Methyl and Isopropyl N-Methylanthranilates Affect Primary Macrophage Function - An Insight into the Possible Immunomodulatory Mode of Action.

To complement the knowledge on the anti-inflammatory activity of methyl and isopropyl N-methylanthranilates, two natural products with panacea-like properties, we investigated their effects on thioglycolate-elicited macrophages by evaluating macrophage ability to metabolize MTT, macrophage membrane function, and macrophage myeloperoxidase and phagocytic activities. Moreover, two additional aspects of the inflammatory response of these compounds, their inhibitory activity on xanthine oxidase and catalase, were studied. It was found that these two compounds regulate elicited macrophage functions, most probably by interfering with the function of cell membranes and changing the reducing cellular capacity or enzyme activity of macrophages. Nonetheless, no significant inhibitory action either towards xanthine oxidase or catalase was found, suggesting that the inhibition of these enzymes is not involved in the anti-inflammatory mode of action of these two esters.

Assessment of the biological activity of Marrubium friwaldskyanum Boiss. (Lamiaceae).

Present scientific evidences about the biological activity and potential medical application of extracts derived from Marrubium friwaldskyanum Boiss. are limited. Therefore, our study was undertaken to define several main characteristics in this regard - in vitro cytotoxicity and antitumor properties, antibacterial activity and immunomodulatory potential. Extracts were obtained from different aerial parts of Marrubium friwaldskyanum - stems, leaves and flowers. The in vitro cytotoxicity and antitumor activity of the samples were evaluated by tetrazolium salt reduction tests and Neutral red uptake assays using four human cell lines (a normal fibroblastic and three adenocarcinoma cell lines/A549, HeLa, HT-29/) and by experiments with HT-29 tumor spheroids. Antibacterial activity toward Gram-negative (Escherichia coli) and Gram-positive (Bacillus cereus) species was assessed based on estimation of minimal inhibitory and minimal bactericidal concentrations as well as longitudinal studies on bacterial viability. Ex vivo assays with normal leukocytes were performed to define potential immunomodulatory activity of the extracts. Our results demonstrated selective antitumor activity of the extracts directed against colon adenocarcinoma HT-29 cells and cervical adenocarcinoma HeLa cell line. Metabolic activity of A549 lung adenocarcinoma cells was affected only by the sample derived from flowers. M. friwaldskyanum leaf and flower extracts showed the highest activity, which included reduction of HT-29 tumor spheroid growth and viability. The studied samples exhibited antibacterial activity against both bacterial species tested. Treatment with M. friwaldskyanum extracts affected specific leukocyte populations (HLA+, CD19+, CD11b+, CD25+ cells). These results demonstrate for the first time complex biological effects of extracts derived from M. friwaldskyanum and their potential to serve as a source of valuable compounds for the pharmaceutical industry.

In vitro evaluation of the immunomodulatory and wakame assimilation properties of Lactiplantibacillus plantarum strains from swine milk.

The emergence and spread of antibiotic resistance threat forced to explore alternative strategies for improving the resistance to pathogens in livestock production. Probiotic lactic acid bacteria represent an alternative for this objective. In this study, seven Lactiplantibacillus plantarum strains from porcine colostrum and milk were isolated, identified and characterized in terms of their abilities to modulate immunity in porcine intestinal epithelial (PIE) cells. Then, two potential immunoregulatory strains were studied in terms of their ability to utilize and grow in wakame (Undaria pinnafida). Isolates were identified by 16S rRNA gene and evaluated by studying their interaction with PIE cells. The expressions of peptidoglycan recognition proteins (PGRPs), nucleotide-binding oligomerization domain (NODs), host defense peptides (pBD), and type I interferons (IFNs) were evaluated by RT-qPCR. The strain 4M4417 showed a remarkable capacity to differentially regulate the expression of PGRP1, PGRP3, NOD1, NOD2, and pBD1 in PIE cells. On the other hand, the strain 4M4326 was the most efficient to improve the expression of IFN-α and IFN-ß in PIE cells challenged with poly (I:C). Both L. plantarum 4M4326 and 4M4417 were characterized in terms of their ability to utilize wakame. Results demonstrated that both strains efficiently grew in wakame-based broth. Our results suggest that L. planatrum 4M4326 and 4M4417 are interesting candidates to develop immunomodulatory feeds based on wakame utilization. These new immunosynbiotic feeds could help to reduce severity of intestinal infections and improve immune health status in pigs.

