scholarly journals Optimization of Lyophilized Hyperacute Serum (HAS) as a Regenerative Therapeutic in Osteoarthritis

2021 ◽  
Vol 22 (14) ◽  
pp. 7496
Author(s):  
Isabel Olmos Calvo ◽  
Olga Kuten-Pella ◽  
Karina Kramer ◽  
Ágnes Madár ◽  
Szilvia Takács ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Platelet Rich Plasma ◽  
Cell Types ◽  
Inflammatory Conditions ◽  
Osteoarthritic Chondrocytes ◽  
Prp Gene ◽  
Human Explants ◽  
Cytokine Quantification

Hyperacute serum (HAS) is a blood derivative product that promotes the proliferation of various cell types and controls inflammation in vitro. The aim of this study is to investigate the regenerative potential of different formulations of HAS, including lyophilized and hyaluronic acid combined versions, to obtain a stable and standardized therapeutic in osteoarthritis (OA), which may be able to overcome the variability limitations of platelet-rich plasma (PRP). Primary human osteoarthritic chondrocytes were used for testing cellular viability and gene expression of OA-related genes. Moreover, a co-culture of human explants of cartilage, bone and synovium under inflammatory conditions was used for investigating the inflammatory control capacities of the different therapeutics. In this study, one formulation of lyophilized HAS achieved the high cell viability rates of liquid HAS and PRP. Gene expression analysis showed that HAS induced higher Col1a1 expression than PRP. Cytokine quantification from supernatant fluids revealed that HAS treatment of inflamed co-cultures significantly reduced levels of IL-5, IL-15, IL-2, TNFα, IL-7 and IL-12. To conclude, lyophilized HAS is a stable and standardized therapeutic with high potential in joint regeneration.

scholarly journals Platelet-Released Growth Factors Modulate the Secretion of Cytokines in Synoviocytes under Inflammatory Joint Disease

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Mersedeh Tohidnezhad ◽  
Andreas Bayer ◽  
Biljana Rasuo ◽  
Jennifer Vanessa Phi Hock ◽  
Nisreen Kweider ◽  
...  
Keyword(s):  
Gene Expression ◽  
Platelet Rich Plasma ◽  
Joint Disease ◽  
Cytokine Gene Expression ◽  
Inflammatory Conditions ◽  
Autologous Platelet Rich Plasma ◽  
Exact Effect ◽  
Autologous Platelet ◽  
Inflammatory Effect

The etiology and pathogenesis of rheumatoid arthritis (RA) are marked by a complex interplay of various cell populations and is mediated by different signaling pathways. Traditionally, therapies have primarily focused on pain relief, reducing inflammation and the recovery of joint function. More recently, however, researchers have discussed the therapeutic efficacy of autologous platelet-rich plasma (PRP). The main objective of this work is to examine the influences of platelet-released growth factor (PRGF) on human synoviocytes under inflammatory conditions. Additionally, it is checked to which extend treatment with platelet concentrate influences the release of cytokines form synoviocytes. For this purpose, an in vitro RA model was created by stimulating the cells with the TNF-α. The release of cytokines was measured by ELISA. The cytokine gene expression was analyzed by real-time PCR. It has been observed that the stimulation concentration of 10 ng/ml TNF-α resulted in a significantly increased endogenous secretion and gene expression of IL-6 and TNF-α. The anti-inflammatory effect of PRGF could be confirmed through significant reduction of TNF-α and IL-1β. An induced inflammatory condition seems to cause PRGF to inhibit the release of proinflammatory cytokines. Further study is required to understand the exact effect mechanism of PRGF on synoviocytes.

Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

2019 ◽  
Vol 20 (11) ◽  
pp. 920-933 ◽  
Author(s):  
Lucía Gato-Calvo ◽  
Tamara Hermida-Gómez ◽  
Cristina R. Romero ◽  
Elena F. Burguera ◽  
Francisco J. Blanco
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Ex Vivo Model ◽  
Chondrocyte Viability ◽  
Platelet Concentration ◽  
The Absolute ◽  
Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

scholarly journals Control of impulsivity by Gi-protein signalling in layer-5 pyramidal neurons of the anterior cingulate cortex

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Bastiaan van der Veen ◽  
Sampath K. T. Kapanaiah ◽  
Kasyoka Kilonzo ◽  
Peter Steele-Perkins ◽  
Martin M. Jendryka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Anterior Cingulate Cortex ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Pyramidal Neurons ◽  
Treatment Options ◽  
Pyramidal Cells ◽  
Cingulate Cortex ◽  
Cell Types ◽  
Anterior Cingulate

AbstractPathological impulsivity is a debilitating symptom of multiple psychiatric diseases with few effective treatment options. To identify druggable receptors with anti-impulsive action we developed a systematic target discovery approach combining behavioural chemogenetics and gene expression analysis. Spatially restricted inhibition of three subdivisions of the prefrontal cortex of mice revealed that the anterior cingulate cortex (ACC) regulates premature responding, a form of motor impulsivity. Probing three G-protein cascades with designer receptors, we found that the activation of Gi-signalling in layer-5 pyramidal cells (L5-PCs) of the ACC strongly, reproducibly, and selectively decreased challenge-induced impulsivity. Differential gene expression analysis across murine ACC cell-types and 402 GPCRs revealed that - among Gi-coupled receptor-encoding genes - Grm2 is the most selectively expressed in L5-PCs while alternative targets were scarce. Validating our approach, we confirmed that mGluR2 activation reduced premature responding. These results suggest Gi-coupled receptors in ACC L5-PCs as therapeutic targets for impulse control disorders.

