000, respectively. B-FCXM detected 58.3% of class I-II + DSA -positive (≥1000) cases, but detected 76.7% (33/43) and 89.2% (33/37) of class I-II + DSAs if MFI values of sumDSA and immunodominant DSA (iDSA) were above 5000, respectively. Halifax FCXM had sensitivities of 91.5% and 96.2% for detecting VXM (+) having MFI values above 5000 for class I or class II sumDSA and iDSA, respectively.ConclusionHalifax FCXM showed a good correlation, especially with SAB assay-based high MFI DSA or sumDSA. Concurrent application of FCXM with VXM can improve pre-transplant risk assessment and progress organ allocation efficiency.     

Copper is an endogenous modulator of neural circuit spontaneous activity

For reasons that remain insufficiently understood, the brain requires among the highest levels of metals in the body for normal function. The traditional paradigm for this organ and others is that fluxes of alkali and alkaline earth metals are required for signaling, but transition metals are maintained in static, tightly bound reservoirs for metabolism and protection against oxidative stress. Here we show that copper is an endogenous modulator of spontaneous activity, a property of functional neural circuitry. Using Copper Fluor-3 (CF3), a new fluorescent Cu⁺ sensor for one- and two-photon imaging, we show that neurons and neural tissue maintain basal stores of loosely bound copper that can be attenuated by chelation, which define a labile copper pool. Targeted disruption of these labile copper stores by acute chelation or genetic knockdown of the CTR1 (copper transporter 1) copper channel alters the spatiotemporal properties of spontaneous activity in developing hippocampal and retinal circuits. The data identify an essential role for copper neuronal function and suggest broader contributions of this transition metal to cell signaling.The foundation of cellular signal transduction relies on intricate chemical messenger systems that operate through the dynamic spatial and temporal regulation of elements, ions, and molecules. Nowhere is this concept better illustrated than in the brain, which extensively regulates fluxes of alkali and alkaline earth metals such as sodium, potassium, and calcium for a diverse array of signaling processes. Interestingly, the brain also accumulates among the highest levels of transition metals in the body (1–3), including redox-active copper. This high-redox metal load, in combination with the brain''s disproportionately active oxygen metabolism (4), makes this organ particularly susceptible to oxidative stress (4–6). As such, copper has been historically regarded as a tightly sequestered cofactor that must be buried within protein active sites to protect against reactive oxygen species generation and subsequent free radical damage chemistry. Indeed, elegant work continues to identify molecular players that maintain copper homeostasis in the brain (7, 8) and related organs (9–11), and loss of this strict regulation is implicated in neurotoxic stress (12–14) and a variety of neurodegenerative and neurodevelopmental disorders including Menkes (15, 16) and Wilson''s (17) diseases, familial amyotrophic lateral sclerosis (18, 19), Alzheimer''s (6, 14, 20–22) and Huntington''s (23, 24) diseases, and prion-mediated encephalopathies (14, 25, 26).Despite this long-held paradigm, emerging data also link pools of labile copper (defined as dynamic and loosely bound stores that undergo facile ligand exchange relative to static, tightly bound pools buried within protein active sites) to neurophysiology. Included are observations of ⁶⁴Cu efflux from stimulated neurons (12, 27), reversible trafficking of ATP7A from the perinuclear trans-Golgi to neuronal processes by NMDA receptor activation (12), effects of copper chelation on olfactory response to thiol odorants (28), and direct X-ray fluorescence imaging of copper translocation in neurons from somatic cell bodies to peripheral processes upon depolarization (29). Against this backdrop, we have initiated a program aimed at exploring the potential contributions of loosely bound forms of redox-active metals like copper in cell signaling. In this report, we identify a role for copper in the brain as a modulator of spontaneous activity, a fundamental property of developing neural circuits. The design and synthesis of Copper Fluor-3 (CF3), a fluorescent copper sensor based on a hydrophilic and tunable rhodol scaffold, along with Control Copper Fluor-3 (Ctrl-CF3), a matched control dye based on an identical fluorophore but lacking responsiveness to copper, enabled the visualization of loosely bound Cu⁺ in dissociated neurons and neural tissue by one- and two-photon microscopy. Disruption of Cu⁺ stores by acute application of a copper chelator or genetic knockdown of the copper ion channel CTR1 altered the spatiotemporal properties of spontaneous activity. In dissociated hippocampal cultures, these manipulations increased the correlation of spontaneous calcium transients, whereas in retina, both cell participation and frequency of correlated calcium transients increased.     

Limbic covariance network alterations in patients with transient global amnesia

Journal of Neurology - We compared limbic structure volumes and graph theory parameters of the limbic covariance network between patients with transient global amnesia (TGA) and healthy controls,...     

