Exploring In Vitro Immunomodulatory Properties of Moss Atrichum undulatum Extracts.

Bryophytes are rich sources of diverse secondary metabolites with a wide range of biological activities, including anti-inflammatory, antitumor and antimicrobial effects. The aim of this study was to investigate the chemical composition of extracts from two different genotypes (Serbian and Hungarian) of the axenic moss Atrichum undulatum and evaluate the immunomodulatory potential of the prepared extracts in vitro. Both genotypes of moss samples were cultivated in vitro and subsequently extracted in a Soxhlet apparatus with methanol or ethyl acetate. The highest concentration of total phenolic compounds was found in the methanolic extract of the Serbian genotype (54.25 mg GAE/g extract), while the ethyl acetate extract of the Hungarian genotype showed the highest concentration of phenolic acids (163.20 mg CAE/extract), flavonoids (35.57 mg QE/extract), and flavonols (2.25 mg QE/extract). The extracts showed anti-neuroinflammatory properties by reducing the production of reactive oxygen species, nitric oxide, and tumor necrosis factor alpha by lipopolysaccharide-stimulated microglial cells. Moreover, they mitigated the cytotoxic effects of the pro-inflammatory mediators produced by activated microglia on neurons. The data obtained suggest that extracts from A. undulatum moss have promising anti-neuroinflammatory and neuroprotective properties, making them interesting candidates for further research to combat neuroinflammation.

Microbes as triggers and boosters of Type 1 Diabetes - Mediation by molecular mimicry.

AIMS: Type 1 diabetes is characterized by steadily increasing incidence and largely obscured pathogenesis. Molecular mimicry is well-established as trigger for different autoimmune pathologies, but obscurely explored in the context of T1D. The presented study explores the underestimated role of molecular mimicry in T1D-etiology/progression in search for etiologic factors among human pathogens and commensals. METHODS: A comprehensive immunoinformatics analysis of T1D-specific experimental T-cell epitopes across bacterial, fungal, and viral proteomes was performed, coupled with MHC-restricted mimotope validation and docking of most potent epitopes/mimotopes to T1D-high-risk MHCII molecules. In addition, re-analysis of the publicly available T1D-microbiota dataset was performed, including samples at the pre-T1D disease stage. RESULTS: A number of bacterial pathogens/commensals were tagged as putative T1D triggers/boosters, including ubiquitous gut residents. The prediction of most likely mimicked epitopes revealed heat-shock proteins as most potent autoantigens for autoreactive T-cell priming via molecular mimicry. Docking revealed analogous interactions for predicted bacterial mimotopes and corresponding experimental epitopes. Finally, re-analysis of T1D gut microbiota datasets prompted pre-T1D as most significantly different/dysbiotic, compared to other explored categories (T1D stage/controls). CONCLUSIONS: Obtained results support the unrecognized role of molecular mimicry in T1D, suggesting that autoreactive T-cell priming might be the triggering factor of disease development.

Vitamin B complex suppresses neuroinflammation in activated microglia: in vitro and in silico approach combined with dynamical modeling.

Activated microglia is critically involved in the regulation of neuroinflammation/neurodegradation. Hereby, the anti-inflammatory effects of the vitamin B complex (VBC - B1, B2, B3, B5, B6, and B12) on the function and phenotype of lipopolysaccharide (LPS)-stimulated BV2 microglial cells were examined in vitro. Additionally, VBC-treated microglia supernatants were evaluated on SH-SY5Y cells to investigate the effects on neurons' viability. Further, anti-inflammatory mechanisms of VBC were examined by molecular dockingstudies to determine the binding affinity of each VBC component to Toll-like receptor 4 (TLR4) signalling pathway proteins and inducible nitric oxide synthase. In addition, the dynamical model which simulates VBC inhibition of TLR4 signalling pathway proteins activated by LPS has been constructed and excellent agreement with experimental data has been observed (adjR2 = 0.9715 and 0.9909 for TNF-α and IL-6, respectively). The obtained data demonstrated that VBC treatment reduced the inflammatory mediators secreted by LPS-stimulated microglia, diminished their neurotoxic effects against neurons, and induced changes in phenotype profile toward M2 microglia type. Finally, the constructed dynamical model provides deeper insight into the involvement of each VBC component on the VBC inhibitory potential toward the TLR4 signalling pathway and enables optimization of novel VBC formulations as well as inhibitory potential of new putative inhibitors.

