ABSTRACT
Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.
Subject(s)
Humans , Amino Acid Sequence , Antigens, CD , Chemistry , Genetics , Pharmacology , Autoimmunity , Biological Assay , Cell Line , Cytotoxicity, Immunologic , Genetics , Allergy and Immunology , Dose-Response Relationship, Immunologic , Immunoglobulins , Allergy and Immunology , Metabolism , Immunologic Factors , Chemistry , Genetics , Pharmacology , Kinetics , Leukocyte Immunoglobulin-like Receptor B1 , Lymphocyte Activation , Genetics , Allergy and Immunology , Major Histocompatibility Complex , Genetics , Allergy and Immunology , Molecular Sequence Data , Molecular Targeted Therapy , Mutagenesis, Site-Directed , Peptide Library , Polyethylene Glycols , Protein Binding , Genetics , Allergy and Immunology , Receptors, Immunologic , Chemistry , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , T-Lymphocytes, Cytotoxic , Allergy and Immunology , MetabolismABSTRACT
There is growing evidence indicating the benefits of Lactobacilli and Bifidobacteria in attenuation of colitis. On the other hand, some studies have shown that n-3 fatty acids can ameliorate inflammation in colitis. The aim of this study was to examine the interaction between different dietary oils and intestinal microflora in an experimental model of colitis. Eight week-old BALB/C mice [n = 9] were fed isocaloric diets varying only in fat composition for 4 weeks. A group fed the chow diet served as control. The diets contained 20% fat from fish oil, canola oil, safflower oil, or beef tallow. Colitis was induced by intracolonic administration of acetic acid on day 21. Inflammation, fecal microflora and serum lipid profile were compared among the groups one week after induction. The highest degree of inflammation was seen in the chow-diet group, followed by safflower, canola-, and fish oil-fed groups [p < 0.05]. As compared to the experimental groups, the number of fecal bacteroideceae was significantly higher [p < 0.05], and the number of fecal bifidobacteria significantly lower, in the control group [p < 0.05]. Moreover, fish oil could reduce the plasma level of triacylglycerole significantly [p < 0.05]. These results indicate that n-3 fatty acids can affect intestinal microfloral populations in favor of increasing the number of bifidobacteria. They might be recommended as an adjunct therapy to patients with colitis