Cartilage Oligomeric Matrix Protein in Osteoarthritis and Obesity-Do New Considerations Emerge?

The diagnosis of osteoarthritis (OA) is based on radiological changes that are delayed, along with clinical symptoms. Early and very early diagnosis at the stage of molecular pathology may eventually offer an opportunity for early therapeutic intervention that may retard and prevent future damage. Cartilage oligomeric matrix protein (COMP) is a non-collagenous extracellular matrix protein that promotes the secretion and aggregation of collagen and contributes to the stability of the extracellular matrix. There are contradictory literature data and currently, the parameter is used only for scientific purposes and its significance is not well-determined. The serum level of COMP in patients with metabolic type OA of the knee has not been evaluated. The aim of the study was to analyze serum COMP levels in metabolic knee OA and controls with different BMI. Our results showed that the mean COMP values were significantly higher in the control group (1518.69 ± 232.76 ng/mL) compared to the knee OA patients (1294.58 ± 360.77 ng/mL) (p = 0.0012). This may be related to the smaller cartilage volume in OA patients. Additionally, COMP levels negatively correlated with disease duration (p = 0.04). The COMP level in knee OA with BMI below 30 kg/m2 (n = 61, 1304.50 ± 350.60 ng/mL) was higher compared to cases with BMI ≥ 30 kg/m2 (n = 76, 1286.63 ± 370.86 ng/mL), but the difference was not significant (p = 0.68). Whether this finding is related to specific features in the evolution of the metabolic type of knee OA remains to be determined. Interestingly, comparison of COMP levels in the controls with different BMI revealed significantly higher values in overweight and obese individuals (1618.36 ± 203.76 ng/mL in controls with BMI ≥ 25 kg/m2, n = 18, 1406.61 ± 216.41 ng/mL, n = 16; p = 0.0092). Whether this finding is associated with increased expression of COMP in the adipose tissue or with more intensive cartilage metabolism in relation to higher biomechanical overload in obese patients, considering the earlier development of metabolic type knee OA as an isolated finding, remains to be determined.

Significance of Type II Collagen Posttranslational Modifications: From Autoantigenesis to Improved Diagnosis and Treatment of Rheumatoid Arthritis.

Collagen type II (COL2), the main structural protein of hyaline cartilage, is considerably affected by autoimmune responses associated with the pathogenesis of rheumatoid arthritis (RA). Posttranslational modifications (PTMs) play a significant role in the formation of the COL2 molecule and supramolecular fibril organization, and thus, support COL2 function, which is crucial for normal cartilage structure and physiology. Conversely, the specific PTMs of the protein (carbamylation, glycosylation, citrullination, oxidative modifications and others) have been implicated in RA autoimmunity. The discovery of the anti-citrullinated protein response in RA, which includes anti-citrullinated COL2 reactivity, has led to the development of improved diagnostic assays and classification criteria for the disease. The induction of immunological tolerance using modified COL2 peptides has been highlighted as a potentially effective strategy for RA therapy. Therefore, the aim of this review is to summarize the recent knowledge on COL2 posttranslational modifications with relevance to RA pathophysiology, diagnosis and treatment. The significance of COL2 PTMs as a source of neo-antigens that activate immunity leading to or sustaining RA autoimmunity is discussed.

NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

It has been shown that synovial fibroblasts (SF) play a key role in the initiation of inflammation and joint destruction, leading to arthritis progression. Fibroblasts may express major histocompatibility complex class II region (MHCII) molecules, and thus, they could be able to process and present antigens to immunocompetent cells. Here we examine whether different types of fibroblasts (synovial, dermal, and thymic murine fibroblasts, destructive LS48 fibroblasts, and noninvasive NIH/3T3 fibroblasts) may be involved in the initiation of rheumatoid arthritis (RA) pathogenesis and can process and present type II collagen (COL2)-an autoantigen associated with RA. Using a panel of MHCII/Aq-restricted T-cell hybridoma lines that specifically recognize an immunodominant COL2 epitope (COL2259-273), we found that NIH/3T3 fibroblasts activate several T-cell clones that recognize the posttranslationally glycosylated or hydroxylated COL2259-273 epitope. The HCQ.3 hybridoma, which is specific for the glycosylated immunodominant COL2 epitope 259-273 (Gal264), showed the strongest response. Interestingly, NIH/3T3 cells, but not destructive LS48 fibroblasts, synovial, dermal, or thymic fibroblasts, were able to stimulate the HCQ.3 hybridoma and other COL2-specific T-cell hybridomas. Our experiments revealed that NIH/3T3 fibroblasts are able to activate COL2-specific T-cell hybridomas even in the absence of COL2 or a posttranslationally modified COL2 peptide. The mechanism of this unusual activation is contact-dependent and involves the T-cell receptor (TCR) complex.

