A Novel Approach for Fast Screening of a Complex Cyanobacterial Extract for Immunomodulatory Properties and Antibacterial Activity

The filamentous cyanobacteria from genus Phormidium are rich natural sources of bioactive compounds that could be exploited as pharmaceuticals or nutraceuticals. In this study, we suggest a novel approach for assessing the immunomodulatory properties of the products derived from cyanobacteria. The influence of Phormidium papyraceum extract on the human leukocyte immunophenotype was evaluated by attempting to link this activity to certain putative compounds identified in the extract. By using three staining panels and flow cytometry, we found that the cyanobacterial extract affected mainly CD4+ T cells upregulating activated CD4+CD152+ T cells (15.75 ± 1.93% treated vs. 4.65 ± 1.41% control) and regulatory CD4+CD25+ T cells (5.36 ± 0.64% treated vs. 1.03 ± 0.08% control). Furthermore, P. papyraceum extract can modulate T cell subpopulations with a CD4+ effector/memory phenotype. Extract-treated cells showed increased production of IL-2 (55 ± 12 pg/mL) and IL-6 (493 ± 64 pg/mL) compared to the untreated, 21 ± 7 pg/mL and 250 ± 39 pg/mL, respectively. No significant changes were observed in the secretion of TNF-α. In addition, P. papyraceum extract displayed antibacterial activity against both Gram-negative (inhibition zone from 18.25 ± 0.50 mm to 20.28 ± 1.50 mm) and Gram-positive (inhibition zone from 10.86 ± 0.85 mm to 17.00 ± 0.82 mm) bacteria. The chemical profile of the cyanobacterial extract was determined using LC–ESI–MS/MS analysis, where at least 112 putative compounds were detected. Many of these compounds have proven different biological activities. We speculated that compounds such as betulin and the macrolide azithromycin (or their analogues) could be responsible for the immunomodulatory potential of the investigated extract. More studies are needed to determine and validate the biological activities of the determined putative compounds

Natural Xylooligosaccharides Exert Antitumor Activity via Modulation of Cellular Antioxidant State and TLR4

It has been recently proven that xylooligosaccharides (XOS) with prebiotic properties have diverse beneficial biological effects including immunomodulatory and antitumor activities. The present article focused on the chemical and biological evaluation of corn-derived commercially available XOS and aimed to elucidate their cytotoxicity and inhibitory potential against tumor cells. Spectrophotometric chemical analyses, Fourier transform infrared spectroscopy, and high-performance liquid chromatography analyses were performed. Antioxidant activity was determined by measuring the oxygen radical absorbance capacity and hydroxyl radical averting capacity. In vitro cytotoxicity assays with human cell lines derived from normal and tumor tissues, assessments of ATP production, mitochondrial membrane potential specific staining, cytokine assays, and molecular docking were used to evaluate the biological activity of XOS. The sample showed significant antioxidant activity, and it was determined that most xylose oligomers in it are composed of six units. XOS exhibited antitumor activity with pronounced inhibitory effect on lysosomes, but mitochondrial functionality was also affected. The production of proinflammatory cytokines by lipopolysaccharide-stimulated U-937 cells was reduced by XOS treatment, which suggested the involvement of Toll-like receptor 4 (TLR4)-mediated signaling in the mechanism of XOS action. Molecular docking analyses confirmed the potential inhibitory interaction between the sample and TLR4. In addition, XOS treatment had significant tumor-cell-specific influence on the glutathione antioxidant system, affecting its balance and thus contributing to the inhibition of cellular viability. The present study elucidated the tumor-inhibitory potential of commercially available XOS that could be utilized in pharmaceutical and food industry providing disease-preventive and therapeutic benefits