Advanced rhythm discrimination for implantable cardioverter defibrillators using electrogram vector timing and correlation

INTRODUCTION: Discrimination of ventricular and supraventricular arrhythmias remains one of the major challenges for appropriate implantable defibrillator (ICD) therapy delivery. The electrogram vector timing and correlation (VTC) algorithm was developed for such rhythm discrimination. The VTC algorithm differentiates normally conducted supraventricular beats from abnormally conducted ventricular beats by comparing the timing and correlation of rate and shock channel electrograms. METHODS AND RESULTS: Rate and shock channel electrograms of sinus rhythm and induced arrhythmias were collected from 93 patients during ICD placement. The algorithm was developed using data from 50 patients and prospectively tested in a software model with the remaining 43 patients. A sinus rhythm reference was formed by averaging complexes of the shock channel signal aligned by the peak amplitude of the rate channel. Eight features measuring the amplitude and timing of shock channel signal characteristics were extracted from the reference for comparison. When a high-rate rhythm was detected, the VTC algorithm computed the correlation of the arrhythmia complex features with the reference. Rhythms with a sufficient number of uncorrelated beats were classified as ventricular tachycardia (VT). In a dual-chamber implementation, the VTC algorithm is integrated with ventricular and atrial rate comparison (V>A) and stability above an atrial fibrillation rate threshold. The test set consisted of 117 arrhythmias. Dual-chamber sensitivity was 100% (81/81 VT) and specificity was 97% (35/36 supraventricular tachycardia). Single-chamber analysis demonstrated 99% sensitivity and 97% specificity. CONCLUSION: The VTC algorithm demonstrated high sensitivity and specificity in discriminating between ventricular and supraventricular arrhythmias.     

Atopic Dermatitis and Cytokines: The Immunoregulatory and Therapeutic Implications of Cytokines in Atopic Dermatitis - Part II: Negative Regulation and Cytokine Therapy in Atopic Dermatitis

Atopic dermatitis is an immunologic disease that results in allergic inflammations of the skin. Cytokines are involved in the negative regulation of immunopathogenesis of atopic dermatitis. Negative immune regulation is also achieved by immune cells in addition to cytokines which are subsequently regulated by a counter-regulatory mechanism. Allergen tolerance is an important aspect of the treatment of atopic dermatitis. Recently, the IL-27, IL-21, and IL-10 cytokines were found to be important components of the counter regulatory mechanism that terminates immune response, and protects the host from excessive immune responses. IL-10 and TGF-β are well-known to be involved in the immune tolerance. IL-10 and IFN-γ are promising cytokines with respect to the prevention of allergen sensitization and the induction of allergen-specific tolerance. In particular, IFN-γ has unique tolerogenic effects with respect to pre-sensitized allergens, especially in atopic dermatitis. In this review, the role of cytokines in the immune tolerance and relevant patents are reviewed, and therapeutic strategies are presented based on the immunologic architecture of AD.     

SERPINE2 Polymorphisms and Chronic Obstructive Pulmonary Disease

A number of genome-wide linkage analyses have identified the 2q33.3-2q37.2 region as most likely to contain the genes that contribute to the susceptibility to chronic obstructive pulmonary disease (COPD). It was hypothesized that the SERPINE2 gene, which is one of the genes located at the 2q33.3-2q37.2 region, may act as a low-penetrance susceptibility gene for COPD. To test this hypothesis, the association of four SERPINE2 single nucleotide polymorphisms (SNPs; rs16865421A>G, rs7583463A>C, rs729631C>G, and rs6734100C>G) with the risk of COPD was investigated in a case-control study of 311 COPD patients and 386 controls. The SNP rs16865421 was associated with a significantly decreased risk of COPD in a dominant model for the polymorphic allele (adjusted odds ratio [OR]=0.66, 95% confidence interval [CI]=0.45-0.97, P=0.03). In haplotype analysis, the GACC haplotype carrying the polymorphic allele at the rs16865421 was associated with a significantly decreased risk of COPD when compared to the AACC haplotype (adjusted OR=0.58, 95% CI=0.38-0.89, P=0.01), and this effect was evident in younger individuals (adjusted OR=0.30, 95% CI=0.14-0.64, P=0.002). This study suggests that the SERPINE2 gene contributes to the susceptibility to COPD.     

Fibrinolytic and antiplatelet aggregation properties of a recombinant Cheonggukjang kinase

This study characterized the efficacy of recombinant Cheonggukjang kinase (CGK) 3-5-rich fraction as a thrombolytic agent, which we overexpressed in Bacillus licheniformis ATCC10716, a strain normally lacking fibrinolytic activity. We found that CGK3-5 is a plasmin-like protease that directly degrades fibrin clots and does not activate plasminogen during fibrin clot lysis and platelet-rich clot lysis assays. We also confirmed antiplatelet and antithrombotic activity by CGK3-5-rich fraction both in vitro and in vivo. CGK3-5-rich fraction inhibited collagen-induced platelet aggregation in platelet-rich plasma in a concentration-dependent manner. The concentration of 1.5?mg/mL CGK3-5-rich fraction completely inhibited collagen-induced platelet aggregation. Furthermore, injection of CGK3-5-rich fraction into tail veins dose-dependently protected mice from death by pulmonary embolism induced by collagen and epinephrine. The survival rates were 30%, 70%, and 100%, respectively, with doses of 130?mg/kg, 260?mg/kg, and 520?mg/kg. These findings suggest that CGK3-5 holds promise as a treatment to mitigate the potentially effects of stroke and heart failure.