Balik Dzhambazov

A novel probiotic mixture exerts a therapeutic effect on experimental autoimmune encephalomyelitis mediated by IL-10 producing regulatory T cells.

Background Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Methodology/Principal Findings The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4+CD25+Foxp3+ regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-β1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4+CD25+ Tregs are involved in the suppressive effect induced by the lactobacilli mixture. Conclusions/Significance Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.

The major T cell epitope on type II collagen is glycosylated in normal cartilage but modified by arthritis in both rats and humans

Type II collagen (CII) is a target for autoreactive T cells in both rheumatoid arthritis and the murine model collagen‐induced arthritis. The determinant core of CII has been identified as CII260–270, and the alteration of this T cell epitope by posttranslational modifications is known to be critical for development of arthritis in mice. Using CII‐specific T cell hybridomas we have now shown that the immunodominant T cell epitope in the normal (healthy) human and rat joint cartilage is O‐glycosylated at the critical T cell receptor recognition position 264 with a mono‐ or di‐saccharide attached to a hydroxylysine. In contrast, in the arthritic human and rat joint cartilage there are both glycosylated and non‐glycosylated CII forms. Glycosylated CII from normal cartilage could not be recognized by T cells reactive to peptides having only lysine or hydroxylysine at position 264, showing that antigen‐presenting cells could not degrade the O‐linked carbohydrate. Thus, the variable forms of the glycosylated epitope are determined by the structures present in cartilage, and these vary during the disease course. We conclude that the chondrocyte determines the structures presented to the immune system and that these structures are different in normal versus arthritic states.

Therapeutic Vaccination of Active Arthritis with a Glycosylated Collagen Type II Peptide in Complex with MHC Class II Molecules

In both collagen-induced arthritis (CIA) and rheumatoid arthritis, T cells recognize a galactosylated peptide from type II collagen (CII). In this study, we demonstrate that the CII259–273 peptide, galactosylated at lysine 264, in complex with Aq molecules prevented development of CIA in mice and ameliorated chronic relapsing disease. In contrast, nonglycosylated CII259–273/Aq complexes had no such effect. CIA dependent on other MHC class II molecules (Ar/Er) was also down-regulated, indicating a bystander vaccination effect. T cells could transfer the amelioration of CIA, showing that the protection is an active process. Thus, a complex between MHC class II molecules and a posttranslationally modified peptide offers a new possibility for treatment of chronically active autoimmune inflammation such as rheumatoid arthritis.

Molecular and phylogenetic characterization of Phormidium species (Cyanoprokaryota) using the cpcB-IGS-cpcA locus

The accurate determination of species of Cyanoprokaryota/Cyanophyceae has many important applications. These include the assessment of risk with regard to blooms in water reservoirs as well as the identification of species capable of producing valuable bioactive compounds. Commonly, Cyanoprokaryota are classified based on their morphology. However, morphological criteria are not always reliable because they may change, for example, due to environmental factors. Thus, genetic and molecular analyses are a promising additional approach, but their application has so far been limited to relatively few genera. In light of this, we present here the first characterization of species and strains of the genus Phormidium Kütz. based on the cpcB‐IGS‐cpcA locus of the phycocyanin operon. In phylogenetic analyses using deduced amino acid sequences of the cpcB‐cpcA regions, Phormidium was found to be polyphyletic. This analysis appeared to be dominated by the cpcB region, which is characterized by a relatively high percentage of informative substitutions. The percentage of variable positions within the cpcB‐IGS‐cpcA locus overall was 16.5%, thereby indicating a level of divergence remarkably higher than that reported for Nodularia and Arthrospira in previous studies relying on cpcB‐IGS‐cpcA. Further, alignment of informative nucleotide substitutions in the cpcB‐IGS‐cpcA sequences revealed a mosaic distribution, which may be indicative of genetic recombination events. Finally, the length and sequences of the IGS region alone proved useful as markers to differentiate the cyanobacterial genus Phormidium. However, whether the IGS region per se is sufficiently discriminatory to differentiate between Phormidium species or even strains requires further investigation using newly identified Phormidium sequence data.