Phylogenetic Relationships of Some Filamentous Cyanoprokaryotic Species

The polyphasic approach is the most progressive system that has been suggested for distinguishing and phylogenetically classifying Cyanoprokaryota (Cyanobacteria/Cyanophyta). Several oscillatorialean genera ( Lyngbya, Phormidium, Plectonema , and Leptolyngbya ) have problematic phylogenetic position and taxonomic state because of their heterogeneity and polyphyletic nature. To accurately resolve the phylogenetic relationship of some filamentous species ( Nodosilinea bijugata, Phormidium molle, Phormidium papyraceum ), we have performed phylogenetic analyses based on 16S rRNA gene and the phycocyanin Operon (PC-IGS) by using maximum-likelihood (ML) tree inference methods. These analyses were combined with morphological re-evaluation. Our phylogenetic analyses support the taxonomic separation of genus Nodosilinea from the polyphyletic genus Leptolyngbya. Investigated Nodosilinea strains always formed a coherent genetic cluster supported with a high bootstrap value. The molecular phylogeny confirmed also the monophyly of the Wilmottia group. In addition, data reveal that although P. papyraceum is morphologically similar to Wilmottia murrayi , this species is genetically distinct. Strains from the newly formed genus Phormidesmis and some Phormidium priestleyi strains were clustered in a separate clade different from the typical Phormidium species, but without strong bootstrap support

Characteristics of T-cell and B-cell immune respoonses to pollen allergens in Bulgarian patients with pollinosis

Characterization of the pollen allergens and identification of the main T-cell and B-cell epitopes is of crucial importance, since the clarification of the cellular and molecular mechanisms will allow long-term control of this type allergic diseases and development of safe immunotherapeutic medication. The objective of this study was to identify which pollen allergens are responsible for the T-cell activation in atopic patients with pollinosis and to determine whether the same allergens are responsible for the IgE-mediated reactions. Thirty-seven patients with pollinosis and thirteen non-allergic subjects were recruited. Peripheral blood samples were collected out of the pollen season. T-cell responses (IFN-γ production) towards different pollen allergens and levels of pollen specific IgE and IgG in the sera were measured by ELISA. The T-cell reactivity in most patients was directed towards the grass pollen B1, tree pollen I and autumn pollen B5. 70% of the allergic individuals responded to allergens from Lolium perenne, 84% to Dactylis glomerata, 11% to Phleum pratense, 65% to Betula pendula and 70% to Taxus baccata. Elevated serum levels of specific IgE in the allergic patients (p<0.01) were measured against the tree pollen I and autumn pollen, which include widespread deciduous trees (birch, willow, poplar and yew) and Artemisia absinthium, respectively. Our results show that the T-cell reactivity and antibody responses may be directed towards different or the same allergens. In addition, we propose that short synthetic peptides, which contain overlapping T- and B-cell epitopes can be used for specific immunotherapy treatment if they lack antibody recognition domains

Molecular Mimicry of the Rheumatoid Arthritis-Related Immunodominant T-Cell Epitope within Type II Collagen (CII260-270) by the Bacterial L-Asparaginase