Exploring clinical applications and long-term effectiveness of benzodiazepines: An integrated perspective on mechanisms, imaging, and personalized medicine.

Benzodiazepines have been long-established treatments for various conditions, including anxiety disorders and insomnia. Recent FDA warnings emphasize the risks of misuse and dependence associated with benzodiazepines. This article highlights their benefits and potential drawbacks from various perspectives. It achieves this by explaining how benzodiazepines work in terms of neuroendocrinology, immunomodulation, sleep, anxiety, cognition, and addiction, ultimately improving their clinical effectiveness. Benzodiazepines play a regulatory role in the HPA axis and impact various systems, including neuropeptide Y and cholecystokinin. Benzodiazepines can facilitate sleep-dependent memory consolidation by promoting spindle wave activity, but they can also lead to memory deficits in older individuals due to reduced slow-wave sleep. The cognitive effects of chronic benzodiazepines use remain uncertain; however, no adverse findings have been reported in clinical imaging studies. This article aims to comprehensively review the evidence on benzodiazepines therapy, emphasizing the need for more clinical studies, especially regarding long-term benzodiazepines use.

The structure, characterization and immunomodulatory potential of exopolysaccharide produced by Planococcus rifietoensis AP-5 from deep-sea sediments of the Northwest Pacific.

Polysaccharides derived from microorganisms exhibit diverse structures and bioactivities, making them promising candidates for the treatment of various diseases. However, marine-derived polysaccharides and their activities are relatively little known. In this work, fifteen marine strains were isolated from surface sediments in the Northwest Pacific Ocean for screening of EPS production. Planococcus rifietoensis AP-5 produced a maximum yield of EPS at 4.80 g/L. The purified EPS (referred to as PPS) had a molecular weight of 51,062 Da and contained amino, hydroxyl, and carbonyl groups as its major functional groups. PPS primarily consisted of →3)-α-D-Galp-(1 â†’ 4)-α-D-Manp-(1 â†’ 2)-α-D-Manp-(1 â†’ 4)-α-D-Manp-(1 â†’ 4,6)-α-D-Glcp-(1 â†’ 6)-ß-D-Galp-(1→, with a branch consisting of T-ß-D-Glcp-(1→. Additionally, surface morphology of PPS was hollow, porous, and sphere-like stack. PPS primarily contained C, N, and O elements, with a surface area of 33.76 m2/g, a pore volume of 0.13 cc/g, and a pore diameter of 1.69 nm, respectively. Based on the TG curve, the degradation temperature of PPS was measured to be 247 °C. Furthermore, PPS demonstrated immunomodulatory activity through dose-dependently upregulating the expression level of cytokines. It significantly enhanced the cytokine secretion at a concentration of 5 µg/mL. To sum up, this study offers valuable insights for screening marine polysaccharide-based immunomodulators.

Umbilical-Cord-Derived Mesenchymal Stromal Cells Modulate 26 Out of 41 T Cell Subsets from Systemic Sclerosis Patients.