Retinoic Acid Receptor Agonists Suppress Muscle Fatty Infiltration in Mice

2021 ◽  
Vol 49 (2) ◽  
pp. 332-339
Author(s):  
Hideyuki Shirasawa ◽  
Noboru Matsumura ◽  
Masaki Yoda ◽  
Kazumasa Okubo ◽  
Masayuki Shimoda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Rotator Cuff ◽  
Rotator Cuff Tear ◽  
Gene Expression Analysis ◽  
Retinoic Acid Receptor ◽  
Adipogenic Differentiation ◽  
Fatty Infiltration ◽  
Cuff Tear

Background: The infiltration of fat tissue into skeletal muscle, a condition referred to as muscle fatty infiltration or fatty degeneration, is regarded as an irreversible event that significantly compromises the motor function of skeletal muscle. Purpose: To investigate the effect of retinoic acid receptor (RAR) agonists in suppressing the adipogenic differentiation of fibroadipogenic progenitors (FAPs) in vitro and fatty infiltration after rotator cuff tear in mice. Study Design: Controlled laboratory study. Methods: FAPs isolated from mouse skeletal muscle were cultured in adipogenic differentiation medium in the presence or absence of an RAR agonist. At the end of cell culture, adipogenic differentiation was evaluated by gene expression analysis and oil red O staining. A mouse model of fatty infiltration—which includes the resection of the rotator cuff, removal of the humeral head, and denervation the supraspinatus muscle—was used to induce fatty infiltration in the supraspinatus muscle. The mice were orally or intramuscularly administered with an RAR agonist after the surgery. Muscle fatty infiltration was evaluated by histology and gene expression analysis. Results: RAR agonists effectively inhibited the adipogenic differentiation of FAPs in vitro. Oral and intramuscular administration of RAR agonists suppressed the development of muscle fatty infiltration in the mice after rotator cuff tear. In accordance, we found a significant decrease in the number of intramuscular fat cells and suppressed expression in adipogenic markers. RAR agonists also increased the expression of the transcripts for collagens; however, an accumulation of collagenous tissues was not histologically evident in the present model. Conclusion: Muscle fatty infiltration can be alleviated by RAR agonists through suppressing the adipogenic differentiation of FAPs. The results also suggest that RAR agonists are potential therapeutic agents for treating patients who are at risk of developing muscle fatty infiltration. The consequence of the increased expression of collagen transcripts by RAR agonists needs to be clarified. Clinical Relevance: RAR agonists can be used to prevent the development of muscle fatty infiltration after rotator cuff tear. Nevertheless, further studies are mandatory in a large animal model to examine the safety and efficacy of intramuscular injection of RAR agonists.

scholarly journals Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

2021 ◽  
Vol 22 (3) ◽  
pp. 1222
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Survival Rates ◽  
Control Group ◽  
P Value ◽  
Transcriptome Profile ◽  
Embryo Viability ◽  
The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

scholarly journals Insulin-Induced Gene 2 Involvement in Human Adipocyte Metabolism and Body Weight Regulation

2008 ◽  
Vol 93 (5) ◽  
pp. 1995-2001 ◽  
Author(s):  
Sergey Krapivner ◽  
Sergej Popov ◽  
Ekaterina Chernogubova ◽  
Mai-Lis Hellénius ◽  
Rachel M. Fisher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Adipocyte Differentiation ◽  
Regulatory Element ◽  
Human Adipose Tissue ◽  
Human Tissues ◽  
Weight Regulation ◽  
Body Weight Regulation ◽  
Sterol Regulatory Element

Abstract Background: Insulin-induced genes (INSIGs) encode proteins that block proteolytic activation of sterol regulatory element-binding proteins, transcription factors that regulate lipogenic enzymes, and adipocyte differentiation. Objective: Here, we analyzed the relative significance of INSIG1 and INSIG2 in human liver and adipocyte metabolism, and defined a novel, functional polymorphism in the promoter of INSIG2 associated with body mass index. Research Methods: Variations in gene expression of different human tissues, of hepatoma cells exposed to INSIG1 and INSIG2 gene silencing probes, and of differentiating 3T3-L1 adipocytes were determined by real-time quantitative PCR. The functional significance of a novel polymorphism in the promoter of INSIG2 was analyzed using in vitro methods and gene expression analysis of human adipose tissue, whereas the phenotype associated with this polymorphism was studied in two cohorts of middle-aged men. Results: Gene expression analysis of 17 human tissues demonstrated that INSIG1 is highly expressed in the liver, whereas INSIG2 is ubiquitously expressed. Gene silencing experiments confirmed that INSIG1, but not INSIG2, regulates the expression of sterol regulatory element-binding proteins target genes in human hepatoma cells. In contrast, adipocyte differentiation of 3T3-L1 cells was associated with a 13-fold increase in expression of INSIG2. Significant relationships between the INSIG2–102G/A polymorphism and body mass index were observed in two cohorts of middle-aged men (ANOVA P = 0.017 and 0.044, respectively). In vitro studies and analysis of allele-specific expression in human adipose tissue substantiated the functional significance of the INSIG2–102G/A polymorphism. Conclusion: INSIG2 is involved in adipocyte metabolism and body weight regulation.

scholarly journals Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

2019 ◽  
Author(s):  
Marcus Alvarez ◽  
Elior Rahmani ◽  
Brandon Jew ◽  
Kristina M. Garske ◽  
Zong Miao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Types ◽  
Supervised Machine Learning ◽  
Data Sets ◽  
Rna Seq ◽  
Novel Approach ◽  
Single Nucleus ◽  
Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

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