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1.
Scand J Immunol ; 78(2): 149-56, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23679222

ABSTRACT

One of the principles behind vaccination, as shown by Edward Jenner in 1796, and host protection is immunological memory, and one of the cells central to this is the antigen-experienced memory B cell that responds rapidly upon re-exposure to the initiating antigen. Classically, memory B cells have been defined as progenies of germinal centre (GC) B cells expressing isotype-switched and substantially mutated B cell receptors (BCRs), that is, membrane-bound antibodies. However, it has become apparent over the last decade that this is not the only pathway to B cell memory. Here, we will discuss memory B cells in mice, as defined by (1) cell surface markers; (2) multiple layers; (3) formation in a T cell-dependent and either GC-dependent or GC-independent manner; (4) formation in a T cell-independent fashion. Lastly, we will touch upon memory B cells in; (5) mouse models of autoimmune diseases.


Subject(s)
Autoimmune Diseases/pathology , B-Lymphocyte Subsets/immunology , Germinal Center/immunology , Immunity, Cellular , Immunologic Memory , Animals , B-Lymphocyte Subsets/pathology , Disease Models, Animal , Gene Expression/immunology , Germinal Center/pathology , Humans , Immunoglobulin Isotypes/biosynthesis , Mice , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , T-Lymphocytes/immunology , T-Lymphocytes/pathology
2.
Gene Ther ; 19(11): 1041-7, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22130447

ABSTRACT

In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.


Subject(s)
Genetic Vectors/genetics , Lentivirus/genetics , Macrophages/metabolism , Promoter Regions, Genetic , Animals , Cell Line , Gene Dosage , Gene Expression , Gene Order , Genetic Therapy , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Humans , Mice , Organ Specificity/genetics , Transduction, Genetic , Transgenes
3.
Cell Mol Life Sci ; 61(1): 4-9, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14704849

ABSTRACT

Atherosclerosis is a multifactorial disease whose pathogenesis is still unclear. Mounting evidence, however, supports the concept that subendothelial retention of apoB100-containing lipoproteins is the initiating event in atherogenesis. Subsequently, a series of biological responses to this retained material leads to specific molecular and cellular processes that promote lesion formation.


Subject(s)
Arteriosclerosis/blood , Arteriosclerosis/pathology , Lipoproteins/blood , Apolipoproteins B/genetics , Arteriosclerosis/etiology , Coronary Disease/blood , Coronary Disease/etiology , Coronary Disease/pathology , Glycosaminoglycans/blood , Humans , Inflammation , Models, Biological
4.
Protein Eng ; 14(1): 67-74, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11287680

ABSTRACT

Antibody binding sites provide an adaptable surface capable of interacting with essentially any molecular target. Using CDR shuffling, residues important for the assembly of mucin-1 specific paratopes were defined by random recombination of the complementarity determining regions derived from a set of mucin-1 specific clones, previously selected from an antibody fragment library. It was found that positions 33 and 50 in the heavy chain and 32, 34, 90, 91 and 96 in the light chain were conserved in many of the clones. These particular residues seem to be located centrally in the binding site as indicated by a structure model analysis. The importance of several of these conserved residues was supported by their presence in a mouse monoclonal antibody with a known structure and the same epitope specificity. Several of these corresponding residues in the mouse monoclonal antibody are known to interact with the antigen. In conclusion, critical residues important for maintaining a human antigen-specific binding site during the process of in vitro antibody evolution were defined. Furthermore, an explanation for the observed restricted germline gene usage in certain antibody responses against protein epitopes is provided.


Subject(s)
Antibodies, Monoclonal/genetics , Complementarity Determining Regions/chemistry , Mucin-1/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Binding Sites , Binding Sites, Antibody , Conserved Sequence , Epitopes/chemistry , Evolution, Molecular , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/chemistry , Mice , Models, Molecular , Molecular Sequence Data , Molecular Structure , Peptide Library , Protein Binding , Sequence Homology, Amino Acid , Tandem Repeat Sequences
5.
Nat Biotechnol ; 18(8): 852-6, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10932154

ABSTRACT

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.


Subject(s)
Germ-Line Mutation , Immunoglobulin Variable Region/genetics , Recombination, Genetic , Humans , Immunoglobulin Fragments/genetics , Recombinant Proteins/genetics
6.
Gene ; 215(2): 471-6, 1998 Jul 30.
Article in English | MEDLINE | ID: mdl-9714846

ABSTRACT

A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germlines. All CDR were randomised in one step and by using in vivo formed CDR, the length, sequence and combination were varied simultaneously.


Subject(s)
Base Sequence , Genes, Immunoglobulin , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Models, Genetic , Sequence Alignment , Amino Acid Sequence , Cloning, Molecular , DNA Primers , Escherichia coli , Gene Library , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , Polymerase Chain Reaction/methods , Sequence Homology, Amino Acid
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