Human papillomavirus-specific antibody status among unvaccinated subjects in the region of Vojvodina, Serbia.

OBJECTIVES: The aim of the study was to evaluate the immune status of young people from the Vojvodina province, Serbia, through the detection of IgG antibodies specific for the L1 protein of HPV types 6, 11, 16, and 18 contained in quadrivalent vaccine. METHODS: The study enrolled 514 healthy persons of both genders, aged between 18 and 30 years. All potential participants were informed about the project's aims by trained interviewers before venous blood collection. Also, participants completed a specially designed anonymous questionnaire to identify socio-demographic characteristics and individual behaviours associated with HPV seroprevalence. VPL HPV L1-specific IgG antibodies were measured using a semi-quantitative HPV IgG ELISA kit (Dia.Pro, Italy). RESULTS: A total of 472 (91.8%) young subjects had no detectable antibodies against high- and low-risk HPV types covered by the quadrivalent vaccine. A slightly higher number of seropositive individuals were detected in the age group of 26-30 years compared to younger than 25. Multivariate analysis showed that the number of lifetime sexual partners was the most powerful predictor of HPV seropositivity (OR = 3.483, 95% CI: 1.294-9.379). CONCLUSIONS: Obtained data point out low levels of naturally induced HPV-specific serum antibodies among the target population in the Vojvodina province. The present work highlights the significance and potential benefits of HPV vaccination. Routine HPV vaccination should be the public health priority in our country and should be included in the national immunization programme as soon as possible.

Vitamin B Complex and Experimental Autoimmune Encephalomyelitis -Attenuation of the Clinical Signs and Gut Microbiota Dysbiosis.

The present study aimed to investigate the neuroprotective effects of the vitamin B complex (B1, B2, B3, B5, B6, and B12-VBC), by studying the changes in the femoral nerve, quadriceps muscle, popliteal lymph nodes and gut microbiota in the rat model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). VBC treatment attenuated clinical signs of EAE during the disease, and reduced the duration of EAE thereby contributing to a faster recovery. In VBC-treated EAE rats, a significant decrease in nerve and muscle nuclear density was revealed during the onset period of the disease, while a marked increase was detected at the end of the disease, compared with untreated EAE rats. In the lymph nodes of VBC-treated EAE rats, a fewer number of lymphoid follicles in the cortical area and smaller epithelioid granulomas were detected. The changes in microbiota composition were examined using 16S rRNA gene sequencing and bioinformatics analysis, which revealed the potential of VBC treatment in establishing and/or maintaining gut microbiota homeostasis. Finally, the present study demonstrated that VBC treatment ameliorated the cellular changes in the affected peripheral nerve, muscles innervated by this nerve, and the gut microbiota dysbiosis which occurred during the EAE.

The Influence of Seasonality on Secondary Metabolite Profiles and Neuroprotective Activities of Moss Hypnum cupressiforme Extracts: In Vitro and In Silico Study.

Numerous representatives of mosses, including Hypnum cupressiforme, have been used to alleviate different inflammation-related conditions. However, the mode of action underlying this anti-inflammatory potential has been poorly understood. Moreover, the influence of seasonality on the chemical composition and biological activity of mosses is generally overlooked. This study aimed to investigate the influence of seasonal changes (spring, summer, and autumn) on secondary metabolite composition and biological activities of ethyl acetate H. cupressiforme extracts. Antioxidant activity was measured using ß-carotene bleaching assay, while MTT, NBT, ELISA, and Griess assays were carried out to explore the anti-neuroinflammatory and neuroprotective potential of extracts. Inhibitory activities on acetylcholinesterase and tyrosinase were assessed experimentally and by docking analysis. The highest content of secondary metabolites and antioxidant activity were observed in moss during the summer. Extracts inhibited the secretion of ROS, NO, TNF-α, and IL-6, alleviating the inflammatory potential of H2O2 and LPS in microglial and neuronal cells. Strong inhibitory effects on acetylcholinesterase and tyrosinase were observed in vitro. Docking analyses revealed high-affinity interactions of secondary metabolites present in H. cupressiforme with important enzyme residues. Altogether, these results reveal the neuroprotective potential and the significance of seasonal fluctuations on secondary metabolite content and biological activities in moss H. cupressiforme.

