Control of Immune Response to Allogeneic Embryonic Stem Cells by CD3 Antibody–Mediated Operational Tolerance Induction

Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC–derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20–25 days. Recipients treated with CD3 antibody showed long‐term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self‐limited. Regulatory CD4⁺FoxP3⁺ T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF‐β and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression.     

Deep Convolutional Neural Network Analysis of Flow Imaging Microscopy Data to Classify Subvisible Particles in Protein Formulations

Flow-imaging microscopy (FIM) is commonly used to characterize subvisible particles in therapeutic protein formulations. Although pharmaceutical companies often collect large repositories of FIM images of protein therapeutic products, current state-of-the-art methods for analyzing these images rely on low-dimensional lists of “morphological features” to characterize particles that ignore much of the information encoded in the existing image databases. Deep convolutional neural networks (sometimes referred to as “CNNs or ConvNets”) have demonstrated the ability to extract predictive information from raw macroscopic image data without requiring the selection or specification of “morphological features” in a variety of tasks. However, the inherent heterogeneity of protein therapeutics and optical phenomena associated with subvisible FIM particle measurements introduces new challenges regarding the application of ConvNets to FIM image analysis. We demonstrate a supervised learning technique leveraging ConvNets to extract information from raw images in order to predict the process conditions or stress states (freeze-thawing, mechanical shaking, etc.) that produced a variety of different protein particles. We demonstrate that our new classifier, in combination with a “data pooling” strategy, can nearly perfectly differentiate between protein formulations in a variety of scenarios of relevance to protein therapeutics quality control and process monitoring using as few as 20 particles imaged via FIM.     

Identification of the Molecular Determinants of the Antibacterial Activity of LmutTX,a Lys49 Phospholipase A2 Homologue Isolated from Lachesis muta muta Snake Venom (Linnaeus, 1766)

Snake venom phospholipases A₂ (PLA₂s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA₂ homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA₂s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram‐positive and Gram‐negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA₂ from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.     

Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonin-transporter-deficient mice

Mood disorders cause much suffering and are the single greatest cause of lost productivity worldwide. Although multiple medications, along with behavioral therapies, have proven effective for some individuals, millions of people lack an effective therapeutic option. A common serotonin (5-HT) transporter (5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression in vivo and elevates risk for multiple mood disorders including anxiety, alcoholism, and major depression. Importantly, this variant is also associated with reduced responsiveness to selective 5-HT reuptake inhibitor antidepressants. We hypothesized that a reduced antidepressant response in individuals with a constitutive reduction in 5-HTT expression could arise because of the compensatory expression of other genes that inactivate 5-HT in the brain. A functionally upregulated alternate transporter for 5-HT may prevent extracellular 5-HT from rising to levels sufficiently high enough to trigger the adaptive neurochemical events necessary for therapeutic benefit. Here we demonstrate that expression of the organic cation transporter type 3 (OCT3, SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22 diminishes 5-HT clearance and exerts antidepressant-like effects in these mice but not in WT animals. OCT3 may be an important transporter mediating serotonergic signaling when 5-HTT expression or function is compromised.     

Surface-active phospholipid as the lubricating component of lubricin

To resolve the apparent conflict between a lubricating glycoprotein, 'lubricin', as the active ingredient in synovial fluid (SF) and surface- active phospholipid (SAPL) present in SF (and adsorbed to articular cartilage) as the boundary lubricant reducing friction to such low physiological levels, lubricin was isolated from bovine SF following the original procedure of Swann et al. (Arthritis Rheum 1981;24:22-30). Analysis of the lipid extract by thin-layer chromatography and phosphorus determination demonstrated a phospholipid component of 11.1 +/- 1.7% (N = 5) which corresponds very closely to the 9.2-13.0% of lubricin which had hitherto remained unidentified and which has previously been shown to be transferable to the articular surface to impart lubrication. These results would appear to resolve any theoretical conflict in that lubricin is, indeed, an active ingredient within SF. Yet, as a large water-soluble molecule, it really functions as a carrier for the highly insoluble SAPL which it deposits on the articular surface as the oligolamellar layer visualized in previous studies. However, it is this deposited SAPL, rather than lubricin, which actually lubricates.      

大学生饮酒模式调查分析

目的分析影响大学生饮酒模式的因素,针对饮酒教育及酒精政策提出建议。方法采用整群抽样方法,选择来自北京和郑州的530名大学生完成有效问卷调查。采用 Epidata 录入数据,SAS 12.0和 R 2.7.2进行数据描述和分析。结果74.5%的大学生在过去1年内饮过酒,啤酒是饮酒者的主要选择（85.9%）,餐馆和家里是饮酒比较频繁的场所,饮用酒多来自同学/朋友及家庭成员提供,25.3%饮酒者并无特别原因饮酒,各有约1/5的饮酒者是出于社交目的或喜欢饮酒的感觉。认为“饮酒有助于社交暠或“饮酒促进交流暠者、父母及同学/朋友饮酒者、来自农村地区或小城镇者及吸烟者更容易发生饮酒行为（均 P 〈0.05）,男生大学生、规律饮酒者更容易遭遇强迫劝酒场面、更易发生醉酒（均 P 〈0.05）。超过一半饮酒者出现酒后脸红、肌肉软弱无力等生理反应,饮酒对被调查大学生产生的社会心理影响依次是记忆丧失（23.9%）、宿醉（14.6%）、做了后悔的事（低于10%）等。结论目前大学生仍保持着一种低风险饮酒模式。大学生饮酒教育应提供关于饮酒利弊的科学信息,使其对饮酒保持一种现实的期望。应制定针对性酒精政策和教育鼓励和支持低风险且满足社交功能的饮酒模式,控制高风险饮酒模式。     

Angioblastic meningioma presenting as a pain in the temporomandibular joint

A case of angioblastic meningioma is presented where the presenting symptom was severe TM pain. Theories based on a "referred pain" mechanism, vascular traction, or both, are discussed and suggested as an explanation.