The etiology of most autoimmune diseases, including rheumatoid arthritis (RA), remains unclear. Both genetic and environmental factors are believed to be involved in pathogenesis. Molecular mimicry is considered one of the mechanisms for the occurrence of autoimmune diseases. The aim of the study was to determine whether the bacterial peptide L-ASNase67-81, which mimics the immunodominant T-cell epitope CII259-273, can induce T-cell reactivity in blood samples from RA patients and healthy subjects through molecular mimicry. Using bioinformatic molecular modeling methods, we first determined whether the L-ASNase67-81 peptide binds to the HLA-DRB1*04:01 molecule and whether the formed MHCII&ndash;peptide complex interacts with the corresponding T-cell receptor. To validate the obtained results, leukocytes isolated from early RA patients and healthy individuals were stimulated in vitro with L-ASNase67-81 and CII259-273 peptides as well as with bacterial L-asparaginase or human type II collagen (huCII). The activated T cells (CD4+CD154+) were analyzed by flow cytometry (FACS), and the levels of cytokines produced (IL-2, IL-17A/F, and IFN-&gamma;) were measured by ELISA. Our in silico analyses showed that the bacterial peptide L-ASNase67-81 binds better to HLA-DRB1*04:01 compared to the immunodominant T-cell epitope CII259-273, mimicking its structure and localization in the binding groove of MHCII. Six contact points were involved in the molecular interaction of the peptide with the TCR. FACS data showed that after in vitro stimulation with the L-ASNase67-81 peptide, the percentage of activated T cells (CD154+CD4+) was significantly increased in both cell cultures isolated from ERA patients and those isolated from healthy individuals, as higher values were observed for the ERA group (9.92 &plusmn; 0.23 vs. 4.82 &plusmn; 0.22). Furthermore, the ELISA assays revealed that after stimulation with L-ASNase67-81, a significant increase in the production of the cytokines IL-2, IL-17A/F, and IFN-&gamma; was detected in the group of ERA patients. Our data showed that the bacterial L-ASNase67-81 peptide can mimic the immunodominant T-cell epitope CII259-273 and activate HLA-DRB1*04:01-restricted T cells as well as induce cytokine production in cells isolated from ERA patients. These results are the first to demonstrate that a specific bacterial antigen could play a role in the pathogenesis of RA, mimicking the immunodominant T-cell epitope from type II collagen

Bimetallic Gold–Iron Oxide Nanoparticles as Carriers of Methotrexate: Perspective Tools for Biomedical Applications

Bimetallic nanoparticles (BMNPs) combine unique and synergistic properties of two metals, allowing new specific applications. In this study, bimetallic AuFe nanoparticles and their conjugates with methotrexate (MTX) were obtained with an environmentally safe method of metal-vapor synthesis. The composition and electronic structure of the particles were investigated with X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray (EDX) spectrum and X-ray absorption spectroscopy (XANES and EXAFS). The effects of BMNP-MTX conjugates on human primary cells and tumor cell lines were evaluated with neutral red uptake and MTT in vitro cytotoxicity assays. Bright-field microscopy analyses of tumor spheroid size and evaluations of tumor spheroid vitality based on SFDA AM staining were carried out. In vitro assays for an antibacterial activity evaluation of the generated samples were performed. The influence of BMNP-MTX on cytokine production with normal leukocytes was assessed using ELISA. X-ray analyses of the samples demonstrated that gold was in the ground state Au0 as well as Au+ and Au3+ states are present in small quantities, whereas iron existed as a mixture of non-histometric oxides with states close to Fe2+ and Fe3+. The modification of the AuFe system with MTX is accompanied by a threefold increase in the relative proportion of the Au+ state. BMNP-MTX conjugates demonstrated significant antitumor activity compared to the drug alone, which proves the ability of the generated nanoconjugates to improve the effectiveness of MTX therapy. This was confirmed by a marked reduction in the size and vitality of AuFe-MTX-treated 3D tumor spheroids. In addition to their selective antitumor activity, AuFe-MTX exhibited moderate antibacterial activity and induced sample-specific cytokine production with normal human leukocytes—which points to an immunostimulatory potential. The present findings indicate important and diverse biological properties of BMNP-MTX conjugates and thus highlight perspectives for their biomedical applications and new immune-specific abilities

Polyphasic characterisation of Microcoleus autumnalis (Gomont, 1892) Strunecky, Komárek & J.R.Johansen, 2013 (Oscillatoriales, Cyanobacteria) using a metabolomic approach as a complementary tool

