Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
Hum Gene Ther ; 11(16): 2207-18, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11084678

ABSTRACT

Dendritic cells (DCs) are the most potent inducers of immune reactions. Genetically modified DCs, which express tumor-associated antigens (TAA), can efficiently induce antitumor immunity and thus have a high potential as tools in cancer therapy. The gene delivery is most efficiently achieved by viral vectors. Here, we explored the capacity of influenza virus vectors to transduce TAA genes. These viruses abortively infect DCs without interfering with their antigen-presenting capacity. In contrast to other viruses used for DC transduction, influenza viruses can be efficiently controlled by antiviral pharmaceuticals, lack the ability to integrate into host chromosomes, and fail to establish persistent infections. Genes encoding a melanoma-derived TAA (MAGE-3), or the green fluorescence protein (GFP), were introduced into a high-expression avian influenza virus vector. Monocyte-derived mature DCs infected by these recombinants efficiently produced GFP or MAGE-3. More than 90% of the infected DCs can express a transduced gene. Importantly, these transduced DCs retained their characteristic phenotype and their potent allogeneic T cell stimulatory capacity, and were able to stimulate MAGE-3-specific CD8(+) cytotoxic T cells. Thus influenza virus vectors provide a highly efficient gene delivery system in order to transduce human DCs with TAA, which consequently stimulate TAA-specific T cells.


Subject(s)
Antigens, Neoplasm/genetics , Dendritic Cells/metabolism , Gene Transfer Techniques , Influenza A virus/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Animals , Antigens, Neoplasm/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Line , Cell Separation , Dogs , Flow Cytometry , Genetic Vectors , Green Fluorescent Proteins , Humans , Immunoblotting , Immunophenotyping , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Phase-Contrast , Phenotype , Plasmids/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Cytotoxic/metabolism , Transduction, Genetic , Tumor Cells, Cultured
2.
Gene Ther ; 7(23): 2028-35, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11175315

ABSTRACT

The use of tumor antigen loaded dendritic cells (DC) is one of the most promising approaches to induce a tumor specific immune response in vivo. Several strategies have been designed to load DC with tumor antigens. In this study, we investigated the delivery of in vitro transcribed RNA and plasmid DNA into monocyte-derived, ie non-proliferating human DC, using several nonviral transfection methods including electroporation and lipofection. Green fluorescent protein (GFP) was used as a reporter gene and influenza matrix protein 1 (M1) as a model antigen for HLA class I restricted antigen presentation. Using electroporation in combination with DNA or with RNA, up to 11% of DC were GFP-positive. Using liposomes as a vehicle for DNA transport up to 10% of the DC were GFP-positive. A significant increase in transfection efficacy, of up to 20%, was observed when GFP RNA was used in combination with liposomes. Importantly, the RNA transfected DC retained their typical morphological and immunophenotypical characteristics. In addition, DC transfected with M1 RNA were able to stimulate autologous peripheral M1-specific memory cytotoxic T lymphocytes (CTL), as well as M1-specific CTL clones. Furthermore, comparison of DNA-transfected DC with RNA-transfected DC revealed the latter to be far better stimulators of antigen-specific T cells. This RNA transfection technique consequently represents a very promising tool for future immunotherapy strategies.


Subject(s)
Antigens, Viral/genetics , Dendritic Cells/immunology , Genetic Therapy/methods , T-Lymphocytes, Cytotoxic/immunology , Transfection/methods , Viral Matrix Proteins/genetics , Clone Cells , DNA/administration & dosage , Electroporation , Gene Expression , Green Fluorescent Proteins , Humans , Immunologic Memory , Immunotherapy , Liposomes , Luminescent Proteins/genetics , Lymphocyte Activation , RNA/administration & dosage
4.
J Dermatol Sci ; 13(1): 71-5, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8902656