The role of vitamin A and vitamin D in modulation of the immune response with a focus on innate lymphoid cells.

The immune system with its numerous and complex interactions helps to protect the host from pathogenic microorganisms, and enables cleaning of damaged tissues. It is also associated with constant "monitoring" of the appearance of malignant cells and their elimination that can occur in the human body. Such a role depends on many factors including adequate intake of nutrients, including vitamins. The effect of vitamin supplementation on the modulation of the immune response has always been the focus of numerous studies. Vitamins A and D have been shown to have the greatest immune-modulatory effect. In this review, we discuss and consider the possible roles of vitamins A and D on the immune response through innate and adaptive immune cells, with special focus on the cell population recently characterized as innate lymphoid cells. Recent literature data indicate that vitamin A and its metabolites modulate the balance between Th1 and Th2 immunity. In addition, vitamin D expresses protective effects on the innate immune system and inhibitory effects on adaptive immunity.

A Study of Phytochemistry, Genoprotective Activity, and Antitumor Effects of Extracts of the Selected Lamiaceae Species.

This study was designed to evaluate the genoprotective, antigenotoxic, as well as antitumor potential of methanolic, ethanolic, and aqueous extracts of Melissa officinalis, Mentha × piperita, Ocimum basilicum, Rosmarinus officinalis, Salvia officinalis, and Satureja montana (Lamiaceae), in different model systems. The polyphenols in these extracts were quantified both spectrophotometrically and using HPLC-DAD technique, while DPPH assay was used to assess the antioxidant activity. The genoprotective potential was tested on pUC19 Escherichia coli XL1-blue, and the antigenotoxicity on Salmonella typhimurium TA1535/pSK1002 and human lung fibroblasts, while the antitumor activity was assessed on colorectal cancer cells. Rosmarinic acid, quercetin, rutin, and luteolin-7-O-glucoside were among the identified compounds. Methanolic extracts had the best DPPH-scavenging and SOS-inducing activities, while ethanolic extracts exhibited the highest antigenotoxicity. Additionally, all extracts exhibited genoprotective potential on plasmid DNA. The antitumor effect was mediated by modulation of reactive oxygen species (ROS), nitric oxide (NO) production, and exhibition of genotoxic effects on tumor cells, especially with O. basilicum ethanolic extract. Generally, the investigated extracts were able to provide antioxidant protection for the acellular, prokaryotic, and normal human DNA, while also modulating the production of ROS and NO in tumor cells, leading to genotoxicity toward these cells and their decrease in proliferation.

Mining the capacity of human-associated microorganisms to trigger rheumatoid arthritis-A systematic immunoinformatics analysis of T cell epitopes.

Autoimmune diseases, often triggered by infection, affect ~5% of the worldwide population. Rheumatoid Arthritis (RA)-a painful condition characterized by the chronic inflammation of joints-comprises up to 20% of known autoimmune pathologies, with the tendency of increasing prevalence. Molecular mimicry is recognized as the leading mechanism underlying infection-mediated autoimmunity, which assumes sequence similarity between microbial and self-peptides driving the activation of autoreactive lymphocytes. T lymphocytes are leading immune cells in the RA-development. Therefore, deeper understanding of the capacity of microorganisms (both pathogens and commensals) to trigger autoreactive T cells is needed, calling for more systematic approaches. In the present study, we address this problem through a comprehensive immunoinformatics analysis of experimentally determined RA-related T cell epitopes against the proteomes of Bacteria, Fungi, and Viruses, to identify the scope of organisms providing homologous antigenic peptide determinants. By this, initial homology screening was complemented with de novo T cell epitope prediction and another round of homology search, to enable: i) the confirmation of homologous microbial peptides as T cell epitopes based on the predicted binding affinity to RA-related HLA polymorphisms; ii) sequence similarity inference for top de novo T cell epitope predictions to the RA-related autoantigens to reveal the robustness of RA-triggering capacity for identified (micro/myco)organisms. Our study reveals a much larger repertoire of candidate RA-triggering organisms, than previously recognized, providing insights into the underestimated role of Fungi in autoimmunity and the possibility of a more direct involvement of bacterial commensals in RA-pathology. Finally, our study pinpoints Endoplasmic reticulum chaperone BiP as the most potent (most likely mimicked) RA-related autoantigen, opening an avenue for identifying the most potent autoantigens in a variety of different autoimmune pathologies, with possible implications in the design of next-generation therapeutics aiming to induce self-tolerance by affecting highly reactive autoantigens.