Systemic sclerosis (SSc) is an immune-mediated disease wherein T cells are particularly implicated, presenting a poor prognosis and limited therapeutic options. Thus, mesenchymal-stem/stromal-cell (MSC)-based therapies can be of great benefit to SSc patients given their immunomodulatory, anti-fibrotic, and pro-angiogenic potential, which is associated with low toxicity. In this study, peripheral blood mononuclear cells from healthy individuals (HC, n = 6) and SSc patients (n = 9) were co-cultured with MSCs in order to assess how MSCs affected the activation and polarization of 58 different T cell subsets, including Th1, Th17, and Treg. It was found that MSCs downregulated the activation of 26 out of the 41 T cell subsets identified within CD4+, CD8+, CD4+CD8+, CD4-CD8-, and γδ T cells in SSc patients (HC: 29/42) and affected the polarization of 13 out of 58 T cell subsets in SSc patients (HC: 22/64). Interestingly, SSc patients displayed some T cell subsets with an increased activation status and MSCs were able to downregulate all of them. This study provides a wide-ranging perspective of how MSCs affect T cells, including minor subsets. The ability to inhibit the activation and modulate the polarization of several T cell subsets, including those implicated in SSc's pathogenesis, further supports the potential of MSC-based therapies to regulate T cells in a disease whose onset/development may be due to immune system's malfunction.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Mesenchymal Stem/Stromal Cells Derived from Human Placenta.

Mesenchymal stem/stromal cells derived from placenta (PMSCs) are an attractive source for regenerative medicine because of their multidifferentiation potential and immunomodulatory capabilities. However, the cellular and molecular heterogeneity of PMSCs has not been fully characterized. Here, we applied single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin sequencing (scATAC-seq) techniques to cultured PMSCs from human full-term placenta. Based on the inferred characteristics of cell clusters, we identify several distinct subsets of PMSCs with specific characteristics, including immunomodulatory-potential and highly proliferative cell states. Furthermore, integrative analysis of gene expression and chromatin accessibility showed a clearer chromatin accessibility signature than those at the transcriptional level on immunomodulatory-related genes. Cell cycle gene-related heterogeneity can be more easily distinguished at the transcriptional than the chromatin accessibility level in PMSCs. We further reveal putative subset-specific cis-regulatory elements regulating the expression of immunomodulatory- and proliferation-related genes in the immunomodulatory-potential and proliferative subpopulations, respectively. Moreover, we infer a novel transcription factor PRDM1, which might play a crucial role in maintaining immunomodulatory capability by activating PRDM1-regulon loop. Collectively, our study first provides a comprehensive and integrative view of the transcriptomic and epigenomic features of PMSCs, which paves the way for a deeper understanding of cellular heterogeneity and offers fundamental biological insight of PMSC subset-based cell therapy.

Sulfaguanidine Hybrid with Some New Pyridine-2-One Derivatives: Design, Synthesis, and Antimicrobial Activity against Multidrug-Resistant Bacteria as Dual DNA Gyrase and DHFR Inhibitors.

Herein, a series of novel hybrid sulfaguanidine moieties, bearing 2-cyanoacrylamide 2a-d, pyridine-2-one 3-10, and 2-imino-2H-chromene-3-carboxamide 11, 12 derivatives, were synthesized, and their structure confirmed by spectral data and elemental analysis. All the synthesized compounds showed moderate to good antimicrobial activity against eight pathogens. The most promising six derivatives, 2a, 2b, 2d, 3a, 8, and 11, revealed to be best in inhibiting bacterial and fungal growth, thus showing bactericidal and fungicidal activity. These derivatives exhibited moderate to potent inhibition against DNA gyrase and DHFR enzymes, with three derivatives 2d, 3a, and 2a demonstrating inhibition of DNA gyrase, with IC50 values of 18.17-23.87 µM, and of DHFR, with IC50 values of 4.33-5.54 µM; their potency is near to that of the positive controls. Further, the six derivatives exhibited immunomodulatory potential and three derivatives, 2d, 8, and 11, were selected for further study and displayed an increase in spleen and thymus weight and enhanced the activation of CD4+ and CD8+ T lymphocytes. Finally, molecular docking and some AMED studies were performed.

Immunomodulatory and Antioxidative potentials of adipose-derived Mesenchymal stem cells isolated from breast versus abdominal tissue: a comparative study.

