Hyaluronic acid-mediated epithelial-mesenchymal transition of human lens epithelial cells via CD44 and TGF-β2

ABSTRACT Purpose: The epithelial-mesenchymal transition of human lens epithelial cells plays a role in posterior capsule opacification, a fibrotic process that leads to a common type of cataract. Hyaluronic acid has been implicated in this fibrosis. Studies have investigated the role of transforming growth factor (TGF)-β2 in epithelial-mesenchymal transition. However, the role of TGF-β2 in hyaluronic acid-mediated fibrosis of lens epithelial cell remains unknown. We here examined the role of TGF-β2 in the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells. Methods: Cultured human lens epithelial cells (HLEB3) were infected with CD44-siRNA by using the Lipofectamine 3000 transfection reagent. The CCK-8 kit was used to measure cell viability, and the scratch assay was used to determine cell migration. Cell oxidative stress was analyzed in a dichloro-dihydro-fluorescein diacetate assay and by using a flow cytometer. The TGF-β2 level in HLEB3 cells was examined through immunohistochemical staining. The TGF-β2 protein level was determined through western blotting. mRNA expression levels were determined through quantitative real-time polymerase chain reaction. Results: Treatment with hyaluronic acid (1.0 μM, 24 h) increased the epithelial-mesenchymal transition of HLEB3 cells. The increase in TGF-β2 levels corresponded to an increase in CD44 levels in the culture medium. However, blocking the CD44 function significantly reduced the TGF-β2-mediated epithelial-mesenchymal transition response of HLEB3 cells. Conclusions: Our study showed that both CD44 and TGF-β2 are critical contributors to the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells, and that TGF-β2 in epithelial-mesenchymal transition is regulated by CD44. These results suggest that CD44 could be used as a target for preventing hyaluronic acid-induced posterior capsule opacification. Our findings suggest that CD44/TGF-β2 is crucial for the hyaluronic acid-induced epithelial-mesenchymal transition of lens epithelial cells.

The association of isocarbophos and isofenphos with different types of glucose metabolism: The role of inflammatory cells.

To investigate the associations between isocarbophos and isofenphos with impaired fasting glucose (IFG) and type 2 diabetes mellitus (T2DM), and to assess the mediation roles of inflammation cells. There were 2701 participants in the case-control study, including 896 patients with T2DM, 900 patients with IFG, 905 subjects with NGT. Plasma isocarbophos and isofenphos concentrations were measured using gas chromatography and triple quadrupole tandem mass spectrometry. Generalized linear models were used to calculate the relationships between plasma isofenphos and isocarbophos levels with inflammatory factor levels and T2DM. Inflammatory cell was used as mediators to estimate the mediating effects on the above associations. Isocarbophos and isofenphos were positively related with T2DM after adjusting for other factors. The odds ratio (95% confidence interval) (OR (95%CI)) for T2DM was 1.041 (1.015, 1.068) and for IFG was 1.066 (1.009, 1.127) per unit rise in ln-isocarbophos. The prevalence of T2DM increased by 6.4% for every 1 unit more of ln-isofenphos (OR (95% CI): 1.064 (1.041, 1.087)). Additionally, a 100% rise in ln-isocarbophos was linked to 3.3% higher ln-HOMA2IR and a 0.029 mmol/L higher glycosylated hemoglobin (HbA1c) (95% CI: 0.007, 0.051). While a 100% rise in ln-isofenphos was linked to increase in ln-HOMA2 and ln-HOMA2IR of 5.8% and 3.4%, respectively. Furthermore, white blood cell (WBC) and neutrophilic (NE) were found to be mediators in the relationship between isocarbophos and T2DM, and the corresponding proportions were 17.12% and 17.67%, respectively. Isofenphos and isocarbophos are associated with IFG and T2DM in the rural Chinese population, WBC and NE have a significant role in this relationship.

The impact of endocrine-disrupting chemicals on stem cells: Mechanisms and implications for human health.

Endocrine-disrupting chemicals (EDCs) are compounds, either natural or man-made, that interfere with the normal functioning of the endocrine system. There is increasing evidence that exposure to EDCs can have profound adverse effects on reproduction, metabolic disorders, neurological alterations, and increased risk of hormone-dependent cancer. Stem cells (SCs) are integral to these pathological processes, and it is therefore crucial to understand how EDCs may influence SC functionality. This review examines the literature on different types of EDCs and their effects on various types of SCs, including embryonic, adult, and cancer SCs. Possible molecular mechanisms through which EDCs may influence the phenotype of SCs are also evaluated. Finally, the possible implications of these effects on human health are discussed. The available literature demonstrates that EDCs can influence the biology of SCs in a variety of ways, including by altering hormonal pathways, DNA damage, epigenetic changes, reactive oxygen species production and alterations in the gene expression patterns. These disruptions may lead to a variety of cell fates and diseases later in adulthood including increased risk of endocrine disorders, obesity, infertility, reproductive abnormalities, and cancer. Therefore, the review emphasizes the importance of raising broader awareness regarding the intricate impact of EDCs on human health.

Stem Cell-Derived Exosomes as a Therapeutic Option for Spinal Cord Injuries; a Systematic Review and Meta-Analysis.