Changes in the Subchondral Bone, Visfatin, and Cartilage Biomarkers after Pharmacological Treatment of Experimental Osteoarthritis with Metformin and Alendronate.

Subchondral bone that has intense communication with the articular cartilage might be a potential target for pharmacological treatment in the early stages of osteoarthritis (OA). Considering the emerging data about the role of adipokines in the pathogenesis of OA, the administration of drugs that influence their level is also intriguing. Metformin and alendronate were administered in mice with collagenase-induced OA (CIOA) as a monotherapy and in combination. Safranin O staining was used for the assessment of changes in subchondral bone and articular cartilage. Before and after treatment, serum levels of visfatin and biomarkers of cartilage turnover (CTX-II, MMP-13, and COMP) were assessed. In the current study, the combined administration of alendronate and metformin in mice with CIOA led to the protection against cartilage and subchondral bone damage. In mice with CIOA, metformin led to a decrease in visfatin level. In addition, treatment with metformin, alendronate, or their combination lowered the level of cartilage biomarkers (CTX-II and COMP), while the level of MMP-13 was not influenced. In conclusion, personalized combination treatment in OA according to clinical phenotype, especially in the early stages of the disease, might lead to the identification of a successful disease-modifying therapeutic protocol in OA.

Anticitrullinated antibodies recognize rheumatoid arthritis associated T-cell epitopes modified by bacterial L-asparaginase.

Citrullinated proteins and anti-citrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). It has been suggested that during inflammation or dysbiosis, bacteria could initiate production of ACPAs. Most patients with RA are seropositive for ACPAs, but these antibodies have overlapping reactivity to different posttranslational modifications (PTMs). For initiation and development of RA, T lymphocytes and T cell epitopes are still required. In this study, we evaluated the ability of bacterial L-asparaginase to modify RA-related T cell epitopes within type II collagen (CII259-273 and CII311-325), as well as whether these modified epitopes are recognized by ACPAs from RA patients. We included 12 patients with early RA and 11 healthy subjects selected according to predefined specific criteria. LC-MS/MS analyses revealed that the bacterial L-asparaginase can modify investigated T cell epitopes. ELISA tests showed cross-reactivity of ACPA positive sera from early RA patients towards the enzymatically modified immunodominant T cell epitopes within type II collagen (CII), but not to the modified irrelevant peptides. These data suggest that the cross-reactive ACPAs recognize the "carbonyl-Gly-Pro" motif in CII. Moreover, the T cell recognition of the modified major immunodominant T cell epitope Gal264-CII259-273 was not affected. This epitope was still able to activate autoreactive T cells from early RA patients. It is likely that such modifications are the missing link between the T cell priming and the development of anti-modified protein antibodies (AMPAs). Our results provide additional information on the etiology and pathogenesis of RA.

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-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-γ) 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 ± 0.23 vs. 4.82 ± 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-γ 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.

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.

Prebiotics can change immunomodulatory properties of probiotics.