Translocator Protein Modulation by 4'-Chlorodiazepam and NO Synthase Inhibition Affect Cardiac Oxidative Stress, Cardiometabolic and Inflammatory Markers in Isoprenaline-Induced Rat Myocardial Infarction.

The possible cardioprotective effects of translocator protein (TSPO) modulation with its ligand 4'-Chlorodiazepam (4'-ClDzp) in isoprenaline (ISO)-induced rat myocardial infarction (MI) were evaluated, alone or in the presence of L-NAME. Wistar albino male rats (b.w. 200-250 g, age 6-8 weeks) were divided into 4 groups (10 per group, total number N = 40), and certain substances were applied: 1. ISO 85 mg/kg b.w. (twice), 2. ISO 85 mg/kg b.w. (twice) + L-NAME 50 mg/kg b.w., 3. ISO 85 mg/kg b.w. (twice) + 4'-ClDzp 0.5 mg/kg b.w., 4. ISO 85 mg/kg b.w. (twice) + 4'-ClDzp 0.5 mg/kg b.w. + L-NAME 50 mg/kg b.w. Blood and cardiac tissue were sampled for myocardial injury and other biochemical markers, cardiac oxidative stress, and for histopathological evaluation. The reduction of serum levels of high-sensitive cardiac troponin T hs cTnT and tumor necrosis factor alpha (TNF-α), then significantly decreased levels of serum homocysteine Hcy, urea, and creatinine, and decreased levels of myocardial injury enzymes activities superoxide dismutase (SOD) and glutathione peroxidase (GPx) as well as lower grades of cardiac ischemic changes were demonstrated in ISO-induced MI treated with 4'-ClDzp. It has been detected that co-treatment with 4'-ClDzp + L-NAME changed the number of registered parameters in comparison to 4'-ClDzp group, indicating that NO (nitric oxide) should be important in the effects of 4'-ClDzp.

Chemical characterization and in vitro immunomodulatory effects of different extracts of moss Hedwigia ciliata (Hedw.) P. Beauv. from the Vrsacke Planine Mts., Serbia.

Bioactive compounds from natural sources are of great importance because of their potential pharmacological activity and tremendous structural diversity. In this study, the chemical composition of different moss extracts of Hedwigia ciliata P. Beauv. have been examined, as well as their antioxidant, antineurodegenerative/anti-neuroinflammatory, antidiabetic, and antiproliferative potential. The extracts were prepared by Soxhlet extractor using solvents of different polarity. Chemical characterization of the extracts revealed the presence of phenolics and flavonoid compounds, together with triterpenoids as secondary metabolites of high biological activity. Significant antioxidant properties of all the extracts were exhibited using the ß-carotene assay. The highest activities were found for water:ethanol extract (with the highest inhibition rate of 96%), but also significant inhibition was measured for ethanol and ethyl acetate extracts (80% and 70%, respectively). Confirmation of biocompatibility of investigated moss extracts has been performed using normal human fibroblast cell line, MRC-5. The H. ciliata extracts exhibited significant antiproliferative activity (~ 50%) against the MDA-MB-231 (human breast adenocarcinoma cell line), which has not previously been reported elsewhere. The Griess assay confirmed the potential anti-neuroinflammatory activity of the extracts, as significant effects in reducing NO production by LPS-stimulated BV2 (normal murine microglia cell line) was observed. This data is in line with noted antineurodegenerative potential measured by the inhibition of acetylcholinesterase (with the highest inhibition rate of 60% for ethyl acetate extract) and tyrosinase (with the highest inhibition rate of 70% for ethanol extract). Additionally, the H. ciliata extracts exhibited significant antidiabetic effect mediated by α-glucosidase inhibition (with the highest inhibition rate of 80% for ethyl acetate extract). The obtained data suggest the presence of immunomodulatory effects of the moss extracts in vitro, which allows the design of new experiments aimed at detecting and characterizing bioactive compounds of the extracts and additionally elucidate detailed mechanisms of their effects.