As a result of the continuous revision of cyanobacterial taxonomy, Phormidium autumnale (Agardh) Trevisan ex Gomont, 1892 has been transferred to the genus Microcoleus as Microcoleus autumnalis (Gomont, 1892) Strunecky, Komárek & J.R.Johansen, 2013. This transfer was based on a single strain and literature data. In the present study, we revise the taxonomic position of Microcoleus autumnalis by applying the classical approach of polyphasic taxonomy and additionally using metabolomics. Cyanobacterial strains identified as Phormidium autumnale and Microcoleus vaginatus (type species of the genus Microcoleus) were used for comparative analyses. In addition, the taxonomic relationship between the species Phormidium autumnale and Phormidium uncinatum was determined on the basis of polyphasic characteristics. Monitoring of the morphological variability of Phormidium autumnale and Microcoleus vaginatus strains showed a difference in the morphology concerning the ends of the trichomes, the shape of the apical cells, as well as the presence/absence of the calyptra and its shape. The performed TEM analysis of the thylakoid arrangement of the studied strains showed parietal arrangement of the thylakoids in the representatives of genus Phormidium and fascicular arrangement in genus Microcoleus. Molecular genetic analyses, based on 16S rDNA, revealed grouping of the investigated P. autumnale strains in a separate clade. This clade is far from the subtree, which is very clearly formed by the representatives of the type species of genus Microcoleus, namely M. vaginatus. The metabolomic analysis involving P. autumnale and M. vaginatus strains identified 39 compounds that could be used as potential biochemical markers to distinguish the two cyanobacterial species. Based on the data obtained, we suggest changing of the current status of Microcoleus autumnalis by restoring its previous appurtenance to the genus Phormidium under the name Phormidium autumnale (Agardh) Trevisan ex Gomont, 1892 and distinguishing this species from genus Microcoleus

Phytoplankton composition with an emphasis of Cyanobacteria and their toxins as an indicator for the ecological status of Lake Vaya (Bulgaria) – part of the Via Pontica migration route

As producers of biomass, cyanobacteria are a major part of the phytoplankton in a large number of water basins. Due to the cyanobacterial blooms and cyanotoxins produced, these organisms are recognized as a threat and ecological risk for water bodies. Released cyanotoxins may cause death of many organisms including birds and fish. Vaya Lake is the largest natural lake in Bulgaria. It is located on the Via Pontica migration route of birds between Europe and Africa. Since 2003, the lake has been declared as a "Wetland of international importance” under the Ramsar Convention. According to the literature data from 2002-2006, the Lake is defined as highly eutrophied due to strong anthropogenic pressure, but regular monitoring of the cyanobacterial blooms and presence of cyanotoxins after this period is missing. Taking into account the importance of this unique, protected ecosystem, our aim was to make a complete ecological assessment of the present state of Lake Vaya by using the phytoplankton, with an emphasis on cyanobacterial blooms and the presence of cyanotoxins. As results of the study, we 1) characterized the phytoplankton composition qualitatively and quantitatively; 2) evaluated the ecological status of the western and eastern part of the Lake; 3) identified the potential producers of cyanotoxins; 4) observed cyanobacterial blooms and discussed the influence of macrophytes on their spread; 5) measured the concentrations of the cyanotoxins MCs, CYL and STXs in water samples from both parts of the Lake. Our results indicated the need for continued observation of cyanobacterial composition, blooming and the presence of cyanotoxins in Lake Vaya

Serum Leptin and Resistin Levels in Knee Osteoarthritis—Clinical and Radiologic Links: Towards Precise Definition of Metabolic Type Knee Osteoarthritis