Beneficial effects of probiotics and prebiotics are mainly related to modulation of compositions and activities of gut microbiota as well as manipulation of immunological reactivity in autoimmune diseases. In the present study, we examined whether metabolic products from different strains of Lactobacillus brevis cultured with different prebiotics have similar immunomodulating properties on immune cells under normal and inflammatory conditions, using mouse model of collagen-induced arthritis (CIA). Two strains of Lactobacillus brevis (3448 and 8429) were cultured with four different prebiotics, such as xylooligosaccharides, inulin, pectin, and chitosan. Sterile supernatants containing different metabolic products have been used for direct treatment of cell cultures prepared from CII-immunized mice and non-immunized (control mice). Our results showed that metabolic products from XOS decreased levels of IFN-γ, IL-6, IL-17, and TNF-α in both cultures from immunized and non-immunized mice. In contrast, metabolic products from inulin, pectin, and chitosan increased concentrations of these cytokines with highest values for pectin. Neither of investigated prebiotics influenced the secretion of IL-10. In addition, we found changes in the percentage of macrophages, which were different for the tested prebiotics. Also, metabolic products from pectin and chitosan caused loss of T-cells (CD3+) and increased percentages of CD4+CD25+ regulatory T cells and CD8+CD279+ anergic T cells. Hence, our data indicate that immunomodulating properties of probiotics are strain-specific and prebiotic-dependent.

Ragweed-allergic subjects have decreased serum levels of chemokines CCL2, CCL3, CCL4 and CCL5 out of the pollen season.

CC-chemokines are important mediators of the allergic responses and regulate the cell trafficking. The aim of this study was to examine the serum levels of CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß and CCL5/RANTES, and to determine whether there are differences between ragweed-allergic subjects and healthy individuals out of the pollen season. Peripheral blood samples were collected from 24 subjects allergic to ragweed pollen and 12 healthy controls. Serum concentrations of chemokines/cytokines were measured by an enzyme-linked immunosorbent assay. We observed significantly decreased concentrations of CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß and CCL5/RANTES in the sera of ragweed-allergic patients compared to the healthy individuals (32.2 vs. 106.4 pg/ml, 89.5 vs. 135.7 pg/ml, 63.4 vs. 119.2 pg/ml and 11.2 vs. 18.1 ng/ml, respectively, p < 0.01). In contrast to the CC-chemokines, the serum levels of IL-8/CXCL8 showed a significant increase (p < 0.05) in the allergic group compared to the non-allergic subjects. Interleukin 4 levels were similar in both groups. In the sera of allergic patients, we have also detected significantly elevated levels of ragweed-specific IgE and IgG. However, decreased serum concentrations of the four CC-chemokines and elevated levels of IL-8/CXCL8 can be used as biomarkers for more accurate evaluation of the allergic status of patients with pollen allergy out of the season, to study the mechanisms for activation/inhibition of the subclinical allergic responses and for development of therapeutic strategies.

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.

Allelopathic Potential of the Cyanotoxins Microcystin-LR and Cylindrospermopsin on Green Algae.

Allelopathic interactions are widespread in all aquatic habitats, among all groups of aquatic primary biomass producers, including cyanobacteria. Cyanobacteria are producers of potent toxins called cyanotoxins, whose biological and ecological roles, including their allelopathic influence, are still incompletely understood. The allelopathic potential of the cyanotoxins microcystin-LR (MC-LR) and cylindrospermopsin (CYL) on green algae (Chlamydomonas asymmetrica, Dunaliella salina, and Scenedesmus obtusiusculus) was established. Time-dependent inhibitory effects on the growth and motility of the green algae exposed to cyanotoxins were detected. Changes in their morphology (cell shape, granulation of the cytoplasm, and loss of flagella) were also observed. The cyanotoxins MC-LR and CYL were found to affect photosynthesis to varying degrees in the green algae Chlamydomonas asymmetrica, Dunaliella salina, and Scenedesmus obtusiusculus, affecting chlorophyll fluorescence parameters such as the maximum photochemical activity (Fv/Fm) of photosystem II (PSII), the non-photochemical quenching of chlorophyll fluorescence (NPQ), and the quantum yield of the unregulated energy dissipation Y(NO) in PSII. In the context of ongoing climate change and the associated expectations of the increased frequency of cyanobacterial blooms and released cyanotoxins, our results demonstrated the possible allelopathic role of cyanotoxins on competing autotrophs in the phytoplankton communities.

In Silico Design of a New Epitope-Based Vaccine against Grass Group 1 Allergens.

