The Effect of Immune Cell Activation on Glycogen Storage in the Context of a Nutrient Rich Microenvironment

Lymphocytes of the immune system become activated in order to fight pathogens. Activated lymphocytes absorb more glucose due to their high-energy demand. Glycogen is a branched polymer of glucose units that is formed in times of nutrient sufficiency and it is utilized in times of need. In the presence of high glucose, lymphocytes build up glycogen stores, but the fate of this content is not very well understood. The objective of this work is to demonstrate the presence of glycogen in activated human peripheral blood mononuclear cells (PBMCs) and to investigate the impact of low nutrient levels on the glycogen content of these cells. This was achieved by isolation of PBMCs from human blood, followed by in-vitro activation of the cells by a general activator and a T cell-specific activator. Glycogen concentrations were measured through periodic acid Schiff’s staining (PAS) method and by using an enzymatic detection kit, in various time points. The role of glycogen in times of low nutrient availability was also examined. PBMC were found to contain glycogen by both methods. Upon stimulation of PBMCs with the general activator, there was an increase in glycogen formation in the activated lymphocytes as compared to the non-activated group using both techniques (p<0.05). The effect of T cell-specific activator was consistent with the effects of the general activator. This was confirmed through PAS staining and enzymatic detection kit techniques (p<0.05). Additionally, when the amount of nutrients was lowered, less glycogen was stored in PBMCs. This study demonstrated that activated PBMCs contain more glycogen stores as compared to non-activated cells. The excess glucose that is converted into glycogen may be used by the immune system when nutrients are low

A Multigraph Approach to Social Network Analysis

Multigraphs are graphs where multiple edges and edge loops are permitted. The main purpose of this article is to show the versatility of a multigraph approach when analysing social networks. Multigraph data structures are described and it is exemplified how they naturally occur in many contexts but also how they can be constructed by different kinds of aggregation in graphs. Special attention is given to a random multigraph model based on independent edge assignments to sites of vertex pairs and some useful measures of the local and global structure under this model are presented. Further, it is shown how some general measures of simplicity and complexity of multigraphs are easily handled under the present model.publishe

Characterization of matrix changes associated with disc degeneration in low back pain using multichromatic FAST staining

Introduction: One of the many sources of low back pain (LBP) is the degeneration of intervertebral discs (IVDs), a condition called degenerative disc disease (DDD). Degeneration of IVDs can occur as result of aging, trauma, or genetic predisposition. Better insight into the complex histological profile in healthy and degenerated IVDs will contribute to our understanding of the interplay between the degenerative processes and chronic pain. The knowledge gained will ultimately allow for the development of better diagnostic and therapeutic strategies towards the management of LBP. Standard hematoxylin–eosin (H&E) staining, alone or in combination with other methods, has been traditionally used to assess the normal biology and degeneration of IVDs. A novel technique has recently been developed that provides additional information than previous methods; specifically, a clear differentiation between various sub-regions of the disc. In this method, termed FAST staining, the samples are exposed to a combination of dyes, starting with Alcian blue that blocks the acidic glycoproteins of the tissue. This is followed by a Safranin-O staining through which all the rest of the neutral proteoglycans will be stained. Finally Tartrazine and Fast green are used in order to stain the remaining mucin and collagen moieties. FAST and H&E provide complementary information regarding the compartmental organization and matrix composition of the discs in different conditions of health and degeneration.The principal objective of the current study is to characterize the histological profile of healthy and degenerated human IVDs with a combination of H&E and FAST staining. We will also present some preliminary data regarding the study of innervation in these samples using fluorescent immunohistochemistry (IHC). This will allow future investigations on the relationship between pain and the innervation pattern. Methods: Tissue samples: Samples of degenerating IVDs were collected from chronic low back pain patients. Healthy control discs were obtained post-mortem from donors. Histology: FAST and H&E were performed on cryostat sections obtained from fixed, frozen disc samples. Assessment of innervation: The analysis of the innervation and vascularization in different parts of the discs was done through immunohistochemical mapping studies using specific markers for nerve fibers (anti-PGP 9.5 and anti- CGRP) and blood vessels (anti-PECAM 1). Results: FAST staining enables differentiation of the three regions of the disc: inner annulus fibrosus, outer annulus and nucleous pulposus. In addition, a promising protocol for IHC was adapted to this type of tissue.Conclusion: FAST staining provides detailed information about disorganization of extracellular matrix, proteoglycan changes and calcification during the process of degeneration. H&E and FAST are complementary as one gives more information about the cellular content while the other better identifies the disc compartments, respectively. In addition, sparse sensory innervation can be observed in the very outer regions of healthy human IVDs using immunohistochemical methods.Introduction : La dégénérescence des disques intervertébraux (DIVs) est une des sources connues de lombalgie. Cette condition, reconnue sous le nom de discopathie dégénérative, peut être conséquente du vieillissement, d'un traumatisme ou de prédispositions génétiques. Un meilleur aperçu du profil histologique de DIVs sains et dégénérés contribuera à approfondir notre compréhension des effets du processus dégénératif sur la douleur chronique. De plus, l'avancée des connaissances contribuera au développement de meilleurs diagnostics ainsi qu'à de meilleures stratégies thérapeutiques lors de la prise en charge de patients souffrant de lombalgie. La méthode standard de coloration tissulaire à base d'hématoxyline-éosine (H&E), utilisée seule ou en combinaison avec d'autres colorants, est couramment employée pour évaluer la biologie des DIVs sains ou dégénérés. Récemment, une nouvelle méthode a été développée afin de fournir de l'information additionnelle comme la différentiation définie entre les diverses sous-régions du disque. En utilisant la coloration FAST, les échantillons sont exposés à une combinaison d'agents colorants. Dans un premier temps, le bleu alcien bloque les glycoprotéines acides du tissu. Les protéoglycanes neutres restants sont ensuite colorés par la solution de Safranine-O. Finalement, les colorants Tartrazine et Fast green sont utilisés afin de colorer les fragments moléculaires de mucines et de collagènes. Les colorations FAST et H&E procurent de l'information complémentaire en ce qui concerne la composition de la matrice ainsi que l'organisation structurale des disques et ce, en présence ou non de dégénérescence tissulaire. L'objectif primaire de l'étude est de caractériser les profils histologiques de DIVs humains, sains ou dégénérés, à l'aide des colorations FAST et H&E. De plus, des données préliminaires d'une étude complémentaire sur l'innervation de ces mêmes échantillons observée par immunohistochimie (IHC) sont présentées à la toute fin. Ces données ouvrent la porte pour de futures recherches afin de comprendre la relation entre le patron d'innervation des DIVs et la douleur. Méthodes : Échantillons : Des échantillons tissulaires de DIVs dégénérés ont été recueillis chez des patients ayant de la douleur chronique lombaire. Des disques sains contrôles ont été obtenus de donneurs post-mortem. Histologie : Des coupes de tissus congelés et fixés ont été obtenues à l'aide d'un cryostat, puis colorées à l'aide des méthodes FAST et H&E. Évaluation de l'innervation : L'analyse de l'innervation et de la vascularisation des diverses régions des disques s'est effectuée par une étude de localisation immunohistochimique utilisant des marqueurs spécifiques pour les fibres nerveuses (anti-PGP 9.5 et anti-CGRP) et les vaisseaux sanguins (anti-PECAM 1). Résultats : La coloration FAST permet la différentiation des trois régions principales du disque : l'anneau fibreux interne, l'anneau fibreux externe et le noyau pulpeux. Un protocole d'IHC efficace a été adapté spécifiquement pour ce tissu. Conclusion : La coloration FAST fournit de l'information détaillée sur la désorganisation de la matrice extracellulaire, les changements au niveau des protéoglycanes ainsi que sur la calcification discale survenant lors du processus de dégénérescence. Les colorations H&E et FAST sont complémentaires puisque, de façon respective, une donne de l'information sur le contenu cellulaire alors que l'autre identifie clairement les sous-régions composant le disque. À l'aide de méthodes d'immunohistochimie, il est possible d'identifier une innervation sensorielle dispersée dans les régions les plus externes de disques humains sains