ABSTRACT

The recent structural and functional analysis of the high affinity receptor for IgE (Fc epsilon RI) expressed on human epidermal Langerhans cells (LC) revealed new aspects of the biology of this structure. In contrast to basophils and mast cells where this receptor seems to be expressed constitutively at a constant level, the expression of Fc epsilon RI on LC varies on the donor and the inflammatory environment of the cells and lacks the classical beta-chain. This also implies functional differences most probably related to the expression level. Although the signalling pathway seems to be similar to that of basophils or mast cells, LC from individuals with atopic dermatitis are fully activated by receptor ligation while LC from normal individuals fail to exhibit calcium mobilization under the same conditions. Finally, LC from normal and atopic individuals use Fc epsilon RI to maximize antigen uptake via specific IgE and subsequent presentation to T cells. Thus, Fc epsilon RI expressed on LC differs in terms of structure and function from that expressed on effector cells of anaphylaxis.


Subject(s)
Langerhans Cells/immunology , Receptors, IgE/chemistry , Receptors, IgE/metabolism , Antigen Presentation , Basophils/immunology , Calcium/metabolism , Dermatitis, Atopic/immunology , Dermatitis, Atopic/metabolism , Humans , In Vitro Techniques , Langerhans Cells/metabolism , Mast Cells/immunology , Molecular Structure , Signal Transduction
5.
Ophthalmologe ; 93(1): 68-72, 1996 Feb.
Article in German | MEDLINE | ID: mdl-8867164

ABSTRACT

Retro- und peribulbar anesthetic injections, the common techniques in cataract surgery, have persistently reported complications. Recently topical anesthesia has been mentioned as a possible alternative. The effectiveness of anesthesia, the acceptance on the part of the patients and the consequences of the lack of akinesia were analysed in a prospective study. We compared two groups of 27 patients. Patients in the first group had only topical anesthesia, while patients in the control group had a peribulbar injection. None of the patients included had such conditions as deafness or dementia or felt overanxious. Both methods were accepted very well by the patients. There were no significant differences in the improvement of visual acuity and the opinion of the patients about pain during the operation. The surgeon's assessment revealed a few cases of increased voluntary eye movements in the topical anesthesia group, but this did not affect the complication rate significantly. There was one case of vitreous loss in each group and in the peribulbar group one case of zonular defect. Topical anesthesia should be seen as an alternative to injection anesthesia.


Subject(s)
Anesthesia, Local , Phacoemulsification , Aged , Female , Humans , Lenses, Intraocular , Male , Pain Measurement , Patient Acceptance of Health Care , Visual Acuity/drug effects
6.
Eur J Immunol ; 24(3): 593-8, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8125129

ABSTRACT

Psoriasis vulgaris has been recognized lately as an immunologically mediated inflammatory skin disease. To analyze the pathogenetic role of T lymphocytes in the generation of psoriatic skin lesions, 105 T cell clones (TCC) and 10 T cell lines (TCL) were differentially isolated from dermis and epidermis of psoriatic skin specimens. Supernatants prepared from these T cells were studied for their effects on keratinocyte proliferation in vitro. Conditioned media from 14 of 77 epidermal TCC, 7 of which were CD8+, and from 8 of 28 dermal TCC, 5 of which were CD8+, reproducibly enhanced keratinocyte proliferation, with more pronounced mitogenic activities found in dermal TCC. Another 9 epidermal and 3 dermal TCC did not affect keratinocyte growth and supernatants from the remaining clones, as well as from the 5 epidermal and 5 dermal TCL, inhibited keratinocyte replication to varying degrees. Both mitogenic and suppressive activities were largely abolished by addition of an antiserum to interferon-gamma (IFN-gamma), while addition of epidermal growth factor or irradiated psoriatic TCL had little effect on the activities of the supernatants. These studies reveal that a subpopulation of lesional psoriatic T lymphocytes is capable of enhancing keratinocyte proliferation in vitro via secreted products. Their mitogenic capacity most likely requires IFN-gamma, but the ultimate effect is apparently determined by the presence of additional cytokines. Activation of T cells secreting such combinations of factors in vivo may contribute to the keratinocyte alterations characteristic of psoriatic skin lesions.