Extracts Characterization and In Vitro Evaluation of Potential Immunomodulatory Activities of the Moss Hypnum cupressiforme Hedw.

Recently, there has been an increasing interest in the chemistry and biological potential of mosses, since a large number of biologically active compounds have been found within these species. This study aimed at examining the chemical composition and immunomodulatory potential (antioxidant, antidiabetic, anti-neuroinflammatory/antineurodegenerative, and antitumor activities) of moss Hypnum cupressiforme Hedw. extracts. Corresponding extracts have been obtained applying Soxhlet extractor. The chemical characterization was performed using spectrophotometric assays and liquid chromatography-mass spectrometry (LC-MS). The extracts were analyzed for antioxidant activity and for inhibitory activities on α-glucosidase, α-amylase, acetylcholinesterase, and tyrosinase. Additionally, extracts were tested against four cell lines-MRC-5, BV2, HCT-116, and MDA-MB-231-for antitumor and anti-inflammatory activities. Chemical analysis of extracts revealed the presence of flavonoids, phenolic acids, and triterpenoids. Major compounds identified by LC-MS in H. cupressiforme were kaempferol and five phenolic acids: p-hydroxybenzoic, protocatechuic, p-coumaric, gallic, and caffeic acid. According to biochemical assays the investigated extracts exhibited significant immunomodulatory potential. Significant antiproliferative potential against MDA-MB-231 cells has been observed together with the promising anti-neuroinflammatory application. The obtained data suggest that moss H. cupressiforme is a valuable natural source of biologically active compounds with potential application in the pharmaceutical industry.

Antiproliferative and antimigratory effects of 3-(4-substituted benzyl)-5- isopropyl-5-phenylhydantoin derivatives in human breast cancer cells.

In this study, a series of synthesized 3-(4-substituted benzyl)-5-isopropyl-5-phenylhydantoin derivatives as a potential antiproliferative and antimigratory agents were investigated. The possible antitumor mechanisms of investigated hydantoin derivatives were examined on human breast cancer cell line MDA-MB-231. The cells were treated with different concentrations of compounds (from 0.01 µM to 100 µM) during 24 h and 72 h. The proliferation index, nitric oxide production, apoptosis rate, and migration capacity were measured. The cell invasion potential was examined by measuring the level of MMP-9 and COX-2 gene expression. All tested compounds expressed antiproliferative activity and induced dose- and time-dependent increase in the level of nitrites. The investigated molecules significantly decreased cell survival rate, migration capacity and the expression levels of genes included in the process of tumor invasion. Obtained data suggest that the tested hydantoin derivatives express considerable antitumor activity by reducing cell division rate, elevating apoptosis level, and inhibiting the motility and invasiveness of breast cancer cells. The results obtained in this study indicate that investigated compounds express potential as a novel chemotherapeutic agents against breast cancer growth and progression.

Effects of galectin-1 on immunomodulatory properties of human monocyte-derived dendritic cells.