Allergic diseases are a global public health problem that affects up to 30% of the population in industrialized societies. More than 40% of allergic patients suffer from grass pollen allergy. Grass pollen allergens of group 1 and group 5 are the major allergens, since they induce allergic reactions in patients at high rates. In this study, we used immunoinformatic approaches to design an effective epitope-based vaccine against the grass group 1 allergens. After the alignment of all known pollen T-cell and B-cell epitopes from pollen allergens available in the public databases, the epitope GTKSEVEDVIPEGWKADTSY was identified as the most suitable for further analyses. The target sequence was subjected to immunoinformatics analyses to predict antigenic T-cell and B-cell epitopes. Population coverage analysis was performed for CD8+ and CD4+ T-cell epitopes. The selected T-cell epitopes (VEDVIPEGW and TKSEVEDVIPEGWKA) covered 78.87% and 98.20% of the global population and 84.57% and 99.86% of the population of Europe. Selected CD8+, CD4+ T-cell and B-cell epitopes have been validated by molecular docking analysis. CD8+ and CD4+ T-cell epitopes showed a very strong binding affinity to major histocompatibility complex (MHC) class I (MHC I) molecules and MHC class II (MHC II) molecules with global energy scores of -72.1 kcal/mol and -89.59 kcal/mol, respectively. The human IgE-Fc (PDB ID 4J4P) showed a lower affinity with B-cell epitope (ΔG = -34.4 kcal/mol), while the Phl p 2-specific human IgE Fab (PDB ID 2VXQ) had the lowest binding with the B-cell epitope (ΔG = -29.9 kcal/mol). Our immunoinformatics results demonstrated that the peptide GTKSEVEDVIPEGWKADTSY could stimulate the immune system and we performed ex vivo tests showed that the investigated epitope activates T cells isolated from patients with grass pollen allergy, but it is not recognized by IgE antibodies specific for grass pollen allergens. This confirms the importance of such studies to establish universal epitopes to serve as a basis for developing an effective vaccine against a particular group of allergens. Further in vivo studies are needed to validate the effectiveness of such a vaccine against grass pollen allergens.

Polyphasic characterisation of Microcoleusautumnalis (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, Phormidiumautumnale (Agardh) Trevisan ex Gomont, 1892 has been transferred to the genus Microcoleus as Microcoleusautumnalis (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 Microcoleusautumnalis by applying the classical approach of polyphasic taxonomy and additionally using metabolomics. Cyanobacterial strains identified as Phormidiumautumnale and Microcoleusvaginatus (type species of the genus Microcoleus) were used for comparative analyses. In addition, the taxonomic relationship between the species Phormidiumautumnale and Phormidiumuncinatum was determined on the basis of polyphasic characteristics. Monitoring of the morphological variability of Phormidiumautumnale and Microcoleusvaginatus 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 Microcoleusautumnalis by restoring its previous appurtenance to the genus Phormidium under the name Phormidiumautumnale (Agardh) Trevisan ex Gomont, 1892 and distinguishing this species from genus Microcoleus.

Evolution of Gold and Iron Oxide Nanoparticles in Conjugates with Methotrexate: Synthesis and Anticancer Effects.

Au and Fe nanoparticles and their conjugates with the drug methotrexate were obtained by an environmentally safe method of metal-vapor synthesis (MVS). The materials were characterized by transmission and scanning electron microscopy (TEM, SEM), X-ray photoelectron spectroscopy (XPS), and small-angle X-ray scattering using synchrotron radiation (SAXS). The use of acetone as an organic reagent in the MVS makes it possible to obtain Au and Fe particles with an average size of 8.3 and 1.8 nm, respectively, which was established by TEM. It was found that Au, both in the NPs and the composite with methotrexate, was in the Au0, Au+ and Au3+ states. The Au 4f spectra for Au-containing systems are very close. The effect of methotrexate was manifested in a slight decrease in the proportion of the Au0 state-from 0.81 to 0.76. In the Fe NPs, the main state is the Fe3+ state, and the Fe2+ state is also present in a small amount. The analysis of samples by SAXS registered highly heterogeneous populations of metal nanoparticles coexisting with a wide proportion of large aggregates, the number of which increased significantly in the presence of methotrexate. For Au conjugates with methotrexate, a very wide asymmetric fraction with sizes up to 60 nm and a maximum of ~4 nm has been registered. In the case of Fe, the main fraction consists of particles with a radius of 4.6 nm. The main fraction consists of aggregates up to 10 nm. The size of the aggregates varies in the range of 20-50 nm. In the presence of methotrexate, the number of aggregates increases. The cytotoxicity and anticancer activity of the obtained nanomaterials were determined by MTT and NR assays. Fe conjugates with methotrexate showed the highest toxicity against the lung adenocarcinoma cell line and Au nanoparticles loaded with methotrexate affected the human colon adenocarcinoma cell line. Both conjugates displayed lysosome-specific toxicity against the A549 cancer cell line after 120 h of culture. The obtained materials may be promising for the creation of improved agents for cancer treatment.

