Visualization and phenotyping of proinflammatory antigen-specific T cells during collagen-induced arthritis in a mouse with a fixed collagen type II-specific transgenic T-cell receptor beta-chain

Introduction: The Vbeta12-transgenic mouse was previously generated to investigate the role of antigen-specific T cells in collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. This mouse expresses a transgenic collagen type II (CII)-specific T-cell receptor (TCR) beta-chain and consequently displays an increased immunity to CII and increased susceptibility to CIA. However, while the transgenic Vbeta12 chain recombines with endogenous alpha-chains, the frequency and distribution of CII-specific T cells in the Vbeta12-transgenic mouse has not been determined. The aim of the present report was to establish a system enabling identification of CII-specific T cells in the Vbeta12-transgenic mouse in order to determine to what extent the transgenic expression of the CII-specific beta-chain would skew the response towards the immunodominant galactosylated T-cell epitope and to use this system to monitor these cells throughout development of CIA. Methods: We have generated and thoroughly characterized a clonotypic antibody, which recognizes a TCR specific for the galactosylated CII(260-270) peptide in the Vbeta12-transgenic mouse. Hereby, CII-specific T cells could be quantified and followed throughout development of CIA, and their phenotype was determined by combinatorial analysis with the early activation marker CD154 (CD40L) and production of cytokines. Results: The Vbeta12-transgenic mouse expresses several related but distinct T-cell clones specific for the galactosylated CII peptide. The clonotypic antibody could specifically recognize the majority (80%) of these. Clonotypic T cells occurred at low levels in the naïve mouse, but rapidly expanded to around 4% of the CD4+ T cells, whereupon the frequency declined with developing disease. Analysis of the cytokine profile revealed an early Th1-biased response in the draining lymph nodes that would shift to also include Th17 around the onset of arthritis. Data showed that Th1 and Th17 constitute a minority among the CII-specific population, however, indicating that additional subpopulations of antigen-specific T cells regulate the development of CIA. Conclusions: The established system enables the detection and detailed phenotyping of T cells specific for the galactosylated CII peptide and constitutes a powerful tool for analysis of the importance of these cells and their effector functions throughout the different phases of arthritis

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

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

Breaking T cell tolerance against self type II collagen in HLA-DR4-transgenic mice and development of autoimmune arthritis. Arthritis Rheum62:1911-1920

epitope is clearly immunodominant in both tolerized and nontolerized DR4 mice

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

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

Preparation and Preliminary Evaluation of Silver-Modified Anodic Alumina for Biomedical Applications

The present study reports a specific method for preparation of silver-modified anodic alumina substrates intended for biomaterial applications. Al2O3 coatings were obtained by anodization of technically pure aluminum alloy in sulfuric acid electrolyte. Silver deposition into the pores of the anodic structures was carried out employing in situ thermal reduction for different time periods. The obtained coatings were characterized using scanning electron microscopy (SEM), potentiodynamic scanning after 168 h in 3.5% NaCl solution and bioassays with human fibroblast and NIH/3T3 cell lines. The modified alumina substrates demonstrated better biocompatibility compared to the control anodic Al2O3 pads indicated by increased percent cell survival following in vitro culture with human and mouse fibroblasts. The Ag-deposition time did not affect considerably the biocompatibility of the investigated anodic layers. SEM analyses indicated that mouse NIH/3T3 cells and human fibroblasts adhere to the silver-coated alumina substrates retaining normal morphology and ability to form cell monolayer. Therefore, the present studies demonstrate that silver coating of anodic alumina substrates improves their biocompatibility and their eventual biomedical application

In vitro Digestion: Exploring the probiotic abilities and metabolization of human milk oligosaccharides by two strains of Limosilactobacillus fermentum isolated from breast milk

Human breast milk (HBM) serves as the most optimal nourishment for infants, not only providing essential nutrition but also boasting a rich array of immune components. These include secretory antibodies, immune cells, antimicrobial proteins (like lactoferrin and lysozyme), cytokines, and human milk oligosaccharides. Today, the presence of a specific microbiome in human milk is known. Our primary research involves the examination of certain probiotic properties displayed by two strains of Limosilactobacillus fermentum, which were isolated from breast milk. Furthermore, our goal was to evaluate their ability to metabolize breast milk-derived oligosaccharides through an in vitro digestion simulation system. The in vitro model simulating gastrointestinal digestion was performed according to INFOGEST method with some modification. We used various molecular techniques to isolate and identify strains from breast milk. Additionally, we employed different biochemical analyses to determine specific enzyme activities, and we also assessed the fundamental probiotic characteristics of two strains Limosilactobacillus fermentum. We found good probiotic characteristics in the examined strains, as well as favorable growth properties in the presence of specific human milk oligosaccharides. In this regard, we believe that breast milk represents a rich source for isolating potential probiotic strains