Breaking T Cell Tolerance Against Self Type II Collagen in HLA-DR4-Transgenic Mice and Development of Autoimmune Arthritis

OBJECTIVE To establish a new animal model in DRB1*0401 (DR4)-transgenic mice in which T cell tolerance to self type II collagen (CII) can be broken and allow for the development of autoimmune arthritis, to investigate the role of posttranslational modifications of the CII(259-273) epitope in the induction and breaking of tolerance of DR4-restricted T cells, and to characterize DR4-restricted T cell recognition of the immunodominant CII(259-273) epitope. METHODS DR4-transgenic mice expressing either the entire human CII protein (HuCII) or only the immunodominant T cell epitope of heterologous CII (MMC) in joint cartilage were established on different genetic backgrounds, and susceptibility to collagen-induced arthritis (CIA) was tested. RESULTS HuCII mice displayed stronger T cell tolerance to heterologous CII than did MMC mice. On the B10 background, arthritis developed only in MMC mice with a defective oxidative burst. However, MMC mice on the C3H background were susceptible to arthritis also with a functional oxidative burst. Significant recall responses in tolerized mice were detected only against the nonglycosylated CII(259-273) epitope. Recognition of the CII(259-273) epitope was heterogeneous, but the majority of T cells in DR4 mice specifically recognized the nonglycosylated side chain of lysine at position 264. CONCLUSION It is possible to break tolerance to self CII and induce arthritis in DR4 mice. However, arthritis susceptibility is tightly controlled by the genetic background and by the source of the transgenic element for expressing the heterologous CII peptide as a self CII protein in the joint. In contrast to CIA in A(q)-expressing mice, the nonglycosylated CII(259-273) epitope is clearly immunodominant in both tolerized and nontolerized DR4 mice.

Oxazole-modified glycopeptides that target arthritis-associated class II MHC Aq and DR4 proteins

The glycopeptide CII259-273, a fragment from type II collagen (CII), can induce tolerance in mice susceptible to collagen-induced arthritis (CIA), which is a validated disease model for rheumatoid arthritis (RA). Here, we describe the design and synthesis of a small series of modified CII259-273 glycopeptides with oxazole heterocycles replacing three potentially labile peptide bonds. These glycopeptidomimetics were evaluated for binding to murine CIA-associated A(q) and human RA-associated DR4 class II major histocompatibility complex (MHC) proteins. The oxazole modifications drastically reduced or completely abolished binding to A(q). Two of the glycopeptidomimetics were, however, well tolerated in binding to DR4 and they also induced strong responses by one or two DR4-restricted T-cell hybridomas. This work contributes to the development of an altered glycopeptide for inducing immunological tolerance in CIA, with the long-term goal of developing a therapeutic vaccine for treatment of RA.

Design of Glycopeptides Used to Investigate Class II MHC Binding and T-Cell Responses Associated with Autoimmune Arthritis

The glycopeptide fragment CII259–273 from type II collagen (CII) binds to the murine Aq and human DR4 class II Major Histocompatibility Complex (MHC II) proteins, which are associated with development of murine collagen-induced arthritis (CIA) and rheumatoid arthritis (RA), respectively. It has been shown that CII259–273 can be used in therapeutic vaccination of CIA. This glycopeptide also elicits responses from T-cells obtained from RA patients, which indicates that it has an important role in RA as well. We now present a methodology for studies of (glyco)peptide-receptor interactions based on a combination of structure-based virtual screening, ligand-based statistical molecular design and biological evaluations. This methodology included the design of a CII259–273 glycopeptide library in which two anchor positions crucial for binding in pockets of Aq and DR4 were varied. Synthesis and biological evaluation of the designed glycopeptides provided novel structure-activity relationship (SAR) understanding of binding to Aq and DR4. Glycopeptides that retained high affinities for these MHC II proteins and induced strong responses in panels of T-cell hybridomas were also identified. An analysis of all the responses revealed groups of glycopeptides with different response patterns that are of high interest for vaccination studies in CIA. Moreover, the SAR understanding obtained in this study provides a platform for the design of second-generation glycopeptides with tuned MHC affinities and T-cell responses.

Monoclonal antibody against T-cell receptor alphabeta induces self-tolerance in chronic experimental autoimmune encephalomyelitis.

