Zentralblatt für Bakteriologie. 25 ; Supplement
[1st ed.] 171 s. : obr., tab., grafy ; 25 cm
BACKGROUND: Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose--a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action. RESULTS: In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22-23-24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions--both of afore mentioned have strong effects on the saccharide preferences. CONCLUSION: Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.
- MeSH
- Adhesins, Bacterial genetics chemistry MeSH
- Chromatography, Affinity MeSH
- Financing, Organized MeSH
- Polymorphism, Single Nucleotide MeSH
- Protein Conformation MeSH
- Crystallography, X-Ray MeSH
- Lectins genetics chemistry MeSH
- Models, Molecular MeSH
- Monosaccharides chemistry MeSH
- Mutagenesis, Site-Directed MeSH
- Protein Engineering MeSH
- Pseudomonas aeruginosa genetics MeSH
- Ralstonia solanacearum chemistry MeSH
- Recombinant Proteins genetics isolation & purification MeSH
- Plant Lectins chemistry MeSH
- Amino Acid Substitution MeSH
- Binding Sites MeSH
Ticks are important vectors of serious human and animal disease-causing organisms, but their innate immunity can fight invading pathogens and may have the ability to reduce or block transmission to mammalian hosts. Lectins, sugar-binding proteins, can distinguish between self and non-self-oligosaccharide motifs on pathogen surfaces. Although tick hemolymph possesses strong lectin activity, and several lectins have already been isolated and characterized, little is known about the implementation of these molecules in tick immunity. Here, we have described and functionally characterized fibrinogen-related protein (FReP) lectins in Ixodes ticks. We have shown that the FReP family contains at least 27 genes (ixoderins, ixo) that could, based on phylogenetic and expression analyses, be divided into three groups with differing degrees of expansion. By using RNA interference-mediated gene silencing (RNAi) we demonstrated that IXO-A was the main lectin in tick hemolymph. Further, we found that ixoderins were important for phagocytosis of Gram-negative bacteria and yeasts by tick hemocytes and that their expression was upregulated upon injection of microbes, wounding, or after blood feeding. However, although the tick hemocytes could swiftly phagocytose Borrelia afzelii spirochetes, their transmission and burst of infection in mice was not altered. Our results demonstrate that tick ixoderins are crucial immune proteins that work as opsonins in the tick hemolymph, targeting microbes for phagocytosis or lysis.
- MeSH
- Phagocytosis MeSH
- Hemocytes immunology MeSH
- Hemolymph immunology MeSH
- Ixodes genetics immunology MeSH
- Lectins genetics metabolism MeSH
- Immunity, Innate * MeSH
- Arthropod Proteins genetics metabolism MeSH
- RNA Interference MeSH
- Gene Silencing MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The proportion of fucosylated glycoconjugate-containing rabbit tracheal goblet cells after intratracheal application of trimecaine was studied to evaluate its possible unfavourable effects. This lapine model is comparable with diagnostic findings in humans because airway epithelia in humans and rabbits are similar; tracheal epithelium is also practically identical to bronchial epithelium in both species. Local trimecaine anaesthesia caused a proportional decrease in percentage of the tracheal goblet cells containing both alpha(1-2)- and alpha(1-6)-, alpha(1-3)- and alpha(1-4)-fucosylated glycoconjugates as revealed 10 min postexposure using lectin histochemistry. In previous studies, only mild ultrastructural damage to the airway's epithelium was revealed, but a conspicuous decrease in sialylated glycoconjugate-containing tracheal goblet cells and the dominance of acidic sulphated glycoconjugates were observed as after-effects of the same treatment. Glycoconjugate changes can influence the inner environment of airways (e.g. viscoelastic properties of the airways' mucus and mucosal barrier functions) and thus the patient's defence barriers in airways may be weakened. Concurrently, the histochemical properties of goblet cells can be altered in bronchoscopic specimens. Since trimecaine is widely used as local anaesthesia in airways in bronchoscopy, it is necessary to heed these aforementioned effects.
- MeSH
- Anesthetics, Local administration & dosage adverse effects MeSH
- Administration, Topical MeSH
- Financing, Organized MeSH
- Fucose metabolism MeSH
- Glycoconjugates analysis metabolism MeSH
- Histocytochemistry methods MeSH
- Anesthesia, Endotracheal adverse effects MeSH
- Rabbits MeSH
- Lectins metabolism MeSH
- Goblet Cells chemistry metabolism drug effects MeSH
- Drug Administration Schedule MeSH
- Trachea MeSH
- Trimecaine administration & dosage adverse effects MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Male MeSH
- Animals MeSH
CD69 is an earliest lymphocyte activation antigen and a universal leukocyte triggering molecule expressed at sites of active immune response. The binding of GlcNAc to the dimeric human CD69 was followed by equilibrium dialysis, fluorescence titration, and NMR. Clear cooperation was observed in the high-affinity binding (K(d) = 4.0 x 10(-7) M) of the carbohydrate to two subunits of the dimeric CD69 (Hill coefficient 1.94). A control monosaccharide ManNAc was not bound by human CD69, and both monosaccharides had no effects on the structure of the receptor. However, a monomeric CD69 obtained by mutating Q93 and R134 at the dimer interface exhibited a much lower affinity for GlcNAc (K(d) = 1.3 x 10(-5) M) and no cooperativity (Hill coefficient 1.07). Perturbation of the dimer interface resulted in a severe impairment of the signaling ability of cellular CD69 when cross-linked with an antibody or with a bivalent high-affinity N-acetylhexosamine dimer-based ligand. The availability of stable preparations of soluble CD69 receptor with well-documented ligand binding properties will be beneficial for immunological experiments evaluating the role of this antigen in the complex environment of the immune system. Moreover, such preparations in combination with efficient ligand mimetics able to both activate CD69(+) lymphocytes and to block undesired hyperactivation caused by other cellular ligands will also become indispensable tools in explaining the exact role of the CD69 antigen in the interaction between the tumor cell and the effector natural killer lymphocyte.