BACKGROUND: Adipose-derived stem cells (ASCs) are considered ideal candidates for both research and cellular therapy due to ease of access, large yield, feasibility, and efficacy in preclinical and clinical studies. Unlike the subcutaneous abdominal fat depot, breast ASCs features are still not well recognized, limiting their possible therapeutic use. ASCs were found to exert immunomodulatory and antioxidative activities for maintaining homeostasis and functionality of diseased/damaged tissues. This study aims to investigate the immunomodulatory and antioxidative potentials of breast versus abdominal isolated ASCs to find out which anatomical site provides ASCs with better immunoregulatory and oxidative stress resistance capabilities. METHODS: ASCs were isolated from abdominal and breast tissues. Gene expression analysis was conducted for a panel of immunomodulatory and antioxidative genes, as well as adipokines and proliferation genes. Flow cytometric analysis of a group of immunomodulatory surface proteins was also performed. Finally, the significantly expressed genes have undergone protein-protein interaction and functional enrichment in silico analyses. RESULTS: Our results revealed similar morphological and phenotypic characteristics for both breast and abdominal ASCs. However, a significant elevation in the expression of two potent immunosuppressive genes, IL-10 and IDO as well as the expression of the multifaceted immunomodulatory adipokine, visfatin, was detected in breast versus abdominal ASCs. Moreover, a significant overexpression of the antioxidative genes, GPX1, SIRT5, and STAT3 and the proliferation marker, Ki67, was also observed in breast ASCs relative to abdominal ones. In silico analysis showed that both of the differentially upregulated immunomodulatory and antioxidative mediators integratively involved in multiple biological processes and pathways indicating their functional association. CONCLUSION: Breast ASCs possess superior immunomodulatory and antioxidative capabilities over abdominal ASCs. Our findings shed light on the possible therapeutic applications of breast ASCs in immune-related and oxidative stress-associated diseases.

Pharmacological characterisation of CR6086, a potent prostaglandin E2 receptor 4 antagonist, as a new potential disease-modifying anti-rheumatic drug.

BACKGROUND: Prostaglandin E2 (PGE2) acts via its EP4 receptor as a cytokine amplifier (e.g., interleukin [IL]-6) and induces the differentiation and expansion of inflammatory T-helper (Th) lymphocytes. These mechanisms play a key role in the onset and progression of rheumatoid arthritis (RA). We present the pharmacological characterisation of CR6086, a novel EP4 receptor antagonist, and provide evidence for its potential as a disease-modifying anti-rheumatic drug (DMARD). METHODS: CR6086 affinity and pharmacodynamics were studied in EP4-expressing HEK293 cells by radioligand binding and cyclic adenosine monophosphate (cAMP) production, respectively. In immune cells, IL-6 and vascular endothelial growth factor (VEGF) expression were analysed by RT-PCR, and IL-23 and IL-17 release were measured by enzyme-linked immunosorbent assay (ELISA). In collagen-induced arthritis (CIA) models, rats or mice were immunised with bovine collagen type II. Drugs were administered orally (etanercept and methotrexate intraperitoneally) starting at disease onset. Arthritis progression was evaluated by oedema, clinical score and histopathology. Anti-collagen II immunoglobulin G antibodies were measured by ELISA. RESULTS: CR6086 showed selectivity and high affinity for the human EP4 receptor (Ki = 16.6 nM) and functioned as a pure antagonist (half-maximal inhibitory concentration, 22 nM) on PGE2-stimulated cAMP production. In models of human immune cells in culture, CR6086 reduced key cytokine players of RA (IL-6 and VEGF expression in macrophages, IL-23 release from dendritic cells, IL-17 release from Th17 cells). In the CIA model of RA in rats and mice, CR6086 significantly improved all features of arthritis: severity, histology, inflammation and pain. In rats, CR6086 was better than the selective cyclooxygenase-2 inhibitor rofecoxib and at least as effective as the Janus kinase inhibitor tofacitinib. In mice, CR6086 and the biologic DMARD etanercept were highly effective, whereas the non-steroidal anti-inflammatory drug naproxen was ineffective. Importantly, in a study of CR6086/methotrexate, combined treatment greatly improved the effect of a fully immunosuppressive dose of methotrexate. CONCLUSIONS: CR6086 is a novel, potent EP4 antagonist showing favourable immunomodulatory properties, striking DMARD effects in rodents, and anti-inflammatory activity targeted to immune-mediated inflammatory diseases and distinct from the general effects of cyclooxygenase inhibitors. These results support the clinical development of CR6086, both as a stand-alone DMARD and as a combination therapy with methotrexate. The proof-of-concept trial in patients with RA is ongoing.