Obesity is considered a major risk factor for the development and progression of knee osteoarthritis (OA). Apart from the mechanical effect of obesity via increase in mechanical overload of weight-bearing joints, an association with hand OA has been observed. There has been increasing interest in the role of adipokines in the pathogenesis of OA in the recent years. It has been suggested that their systemic effects link obesity and OA. In this regard, the aim of the current study was measurement and analysis of serum levels of leptin and resistin in patients with knee OA with different body mass index (BMI). Seventy-three patients with primary symptomatic knee OA at the age between 35 and 87 years (mean age 66 years) were included in the study (67 women and 6 men). The patients were from 2nd to 4th radiographic stage according to Kellgren–Lawrence scale. 43 patients were with concomitant obesity (BMI ≥ 30 kg/m2, mean values 38.34 ± 8.20) and 30 patients with BMI &lt; 30 kg/m2 (mean values 25.07 ± 2.95). Eleven individuals with different BMIs, including cases with obesity but without radiographic knee OA, were examined as a control group. Serum levels of leptin and resistin were measured via ELISA method. In patients with knee OA and BMI ≥ 30 kg/m2, serum levels of leptin (39.546 ± 12.918 ng/mL) were significantly higher as compared with healthy individuals (15.832 ± 16.531 ng/mL, p &lt; 0.05) and the patients with low BMI (p &lt; 0.05). In patients with BMI &lt; 30 kg/m2 the levels of leptin (13.010 ± 10.94 ng/mL) did not differ significantly from the respective values in the control group (p = 0.48). Serum levels of resistin were also higher in knee OA patients in comparison with healthy controls, but the difference was statistically significant only for patients with high BMI (2.452 ± 1.002 ng/mL in the group with BMI ≥ 30 kg/m2; 2.401 ± 1.441 ng/mL in patients with BMI &lt; 30 kg/m2; 1.610 ± 1.001 ng/mL in the control group, p &lt; 0.05). A correlation was found between the serum levels of leptin and radiographic stage of OA, i.e., higher leptin levels were present in the more advanced 3rd and 4th radiographic stage, while for resistin a correlation was observed in the patient subgroup with BMI &lt; 30 kg/m2. Serum leptin and resistin levels and clinical characteristics were analyzed in patients with different clinical forms of OA. Novel clinical correlations have been found in the current study in patients with isolated knee OA vs. cases with presence of other disease localizations. It has been observed that patients with isolated knee OA were significantly younger and had higher BMI as compared with cases in whom OA is combined with other localizations i.e., spondyloarthritis ± presence of hip OA and with generalized OA. This supports the hypothesis that presence of obesity promotes earlier development of knee OA as an isolated localization of the disease in younger patients before appearance of osteoarthritic changes at other sites. The levels of leptin and resistin in isolated knee OA were also higher. Serum levels of leptin and resistin in combination with patients’ clinical characteristics suggest existence of different clinical and laboratory profile through which more precise definition of metabolic phenotype of knee OA would be possible. Considering the fact that obesity is a modifiable risk factor that has an impact on progression of knee OA, different approaches to influence obesity may offer potential for future disease-modifying therapeutic interventions

Phytoplankton composition with an emphasis of Cyanobacteria and their toxins as an indicator for the ecological status of Lake Vaya (Bulgaria) – part of the Via Pontica migration route

As producers of biomass, cyanobacteria are a major part of the phytoplankton in a large number of water basins. Due to the cyanobacterial blooms and cyanotoxins produced, these organisms are recognized as a threat and ecological risk for water bodies. Released cyanotoxins may cause death of many organisms including birds and fish. Vaya Lake is the largest natural lake in Bulgaria. It is located on the Via Pontica migration route of birds between Europe and Africa. Since 2003, the lake has been declared as a "Wetland of international importance” under the Ramsar Convention. According to the literature data from 2002-2006, the Lake is defined as highly eutrophied due to strong anthropogenic pressure, but regular monitoring of the cyanobacterial blooms and presence of cyanotoxins after this period is missing. Taking into account the importance of this unique, protected ecosystem, our aim was to make a complete ecological assessment of the present state of Lake Vaya by using the phytoplankton, with an emphasis on cyanobacterial blooms and the presence of cyanotoxins. As results of the study, we 1) characterized the phytoplankton composition qualitatively and quantitatively; 2) evaluated the ecological status of the western and eastern part of the Lake; 3) identified the potential producers of cyanotoxins; 4) observed cyanobacterial blooms and discussed the influence of macrophytes on their spread; 5) measured the concentrations of the cyanotoxins MCs, CYL and STXs in water samples from both parts of the Lake. Our results indicated the need for continued observation of cyanobacterial composition, blooming and the presence of cyanotoxins in Lake Vaya