Search Results (6)

We developed the Stem Cell Educator therapy among multiple clinical trials based on the immune modulations of multipotent cord blood-derived stem cells (CB-SCs) on different compartments of immune cells, such as T cells and monocytes/macrophages, in type 1 diabetes and other autoimmune diseases. However, the effects of CB-SCs on the B cells remained unclear. To better understand the molecular mechanisms underlying the immune education of CB-SCs, we explored the modulations of CB-SCs on human B cells. CB-SCs were isolated from human cord blood units and confirmed by flow cytometry with different markers for their purity. B cells were purified by using anti-CD19 immunomagnetic beads from human peripheral blood mononuclear cells (PBMCs). Next, the activated B cells were treated in the presence or absence of coculture with CB-SCs for 7 days before undergoing flow cytometry analysis of phenotypic changes with different markers. Reverse transcription-polymerase chain reaction (RT-PCR) was utilized to evaluate the levels of galectin expressions on CB-SCs with or without treatment of activated B cells in order to find the key galectin that was contributing to the B-cell modulation. Flow cytometry demonstrated that the proliferation of activated B cells was markedly suppressed in the presence of CB-SCs, leading to the downregulation of immunoglobulin production from the activated B cells. Phenotypic analysis revealed that treatment with CB-SCs increased the percentage of IgD⁺CD27⁻ naïve B cells, but decreased the percentage of IgD⁻CD27⁺ switched B cells. The transwell assay showed that the immune suppression of CB-SCs on B cells was dependent on the galectin-9 molecule, as confirmed by the blocking experiment with the anti-galectin-9 monoclonal antibody. Mechanistic studies demonstrated that both calcium levels of cytoplasm and mitochondria were downregulated after the treatment with CB-SCs, causing the decline in mitochondrial membrane potential in the activated B cells. Western blot exhibited that the levels of phosphorylated Akt and Erk1/2 signaling proteins in the activated B cells were also markedly reduced in the presence of CB-SCs. CB-SCs displayed multiple immune modulations on B cells through the galectin-9-mediated mechanism and calcium flux/Akt/Erk1/2 signaling pathways. The data advance our current understanding of the molecular mechanisms underlying the Stem Cell Educator therapy to treat autoimmune diseases in clinics. Full article

Clostridioides difficile infection is expected to become the most common healthcare-associated infection worldwide. C. difficile-induced pathogenicity is significantly attributed to its enterotoxin, TcdA, which primarily targets Rho-GTPases involved in regulating cytoskeletal and tight junction (TJ) dynamics, thus leading to cytoskeleton breakdown and ultimately increased intestinal permeability. This study investigated whether two non-digestible oligosaccharides (NDOs), alginate (AOS) and chitosan (COS) oligosaccharides, possess antipathogenic and barrier-protective properties against C. difficile bacteria and TcdA toxin, respectively. Both NDOs significantly reduced C. difficile growth, while cell cytotoxicity assays demonstrated that neither COS nor AOS significantly attenuated the TcdA-induced cell death 24 h post-exposure. The challenge of Caco-2 monolayers with increasing TcdA concentrations increased paracellular permeability, as measured by TEER and LY flux assays. In this experimental setup, COS completely abolished, and AOS mitigated, the deleterious effects of TcdA on the monolayer’s integrity. These events were not accompanied by alterations in ZO-1 and occludin protein levels; however, immunofluorescence microscopy revealed that both AOS and COS prevented the TcdA-induced occludin mislocalization. Finally, both NDOs accelerated TJ reassembly upon a calcium-switch assay. Overall, this study established the antipathogenic and barrier-protective capacity of AOS and COS against C. difficile and its toxin, TcdA, while revealing their ability to promote TJ reassembly in Caco-2 cells. Full article

Parvalbumins (PVALBs) are low molecular weight calcium-binding proteins. In addition to their role in many biological processes, PVALBs play an important role in regulating Ca2+ switching in muscles with fast-twitch fibres in addition to their role in many biological processes. The PVALB gene family is divided into two gene types, alpha (α) and beta (β), with the β gene further divided into two gene types, beta1 (β1) and beta2 (β2), carrying traces of whole genome duplication. A large variety of commonly consumed fish species contain PVALB proteins which are known to cause fish allergies. More than 95% of all fish-induced food allergies are caused by PVALB proteins. The authentication of fish species has become increasingly important as the seafood industry continues to grow and the growth brings with it many cases of food fraud. Since the PVALB gene plays an important role in the initiation of allergic reactions, it has been used for decades to develop alternate assays for fish identification. A brief review of the significance of the fish PVALB genes is presented in this article, which covers evolutionary diversity, allergic properties, and potential use as a forensic marker. Full article

Vascular calcification contributes to cardiovascular morbidity and mortality. A recently developed serum calcification propensity assay is based on the half-transformation time (T50) from primary calciprotein particles (CPPs) to secondary CPPs, reflecting the serum’s endogenous capacity to prevent calcium phosphate precipitation. We sought to identify and review the results of all published studies since the development of the T50-test by Pasch et al. in 2012 (whether performed in vitro, in animals or in the clinic) of serum calcification propensity. To this end, we searched PubMed, Elsevier EMBASE, the Cochrane Library and Google Scholar databases from 2012 onwards. At the end of the selection process, 57 studies were analyzed with regard to the study design, sample size, characteristics of the study population, the intervention and the main results concerning T50. In patients with primary aldosteronism, T50 is associated with the extent of vascular calcification in the abdominal aorta. In chronic kidney disease (CKD), T50 is associated with the severity and progression of coronary artery calcification. T50 is also associated with cardiovascular events and all-cause mortality in CKD patients, patients on dialysis and kidney transplant recipients and with cardiovascular mortality in patients on dialysis, kidney transplant recipients, patients with ischemic heart failure and reduced ejection fraction, and in the general population. Switching from acetate-acidified dialysate to citrate-acidified dialysate led to a longer T50, as did a higher dialysate magnesium concentration. Oral administration of magnesium (in CKD patients), phosphate binders, etelcalcetide and spironolactone (in hemodialysis patients) was associated with a lower serum calcification propensity. Serum calcification propensity is an overall marker of calcification associated with hard outcomes but is currently used in research projects only. This assay might be a valuable tool for screening serum calcification propensity in at-risk populations (such as CKD patients and hemodialyzed patients) and, in particular, for monitoring changes over time in T50. Full article

The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury. Full article

As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C β2 (PKCβ2) at Ser660. Inhibiting (protein kinase C β) PKCβ blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCβ2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection. Full article