- MeSH
- Antigens, CD chemistry metabolism MeSH
- Antigens, Differentiation, T-Lymphocyte chemistry metabolism MeSH
- Dimerization MeSH
- Hexosamines chemistry MeSH
- Jurkat Cells MeSH
- Lectins, C-Type chemistry metabolism MeSH
- Humans MeSH
- Ligands MeSH
- Models, Molecular MeSH
- Protein Subunits chemistry metabolism MeSH
- Binding Sites MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Retracted Publication MeSH
- Research Support, Non-U.S. Gov't MeSH
New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblrTM. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.
- MeSH
- Adjuvants, Immunologic pharmacology MeSH
- Antigens, Surface metabolism MeSH
- Candida glabrata chemistry MeSH
- Click Chemistry MeSH
- Dendritic Cells immunology MeSH
- Hydroxylamines chemical synthesis chemistry MeSH
- Lectins, C-Type immunology MeSH
- Mannose-Binding Lectins immunology MeSH
- Humans MeSH
- Lipids chemical synthesis chemistry MeSH
- Liposomes chemistry immunology pharmacology MeSH
- Mannans chemistry immunology pharmacology MeSH
- Microfluidics methods MeSH
- Mice, Inbred BALB C MeSH
- Nanoparticles chemistry MeSH
- Receptors, Cell Surface immunology MeSH
- Particle Size MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
INTRODUCTION: Thyroid cytology is a widely accepted tool in the clinical triaging of nodular lesions. Cell blocks (CBs) can help in the diagnosis of atypical lesions, namely, thyroid Bethesda category of Atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS). METHODS: In a series of 224 AUS/FLUS thyroid samples with CB, we studied CB cellularity and feasibility of 3 immunohistochemical markers (cytokeratin 19 [CK19], HBME-1, and galectin-3) apart and in combination. RESULTS: The CBs were non-diagnostic in 34 cases. Twenty-four CBs contained <10 cells, 45 CBs 10-50 cells, and 121 CBs >50 cells. Notably, more cellularity was found in CBs performed by plasma-thrombin and in-house techniques (p < 0.001). The diagnostic accuracy to detect malignancy was 65.1% for CK19, 72.1% for HBME-1, and 70.3% for galectin-3. CONCLUSION: In conclusion, CB cellularity is essential for successful immunohistochemistry application and further diagnostic workup of AUS/FLUS cases.
- MeSH
- Tissue Fixation MeSH
- Galectins analysis MeSH
- Immunohistochemistry * MeSH
- Keratin-19 analysis MeSH
- Blood Proteins analysis MeSH
- Humans MeSH
- Biomarkers, Tumor analysis MeSH
- Thyroid Neoplasms chemistry pathology MeSH
- Predictive Value of Tests MeSH
- Reproducibility of Results MeSH
- Retrospective Studies MeSH
- Feasibility Studies MeSH
- Neoplasm Grading MeSH
- Paraffin Embedding * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Galectin-3 (Gal-3) is a β-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a β-galactoside LacdiNAc (GalNAcβ1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by β1 and αV integrins-namely α5β1, αVβ3, and αVβ1 integrins.
- MeSH
- Cell Adhesion * MeSH
- Human Umbilical Vein Endothelial Cells cytology physiology MeSH
- Galectins metabolism MeSH
- Integrins metabolism MeSH
- Blood Proteins metabolism MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mesenchymal Stem Cells cytology physiology MeSH
- Cell-Matrix Junctions metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Relapsed acute myeloid leukemia (AML) is a significant post-transplant complication lacking standard treatment and associated with a poor prognosis. Cellular therapy, which is already widely used as a treatment for several hematological malignancies, could be a potential treatment alternative. Natural killer (NK) cells play an important role in relapse control but can be inhibited by the leukemia cells highly positive for HLA class I. In order to restore NK cell activity after their ex vivo activation, NK cells can be combined with conditioning target cells. In this study, we tested NK cell activity against KG1a (AML cell line) with and without two types of pretreatment-Ara-C treatment that induced NKG2D ligands (increased activating signal) and/or blocking of HLA-KIR (killer-immunoglobulin-like receptors) interaction (decreased inhibitory signal). Both treatments improved NK cell killing activity. Compared with target cell killing of NK cells alone (38%), co-culture with Ara-C treated KG1a target cells increased the killing to 80%. Anti-HLA blocking antibody treatment increased the proportion of dead KG1a cells to 53%. Interestingly, the use of the combination treatment improved the killing potential to led to the death of 85% of KG1a cells. The combination of Ara-C and ex vivo activation of NK cells has the potential to be a feasible approach to treat relapsed AML after hematopoietic stem cell transplantation.
- MeSH
- Leukemia, Myeloid, Acute immunology therapy MeSH
- Killer Cells, Natural drug effects immunology transplantation MeSH
- Cytarabine pharmacology MeSH
- Immunosuppressive Agents pharmacology MeSH
- Immunotherapy methods MeSH
- Clinical Trials as Topic MeSH
- Cells, Cultured MeSH
- NK Cell Lectin-Like Receptor Subfamily K immunology MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Receptors, KIR immunology MeSH
- Signal Transduction MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