The therapeutic effect of monoclonal antibody (H57‐597 MoAb) against T‐cell receptor (TCR) αβ has been investigated on MOG35−55‐induced experimental autoimmune encephalomyelitis (EAE), as a model system for T‐cell‐mediated chronic inflammation in the central nervous system (CNS). Short‐term administration of the anti‐TCR αβ immediately after immunization protected the mice from EAE. Furthermore, anti‐TCR αβ treatment on an established disease restored the self‐tolerance which led to a complete remission of EAE and a dramatic reduction of inflammatory cells in the CNS, while treatment with control antibody (hamster IgG) was ineffective. The remission was durable and not associated with disappearance of autoreactive T cells as measured by persistence of MOG‐reactive T‐cell proliferation in vitro. However, MOG‐reactive T cells from anti‐TCR‐treated animals produced significantly lower amounts of inflammatory TNF‐α and IFN‐γ. In addition, while a transient deletion of CD4+ and CD8+ T cells was observed, a population of T cells expressing CD3, NK1.1 and CD69 (NKT cells) were expanding. By transfer of spleen cells from anti‐TCR MoAb‐treated animals, we could show that the tolerogenic capacity can be transferred to untreated recipients with EAE. The data indicate therapeutic effect of anti‐TCR αβ MoAb (H57‐597), which represents a promising approach in treatment of T‐cell‐mediated autoimmune diseases.

New furostanol saponins from Smilax aspera L. and their in vitro cytotoxicity

The occurrence of the two new cis-fused A/B rings furostanol saponins (25S)-26-O-β-D-glucopyranosyl-5β-furostan-1β,3β,22α,26-tetraol-1-O-β-D-glucopyranoside and (25S)-26-O-β-D-glucopyranosyl-5β-furostan-1β,2β,3β,5β,22α,26-hexaol and the known compounds (25S)-26-O-β-D-glucopyranosyl-5β-furostan-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1 → 2)-O-β-D-glucopyranosyl-(1 → 2)-O-β-D-glucopyranoside and (25S)-26-O-β-D-glucopyranosyl-5β-furostan-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1 → 2)-O-β-D-glucopyranoside, trans-resveratrol, (+) catechin and (-) epicatechin in the rhizomes of Smilax aspera is reported. All saponins have been isolated as their 22-OMe derivatives, which were further subjected to extensive spectroscopic analysis. The isolated furostanol saponins were evaluated for cytotoxic activity against human normal amniotic and human lung carcinoma cell lines using neutral red and MTT assays. In vitro experiments showed significant cytotoxicity in a dose dependent manner with IC(50) values in the range of 32.98-94.53 µM.

Phytoplankton community of the drinking water supply reservoir Borovitsa (South Bulgaria) with an emphasis on cyanotoxins and water quality.

The phytoplankton diversity, algal biomass, and selected physicochemical parameters were investigated in the drinking water reservoir (Borovitsa) located in the Kardzhali region, Bulgaria. Particular attention was given to Cyanoprokaryota and presence of cyanotoxins in the water samples. Twenty-nine species belonging to six divisions (Cyanoprokaryota, Chlorophyta, Zygnemophyta, Dinophyta, Euglenophyta and Bacillariophyta) were identified. The microscopic examination of the phytoplankton samples showed the dominance of Ankyra judayi, Oocystis lacustris (Chlorophyta) and Aphanizomenon flos-aquae (Cyanoprokaryota) in July 2006, and Microcystis pulverea, Synechococcus elongatus (Cyanoprokaryota), Radiococcus planktonicus (Chlorophyta) and Melosira varians (Bacillariophyta) in September 2006. A blooming event due to Aphanizomenon flos-aquae was observed in July 2006. The reservoir exhibits a tendency to shift from an oligotrophic environment to a state of mesotrophy. Presence of cyanotoxins such as anatoxin-a, microcystins and saxitoxins were analyzed by HPLC and ELISA methods. Our results demonstrated the presence of anatoxin-a and microcystins (0.09 µg/L) in the raw water samples from July 2006, and saxitoxins (2.5 µg/L) and microcystins (0.18 µg/L) in the raw water samples from September 2006. The study underlines that permanent monitoring programs of Cyanoprokaryota in the reservoirs used as sources of drinking water and toxicity assessments should be implemented. Indirect exposure and transfer of cyanotoxins through food chains must also be considered.