Mice producing less reactive oxygen species are relatively resistant to collagen glycopeptide vaccination against arthritis.

The bottleneck for the induction of collagen-induced arthritis in mice is the recognition of immunodominant type II collagen (CII) peptide (CII259-273) bound to the MHC class II molecule A(q). We have shown previously that the posttranslationally glycosylated lysine at position 264 in this epitope is of great importance for T cell recognition and tolerance induction to CII as well as for arthritis development. The Ncf1 gene, controlling oxidative burst, has been shown to play an important role for immune tolerance to CII. To investigate the effect of oxidation on the efficiency of immune-specific vaccination with MHC class II/glycosylated-CII peptide complexes, we used Ncf1 mutated mice. We demonstrate that normal reactive oxygen species (ROS) levels contribute to the establishment of tolerance and arthritis protection, because only mice with a functional oxidative burst were completely protected from arthritis after administration of the glycosylated CII259-273 peptide in complex with MHC class II. Transfer of T cells from vaccinated mice with functional Ncf1 protein resulted in strong suppression of clinical signs of arthritis in B10.Q mice, whereas the Ncf1 mutated mice as recipients had a weaker suppressive effect, suggesting that ROS modified the secondary rather than the primary immune response. A milder but still significant effect was also observed in ROS deficient mice. During the primary vaccination response, regulatory T cells, upregulation of negative costimulatory molecules, and increased production of anti-inflammatory versus proinflammatory cytokines in both Ncf1 mutated and wild type B10.Q mice was observed, which could explain the vaccination effect independent of ROS.

Design of an Epitope-Based Peptide Vaccine Against the Major Allergen Amb a 11 Using Immunoinformatic Approaches.

Allergic diseases are a socially significant problem of global importance. The number of people suffering from pollen allergies has increased dramatically in recent decades. Pollen allergies affect up to 30% of the world population. Pollen of the common ragweed (Ambrosia artemisiifolia L.) is one of the most aggressive allergens in the world. We have used a series of immunoinformatics approaches to design an effective epitope-based vaccine, which might induce a competent immunity against a major allergen Amb a 11. CD8+ and CD4+ T-cell epitopes and their corresponding MHC restricted alleles were identified by prediction tools provided by immune epitope database (IEDB). Among T-cell epitopes, MHC class I peptide (GLMEPAFTYV) and MHC class II peptide (LVCFSFSLVLILGLV) were identified as most suitable. From all predicted B-cell epitopes, only one epitope (GKLVKFSEQQLVDC) containing sequence from the conserved region was chosen for next processing. Selected epitopes have been validated by molecular docking analysis. These epitopes showed a very strong binding affinity to MHC I molecule and MHC II molecule with binding energy scores - 729.3 and - 725.0 kcal/mole respectively. Performed experimental validation showed that only the MHC class II peptide (LVCFSFSLVLILGLV) can stimulate T cells from ragweed allergic patients and IgE antibodies specific to the ragweed pollen do not recognize this epitope. Therefore, this peptide could be potentially used as a vaccine against the major allergen Amb a 11. The B-cell epitope GKLVKFSEQQLVDC forms a stable complex with the IgE molecule (energy weighted score - 695,0 kcal/mole). Tested sera from patients with ragweed allergy showed that the ragweed specific IgE antibodies can bind to the identified B-cell epitope. Population coverage analysis was performed for CD8+ and CD4+ T-cell epitopes. It was predicted that CD4+ T-cell epitope (LVCFSFSLVLILGLV) covers 90.56% of the population of Europe and 99.36% of the world population. CD8+ T-cell epitope (GLMEPAFTYV) has a population coverage of 77.37% for Europe and 71.35% for all the world.