Effect of Self-etching Adhesives on the Bond Strength of Glass-Ionomer Cements.

Adequate bond strength between glass ionomer cements and composite resin is necessary for the success of the sandwich technique.This study assessed the micro-shear bond strength of composite resin to glass-ionomer cements (GIC) using self-etch adhesives with different pH values.One hundred specimens (6×4×2 mm) were made using Fuji II and Fuji II LC GICs and treated with different adhesives as follows: Group 1:Fuji II+ Adper Prompt L-Pop, Group-2: Fuji II+SE bond, Group-3: Fuji II + AdheSE, Group-4:Fuji II+ Protect bond, Group-5: Fuji II + Single bond, Group-6:Fuji II LC+ Adper Prompt LPop, Group-7: Fuji II LC+SE bond, Group-8:Fuji II LC+ AdheSE, Group-9: Fuji II LC+ Protect bond, and Group-10: Fuji II LC+ Single bond. Each group consisted of 10 specimens. A cylinder of Z100 composite resin was placed on each sample and light cured. After 24 hours of water storage (37°C), the specimens were subjected to micro-shear bond strength tests (0.5 mm/min). Data were analyzed using two-way ANOVA and Tukey's test.The mean micro-shear bond strength of groups 1-10 was 11.66±1.79, 16.50±1.85, 18.47±1.77, 13.95±1.77, 15.27±1.49, 15.14±0.90, 20.03±1.19, 17.48±3.00, 16.24±1.98 and 16.03±1.49 MPa, respectively. There were significant differences between groups 1 and 7 (P0.05). Fuji II LC showed higher bond strength than Fuji II (

A novel image-based high-throughput screening assay discovers therapeutic candidates for adult polyglucosan body disease

Glycogen storage disorders (GSDs) are caused by excessive accumulation of glycogen. Some GSDs (Adult Polyglucosan Body Disease (APBD), Tarui and Lafora diseases) are caused by intracellular accumulation of insoluble inclusions, called polyglucosan bodies (PB), which are chiefly composed of malconstructed glycogen. We developed an APBD patient skin fibroblast cell-based assay for PB identification, where the bodies are identified as amylase-resistant periodic acid-Schiff's (PAS) stained structures, and quantified. We screened the DIVERSet-CL 10,084 compound library using this assay in high throughput format and discovered 11 dose-dependent and 8 non dose-dependent PB-reducing hits. ~70% of the hits appear to act through reducing glycogen synthase (GS) activity which can elongate glycogen chains and presumably promote PB generation. Some of these GS inhibiting hits were also computationally predicted to be similar to drugs interacting with the GS activator protein phosphatase 1 (PP1). Our work paves the way to discovering medications for the treatment of PB-involving GSD, which are extremely severe or fatal disorders