Thermal treatment of luteolin-7-O-ß-glucoside improves its immunomodulatory and antioxidant potencies.

Phytochemicals extracted from flowers, roots and bark, leaves, and other plant sources have been used extensively throughout human history with varying levels of efficacy in prevention and treatment of disease. Recently, advanced methods for characterization and clinical use of these materials have allowed modern understanding of their properties to be used as immunomodulatory agents that act by enhancement of endogenous cytoprotective mechanisms, avoiding interference with normal physiologic signaling and highly effective medical treatment with minimal adverse side effects. Simple methods have been identified for improving their biological effects, such as thermal conditioning by heating or freezing-prominent example being heat treatment of lycopene and tetrahydrocannabinol. The present investigation shows improvement of the ability of heat to augment splenocyte proliferation, natural killer (NK) cell activities, and antioxidant capacity of the flavonoid luteolin-7-O-ß-glucoside (L7G) in comparison with the native (non heat-treated) molecule, while further demonstrating that both the native and the heat-treated variants exhibit comparable antioxidant properties, as evidenced by their effects in macrophages by inhibition of nitric oxide production and lysosomal enzyme activity in experiments that strengthen lysosomal membrane integrity. Outcomes of these studies suggest that heat-treated L7G shows promise for use in immunotherapy, including anti-cancer regimens, as shown by its improvement of NK cell cytotoxicity.

Increased motility of mesenchymal stem cells is correlated with inhibition of stimulated peripheral blood mononuclear cells in vitro.

Immunomodulatory properties of mesenchymal stem cells (MSC) are key components of their successful applications in clinical setting. However, treatments based on MSC immunomodulation need understanding of cell characteristics before cell transplantation. We used live-imaging to test the suitability of the MSC motility as a parameter for quick prediction of the immunomodulatory potential of human MSC in regulating the activity of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Bone marrow MSC, from various donors and in vitro passages, were cultured with or without stimulated PBMC. After seven days, immunomodulation was assessed by measuring PBMC proliferation, IgG production and cytokine secretion in MSC and PBMC monocultures and co-cultures, and results were correlated to MSC motility. In co-culture, we observed that MSC successfully inhibited PBMC activity, reducing PBMC proliferation and IgG production compared to PBMC monoculture. MSC modulated PBMC to reduce the secretion of TNFα and IL-10, increase IL-6, G-CSF and MCP-1, while GM-CSF was not affected. By live-imaging tracking of cell trajectories, we observed that fast moving MSC were inhibiting more efficiently stimulated PBMC compared to slow ones. In co-culture, fast MSC were more effective in inhibiting IgG production (˜30% less IgG), and secreted higher levels of IL-10 (˜10% increase) and GM-CSF (˜20% increase) compared to slower cells. Furthermore, fast MSC in monocultures produced 2.3-fold more IL-6, 1.5-fold MCP-1 and 1.2-fold G-CSF in comparison to slower cells. In conclusion, live-imaging cell tracking allowed us to develop an indicative assay of the immune-regulatory potential of MSC prior to in vivo administration. Key Words: Human mesenchymal stem cells, Immunomodulatory potential, In vitro cell motility, Stem cell transplantation.