Antitumor and Immunomodulatory Properties of the Bulgarian Endemic Plant Betonica bulgarica Degen et Neic. (Lamiaceae).

Background: Extracts obtained from different Betonica species have been shown to possess important biological properties. The present study aimed to investigate the cytotoxicity, antitumor and immunomodulatory potential of the endemic plant Betonica bulgarica (Lamiaceae) and thus, reveal new aspects of its biological activity. Methods: Methanolic extract obtained from inflorescences was analyzed for cytotoxicity against mammalian cell lines. The antitumor potential of the sample was determined using human cervical and lung adenocarcinoma cells (HeLa and A549). Programmed cell death-inducing effects against HeLa cells and peripheral blood lymphocytes, as well as immunomodulatory properties of the extract were determined by flow cytometry analysis. Results: The research results demonstrated that the extract has significant inhibitory potential against HeLa cells (mean IC50 value 119.2 µg/mL). The sample selectively induced apoptotic death in tumor cells. Cytotoxic effects towards mouse cell lines were detected following treatment with high concentrations of Betonica bulgarica extract (200 and 250 µg/mL). Twenty-four-hour ex vivo incubation of peripheral blood leucocytes in growth medium containing plant extract induced prominent effects in distinct immune cell populations. They included elevated levels of CD25+ and CD56+ T cells' lymphocytes, particularly CD4+CD25+ and CD8+CD56+ cells. Conclusions: The present study demonstrates that Betonica bulgarica inflorescence extract possesses potential beneficial antitumor and immunomodulatory activity and could serve as a source of bioactive compounds with biomedical application.

Structural characterization of polysaccharides from Geranium sanguineum L. and their immunomodulatory effects in response to inflammatory agents.

ETHNOPHARMACOLOGICAL RELEVANCE: Geranium sanguineum L. is used for treatment of inflammations, anemia, malignant diseases of the blood-forming organs, diarrhea, respiratory infections, etc. Only flavonoids in root extracts have been elucidated as immunostimulating and anti-inflammatory compounds, and polysaccharides in the herb have not been examined. AIM OF THE STUDY: to compare the chemical features of polysaccharide complexes (PSCs) from leaves (GSL-PSC) and roots (GSR-PSC) of G. sanguineum, as well as their immunomodulatory activities on leukocytes after inflammation, and effects on the growth of different bacteria. MATERIALS AND METHODS: The samples were isolated by water extraction and their structural features were studied by 2D NMR spectroscopy. The stimulatory effects of both PSCs on human leukocytes were analyzed with flow cytometry. Their suppressive activities on the oxidative burst in blood and derived neutrophils against opsonized zymosan and phorbol myristate acetate were investigated. The effects of the samples on viability, NO and interleukin 6 (IL-6) syntheses in RAW264.7 cells after inflammation with lipopolysaccharides (LPS) were tested. The prebiotic and anti-biofilm activities of the PSCs were evaluated. RESULTS: The total carbohydrate content in the samples was significant (73.6-76.8%). GSL-PSC contained pectins, which were rich in homogalacturonan (HG), and smaller amounts of rhamnogalacturonan (RG) type I, decorated by 1,5-α-L-Araf, 1,4- and 1,6-ß-D-Galp chains. GSR-PSC contained starch, followed by pectins with lower HG content and more RG-I regions, substituted by 1 â†’ 3,5-α-L-arabinans and 1 â†’ 3,6-ß-D-galactans. GSL-PSC and GSR-PSC (200 µg/mL) increased monocyte and granulocyte cell counts, but GSR-PSC also elevated T helper and B cell levels in a normal and activated state. GSR-PSC triggered a dose-dependent (50-200 µg/mL) oxidative burst in blood, but alleviated it after inflammation even in blood-derived neutrophils. It was free of LPS, and activated NO and IL-6 productions in RAW264.7 cells better than GSL-PSC, without affecting their viability. Both PSCs (2.0%, w/v) stimulated probiotic co-cultures between Clostridium beijerinckii strains and Lactobacillus sp. ZK9, and inhibited the growth and biofilm formation of Escherichia coli, Streptococcus mutans and Salmonella enterica. CONCLUSIONS: The PSs in G. sanguineum could be involved in the stimulatory effects on blood-forming organs and anti-inflammatory action of aqueous root extracts in case of infections. These PSs should be included in synbiotic foods to support the treatment of inflammations and infections in the gut.