Introduction: Exosomes function as cell signaling carriers and have drawn much attention to the cell-free treatments of regenerative medicine. This meta-analysis aimed to investigate the efficacy of mesenchymal stem cell-derived (MSC-derived) exosomes in animal models of spinal cord injuries (SCI). Method: A comprehensive search was conducted in Medline, Embase, Scopus, and Web of Science to attain related articles published by January 31, 2023. The eligible keywords were correlated with the spinal cord injury and MSC-derived exosomes. The evaluated outcomes were locomotion, cavity size, cell apoptosis, inflammation, neuro-regeneration, and microglia activation. A standardized mean difference was calculated for each sample and a pooled effect size was reported. Results: 65 papers fully met the inclusion criteria. Treatment with MSC-derived exosomes ultimately improved locomotion and shrunk cavity size (p<0.0001). The administration of MSC-derived exosomes enhanced the expression of beta-tubulin III, NF200, and GAP-43, and increased the number of NeuN-positive and Nissl-positive cells, while reducing the expression of glial fibrillary acidic protein (p<0.0001). The number of apoptotic cells in the treatment group decreased significantly (p<0.0001). Regarding the markers of microglia activation, MSC-derived exosomes increased the number of CD206- and CD68-positive cells (p=0.032 and p<0.0001, respectively). Additionally, MSC-derived exosome administration significantly increased the expression of the anti-inflammatory interleukin (IL)-10 and IL-4 (p<0.001 and p=0.001, respectively) and decreased the expression of the inflammatory IL-1b, IL-6, and TNF-a (p<0.0001). Conclusion: MSC-derived exosome treatment resulted in a significantly improved locomotion of SCI animals through ameliorating neuroinflammation, reducing apoptosis, and inducing neuronal regrowth by facilitating a desirable microenvironment.

γδ T-Cell Immunophenotype for the Study of Human Aging.

Flow cytometry serves as a crucial tool in immunology, allowing for the detailed analysis of immune cell populations. γδ T cells, a subset of T cells, play pivotal roles in immune surveillance and immune aging. Assessing the phenotype and functional capabilities of γδ T cells isolated from whole blood or tissue within the context of human aging yields invaluable insights into the dynamic changes affecting immune function, tissue homeostasis, susceptibility to infections, and inflammatory responses.

In Vitro Interaction Between Yeast Extracellular Vesicles and Human Monocyte-Derived Dendritic Cells.

Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host's immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host's immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.

Phenotypic Characterization of B-Lymphocyte Subpopulations in Human Peripheral Blood: A Cost-Effective Seven-Color One-Tube Protocol.

B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.

Endoscopic assessment of the J pouch in ulcerative colitis: A narrative review.

Patients with ulcerative colitis sometimes need a total colectomy with ileal pouch-anal anastomosis due to medically refractory disease or colitis-associated neoplasia. Up to 50% of patients with ulcerative colitis postoperatively develop pouchitis and the rate of chronic inflammatory pouch conditions requiring pouch excision or diverting ileostomy is reported to be 10%. In order to diagnose and monitor pouchitis, pouchoscopy is essential to assess endoscopic inflammatory findings of the J pouch and to survey neoplasia development, particularly in the remnant distal rectum. However, endoscopic protocols for the evaluation of the pouch may not be standardized worldwide and the reliability of existing disease activity indices for pouchitis has been questioned due to the lack of validation. Recently, reliable endoscopic scoring systems based on an observation of the anatomical location of the J pouch were reported and a significant association between the distribution pattern of endoscopic inflammation (i.e., endoscopic phenotype) and pouch outcomes was also uncovered. In this review, we discuss how to survey the J pouch using pouchoscopy, endoscopic indices for pouchitis disease activity, endoscopic phenotypes and classification, and the pathological mechanisms of pouchitis phenotype in patients with ulcerative colitis.

Quantification of T-Cell Receptor Excision Circles (TRECs).

Aging is a natural process that compromises the immune system's functionality increasing the risk of infectious, tumors, and autoimmune diseases. The thymus involution is an age-dependent process characterized by decreased cellularity, peripheral lymphocyte infiltration into the perivascular space, and expansion of adipose tissue. All those modifications hamper the functionality of the organ and lead to a decline of naïve T-cell production with a shrinking of the T-cell repertoire. Thymus atrophy is described in several disorders including autoimmune diseases. The quantification of T-cell receptor excision circles (TRECs) in recent thymus emigrants is a standard procedure to investigate the thymic function. In this chapter, we discuss the methodology used to quantify this molecule in peripheral blood mononuclear cells and isolated CD4+ and CD8+ T cells.

Transcription Factor Analysis to Investigate Immunosenescence in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is linked to various signs of advanced aging, such as premature immunosenescence which occurs due to decline in regenerative ability of T cells. RA T cells develop a unique aggressive inflammatory senescent phenotype with an imbalance of Th17/T regulatory (Treg) cell homeostasis and presence of CD28- T cells. The phenotypic analysis and characterization of T cell subsets become necessary to ascertain if any functional deficiencies exist within with the help of transcription factor (TF) analysis. These subset-specific TFs dictate the functional characteristics of T-cell populations, leading to the production of distinct effector cytokines and functions. Examining the expression, activity, regulation, and genetic sequence of TFs not only aids researchers in determining their importance in disease processes but also aids in immunological monitoring of patients enrolled in clinical trials, particularly in evaluating various T-cell subsets [Th17 (CD3+CD4+IL17+RORγt+) cells and T regulatory (Treg) (CD3+CD4+CD25+CD127-FOXP3+) cells], markers of T-cell aging [aged Th17 cells (CD3+CD4+IL17+RORγt+CD28-), and aged Treg cells (CD3+CD4+CD25+CD127-FOXP3+CD28-)]. In this context, we propose and outline the protocols for assessing the expression of TFs in aged Th17 and Treg cells, highlighting the crucial aspects of this cytometric approach.