Our study aimed to evaluate the effects of Gal-1 in dose depending manner on maturation and immunomodulatory properties of monocyte-derived (Mo) DCs in-vitro. The effects were analyzed by monitoring their phenotypic characteristics, cytokine profile, and the ability to direct the immune response in the co-culture with allogeneic CD4+T cells. Gal-1 reduced the expression of CD80 and CD86 molecules on MoDCs compared to untreated MoDCs. Gal-1 at concentrations of 1 and 6 µg/mL significantly reduced IL-12 production, while the concentration of 3 µg/mL led to its significant increase. Gal-1 in all concentrations induced a significant increase in the production of IL-10. Treatment of MoDCs with 3 and 6 µg/mL of Gal-1 stimulated the production of IL-2 and IFN-γ in the co-culture with CD4+T lymphocytes. This study demonstrated a dual immunomodulatory effect of Gal-1 on MoDCs in terms of immune stimulation and immune suppression, depending on the applied concentration.

Vitamin B Complex Treatment Attenuates Local Inflammation after Peripheral Nerve Injury.

Peripheral nerve injury (PNI) leads to a series of cellular and molecular events necessary for axon regeneration and reinnervation of target tissues, among which inflammation is crucial for the orchestration of all these processes. Macrophage activation underlies the pathogenesis of PNI and is characterized by morphological/phenotype transformation from proinflammatory (M1) to an anti-inflammatory (M2) type with different functions in the inflammatory and reparative process. The aim of this study was to evaluate influence of the vitamin B (B1, B2, B3, B5, B6, and B12) complex on the process of neuroinflammation that is in part regulated by l-type CaV1.2 calcium channels. A controlled transection of the motor branch of the femoral peripheral nerve was used as an experimental model. Animals were sacrificed after 1, 3, 7, and 14 injections of vitamin B complex. Isolated nerves were used for immunofluorescence analysis. Treatment with vitamin B complex decreased expression of proinflammatory and increased expression of anti-inflammatory cytokines, thus contributing to the resolution of neuroinflammation. In parallel, B vitamins decreased the number of M1 macrophages that expressed the CaV1.2 channel, and increased the number of M2 macrophages that expressed this channel, suggesting their role in M1/M2 transition after PNI. In conclusion, B vitamins had the potential for treatment of neuroinflammation and neuroregeneration and thereby might be an effective therapy for PNI in humans.

Function of innate lymphoid cells in the immune-related disorders.

Innate lymphoid cells (ILCs) are a recently described group of innate immune cells that mirror the characteristics of CD4+ T cell subsets. Based on their transcriptional factor and cytokine profile, ILCs family is divided into main subgroups-ILC1s, ILC2s, and ILC3s. Recently, one new subpopulation of ILCs with immunosuppressive characteristics has been described and named as regulatory ILCs. Various roles of ILCs have been confirmed including the role during the response to microbial signals, the role in inflammation and process of tissue repair. Function of ILCs is mediated through the cytokines production and direct cell-to-cell contact. This article summarizes in detail, the relationship between the ILCs and various immune-related disorders.

Effects of silica-rich water on systemic and peritoneal inflammation in rats exposed to chronic low-level (900-MHz) microwave radiation.

Immunomodulating effect of silica-rich water represents a novel field for research, especially regarding its features toward environmental pollutants. The aim of our study was to evaluate the effects of silica-rich water intake on systemic and peritoneal inflammation in rats that were chronically exposed to the low-level microwave (MW) radiation from mobile phones. Wistar Albino rats were exposed to 900 MHz MW radiation for 3 months. The four-treatment model involved rats with standard water (SW) or experimental silica-rich water intake (EW). Peritoneal macrophages (PMs) were harvested using peritoneal lavage and divided into non-stimulated and lipopolysaccharide (LPS) stimulated subgroups. The MW-exposed rats with silica-rich water (MW+EW) had lower serum tumor necrosis factor α (TNF-α) and interleukin 2 (IL-2) levels, but higher IL-10 levels, than MW+SW rats (p < 0.05). The higher TNF-α production by non-stimulated MW exposed PMs was ameliorated by the silica-rich water (p < 0.01). The MW exposition suppressed LPS potential for TNF-α synthesis in both water type groups, with greater suppression in animals that took standard water. Our results show the modulating effect of silica-rich water toward MW-induced systemic and peritoneal inflammation, which reflects the water ability to shape monocyte plasticity, thereby altering the balance between their proinflammatory and anti-inflammatory properties.