Structural Features and Immunomodulatory Effects of Water-Extractable Polysaccharides from Macrolepiota procera (Scop.) Singer.

Macrolepiota procera (MP) is an edible mushroom used in the treatment of diabetes, hypertension and inflammation. However, the structure and biological effects of its polysaccharides (PSs) are unclear. This study investigates the structural features of a PS complex from MP (MP-PSC), its immunomodulatory activities and effects on probiotic and pathogenic bacteria. MP-PSC was obtained by boiling water, and PSs were characterized by 2D NMR spectroscopy. The immunomodulatory effects on blood and derived neutrophils, other leukocytes, and murine macrophages were studied by flow cytometry, chemiluminescence, spectrophotometry, and ELISA. The total carbohydrate content of MP-PSC was 74.2%, with glycogen occupying 36.7%, followed by ß-D-glucan, α-L-fuco-2-(1,6)-D-galactan, and ß-D-glucomannan. MP-PSC (200 µg/mL) increased the number of CD14+ monocyte cells in the blood, after ex vivo incubation for 24 h. It dose-dependently (50-200 µg/mL) activated the spontaneous oxidative burst of whole blood phagocytes, NO, and interleukin 6 productions in RAW264.7 cells. MP-PSC exhibited a low antioxidant activity and failed to suppress the oxidative burst and NO generation, induced by inflammatory agents. It (2.0%, w/v) stimulated probiotic co-cultures and hindered the growth and biofilm development of Escherichia coli, Streptococcus mutans and Salmonella enterica. MP PSs can be included in synbiotics to test their immunostimulating effects on compromised immune systems and gut health.

(E)-alkene and ethylene isosteres substantially alter the hydrogen-bonding network in class II MHC A(q)/glycopeptide complexes and affect T-cell recognition.

The structural basis for antigen presentation by class II major histocompatibility complex (MHC) proteins to CD4(+) T-cells is important for understanding and possibly treating autoimmune diseases. In the work described in this paper, (E)-alkene and ethylene amide-bond isosteres were used to investigate the effect of removing hydrogen-bonding possibilities from the CII259-270 glycopeptide, which is bound by the arthritis-associated murine A(q) class II MHC protein. The isostere-modified glycopeptides showed varying and unexpectedly large losses of A(q) binding that could be linked to the dynamics of the system. Molecular dynamics (MD) simulations revealed that the backbone of CII259-270 and the A(q) protein are able to form up to 11 hydrogen bonds, but fewer than this number are present at any one time. Most of the strong hydrogen-bond interactions were formed by the N-terminal part of the glycopeptide, i.e., in the region where the isosteric replacements were made. The structural dynamics also revealed that hydrogen bonds were strongly coupled to each other; the loss of one hydrogen-bond interaction had a profound effect on the entire hydrogen-bonding network. The A(q) binding data revealed that an ethylene isostere glycopeptide unexpectedly bound more strongly to A(q) than the corresponding (E)-alkene, which is in contrast to the trend observed for the other isosteres. Analysis of the MD trajectories revealed that the complex conformation of this ethylene isostere was structurally different and had an altered molecular interaction pattern compared to the other A(q)/glycopeptide complexes. The introduced amide-bond isosteres also affected the interactions of the glycopeptide/A(q) complexes with T-cell receptors. The dynamic variation of the patterns and strengths of the hydrogen-bond interactions in the class II MHC system is of critical importance for the class II MHC/peptide/TCR signaling system.