Subject(s)
Keratinocytes/cytology , Psoriasis/immunology , T-Lymphocytes/immunology , Cell Division , Epidermal Cells , Epidermal Growth Factor/physiology , Humans , In Vitro Techniques , Interferon-gamma/physiology , Lymphokines/physiology , Mitogens , Psoriasis/pathology , Skin/cytology
7.
Eur J Immunol ; 24(1): 176-80, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8020553

ABSTRACT

Classical, polymorphic major histocompatibility complex class I molecules are expressed on most nucleated cells. They present peptides at the cell surface and, thus, enable the immune system to scan peptides for their antigenicity. The function of the other, nonclassical class I molecules in man is controversial. HLA-G which has been shown by transfection experiments to be expressed at the cell surface, is only transcribed in placental tissue and in the fetal eye. Therefore, a role of HLA-G in the control of rejection of the allogeneic fetus has been discussed. We found that HLA-G expression is induced in keratinocytes by culture in vitro. Three different alternative splicing products of HLA-G can be detected: a full length transcript, an mRNA lacking exon 3 and a transcript devoid of exon 3 and 4. Reverse transcription followed by polymerase chain reaction also revealed the presence of HLA-G mRNA in vivo in biopsies of either diseased or healthy skin.


Subject(s)
Alternative Splicing/genetics , HLA Antigens/biosynthesis , Histocompatibility Antigens Class I/biosynthesis , Keratinocytes/immunology , Skin/immunology , Base Sequence , Blotting, Northern , Cells, Cultured , HLA Antigens/genetics , HLA-G Antigens , Histocompatibility Antigens Class I/genetics , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Tumor Cells, Cultured
8.
J Invest Dermatol ; 98(5): 771-6, 1992 May.
Article in English | MEDLINE | ID: mdl-1569326

ABSTRACT

When screening skin cryosections with a panel of monoclonal antibodies (MoAb), we found that the anti-CD69 MoAb Leu-23 reacted with a subpopulation of epidermal dendritic cells, presumably Langerhans cells (LC). The staining intensity was enhanced by gentle trypsin pretreatment of the sections. Flow cytometric analysis of LC-enriched epidermal cells (EC) revealed that nearly all CD1a-bearing LC display anti-CD69 reactivity when tested briefly after termination of the enrichment procedure. Immunoprecipitation experiments showed that isolated LC specifically express a disulphide-linked dimer composed of 26/30kDa subunits that therefore slightly differs from the 28/32kDa CD69 complex described on activated T or natural killer (NK) cells. This difference is probably due to a different post-translational glycosylation pattern as evidenced by Endoglycosidase-F treatment of the immunoprecipitate disclosing the 24-kDa core protein of CD69. When freshly isolated LC-enriched EC were kept in culture, anti-CD69 reactivity gradually decreased but the addition of IFN-gamma to the culture medium sustained the CD69 expression on LC in vitro. These results strongly suggest that resident but not LC recovered from EC cultures bear CD69 moieties. It remains to be seen whether the expression of this antigen can be linked to (a) particular functional property (ies) of intraepidermal LC.


Subject(s)
Antigens, CD/physiology , Antigens, Differentiation, T-Lymphocyte/physiology , Langerhans Cells/immunology , Antibodies, Monoclonal , Antigens, CD/chemistry , Antigens, Differentiation, T-Lymphocyte/chemistry , Cells, Cultured , Humans , Interferon-gamma/pharmacology , Lectins, C-Type , Membrane Proteins/analysis , Molecular Structure , Phenotype , Precipitin Tests , Recombinant Proteins
9.
J Bacteriol ; 172(12): 7111-8, 1990 Dec.
Article in English | MEDLINE | ID: mdl-2123862

ABSTRACT

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).


Subject(s)
Archaea/analysis , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins , Membrane Glycoproteins , Amino Acid Sequence , Archaea/genetics , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/isolation & purification , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Genes, Bacterial , Glycoproteins/genetics , Glycoproteins/isolation & purification , Glycosylation , Molecular Sequence Data , Peptide Mapping , Polymerase Chain Reaction , Protein Precursors/metabolism , Restriction Mapping , Solubility
SELECTION OF CITATIONS
SEARCH DETAIL
...