28 research outputs found
Clinical-scale isolation of ‘minimally manipulated’ cytomegalovirus-specific donor lymphocytes for the treatment of refractory cytomegalovirus disease
No full textAbstractBackground aimsReactivation of cytomegalovirus (CMV) after hematopoietic stem cell transplantation remains a major cause of morbidity despite improved antiviral drug therapies. Selective restoration of CMV immunity by adoptive transfer of CMV-specific T cells is the only alternative approach that has been shown to be effective and non-toxic. We describe the results of clinical-scale isolations of CMV-specific donor lymphocytes with the use of a major histocompatibility (MHC) class I peptide streptamer-based isolation method that yields minimally manipulated cytotoxic T cells of high purity.MethodsEnrichment of CMV-specific cytotoxic T lymphocytes (CTLs) was performed by labeling 1 × 1010 leukocytes from a non-mobilized mononuclear cell (MNC) apheresis with MHC class I streptamers and magnetic beads. Thereafter, positively labeled CMV-specific CTLs were isolated through the use of CliniMACS (magnetic-activated cell sorting), and MHC streptamers were released through the use of d-biotin. The purity of enriched CMV-specific CTLs was determined on the basis of MHC streptamer staining and fluorescence-activated cell sorting.ResultsA total of 22 processes were performed with the use of five different MHC class I streptamers. The median frequency of CMV-specific CTLs in the starting apheresis product was 0.41% among CD3+ T cells. The isolation process yielded a total of 7.77 × 106 CMV-specific CTLs, with a median purity of 90.2%. Selection reagents were effectively removed from the final cell product; the CMV-specific CTLs displayed excellent viability and cytotoxicity and were stable for at least 72 h at 4°C after MNC collection.ConclusionsClinical-scale isolation of “minimally manipulated” CMV-specific donor CTLs through the use of MHC class I streptamers is feasible and yields functional CTLs at clinically relevant dosages
The simultaneous isolation of multiple high and low frequent T-cell populations from donor peripheral blood mononuclear cells using the major histocompatibility complex I-Streptamer isolation technology
No full textImmunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease
Molecular cloning, DNA sequence and transcriptional analysis of the Rhodospirillum molischianum B800/850 light-harvesting genes
No full textAbstractThe amino acid sequences of the B800/850 light-harvesting proteins from Rhodospirillum molischianum were determined by Edman degradation. On the basis of these amino acid sequences, two degenerated oligonucleotides were synthesized and used for PCR of genomic DNA. The resulting 150 by DNA fragment was cloned, sequenced and used for subsequent Southern blot analysis of digested genomic DNA. A 2.3 kbp EcoRI fragment strongly hybridized to the probe and a size selected genomic library from genomic DNA was constructed. One clone scored positive during screening of the library with the PCR-fragment and subsequent DNA sequence analysis of the clone revealed the presence of three A-genes (A1A2A3) encoding a-polypetides and of two B-genes (B1B2) encoding β-polypeptides of the 13800/850 complex. The arrangement of the different genes are B1A1, B2A2 and A3 where only B1 and B2 are preceded by typical Shine-Dalgamo sequences. In addition, typical nucleotide sequences for a rho-independent termination of transcription are located downstream of the genes A1 and A2. The deduced amino acid sequences revealed that the α-genes encoded for identical polypeptides, whereas the deduced β-polypeptides differed in their amino acid sequence at four positions. Transcriptional operon analysis revealed that the genes A1B1 and A2B2 are both dicistronically transcribed, whereas the gene A3 is not
Structure of the light harvesting antenna from Rhodospirillum molischianum studied by electron microscopy
No full textThe structure of two types of isolated light-harvesting antenna complexes from Rhodospirillum molischianum was studied by electron microscopy and image analysis. The B870 reaction center complex forms an almost circular particle with a diameter in the plane of the membrane of about 10.7-11.2 nm. A complex consists of a reaction center surrounded most likely by 12 B870 antennae units. An asymmetrical protrusion, consisting of the cytochrome subunit, gives the reaction center a height of 11.5 nm. The peripheral B800-820 complex is cylindrical, with a diameter of 5.3 nm and a small central indentation. At low resolution, it is structurally very similar to the B800-850 complex of Rhodobacter sphaeroides and is considered to be most likely of the α6β6 type.
Identification of peptides inhibiting enzyme I of the bacterial phosphotransferase system using combinatorial cellulose-bound peptide libraries
No full textStarGate®: A High‐Capacity Expression Cloning System to Speed‐Up Biopharmaceutical Development
No full text‐rate assay for fast avidity screening of even very small antigen‐specific T cell populations ex vivo
No full textSequential Nucleophilic Substitution on Halogenated Triazines, Pyrimidines, and Purines: A Novel Approach to Cyclic Peptidomimetics
No full textA novel concept for the synthesis of macrocyclic peptidomimetics which incorporate heteroaromatic
units is reported. The method involves sequential SNAr reactions of orthogonally protected amino
groups of peptides and other linear oligomers on halogenated heterocycles such as 2,4,6-trichloro-[1,3,5]triazine, 2,4,6-trichloropyrimidine, 4,6-dichloro-5-nitropyrimidine, and 2,6,8-trichloro-7-methylpurine. The scope of this novel solid-phase approach was systematically evaluated by means
of the SPOT-synthesis methodology on planar cellulose membranes. Besides the question of the
accessibility of different ring sizes and the compatibility with protecting groups of commonly used
amino acids, the applicability of the technique toward different halogenated heteroaromatics and
peptidomimetics was studied. It was found that the procedure is well suited to assemble a wide
variety of cyclic peptidomimetics differing in both size (11- to 37-membered rings) and chemical
nature of the assembled backbones
Dissociation of the signalling and antiviral properties of SDF-1-derived small peptides
No full textAbstractBackground: The chemokine receptor CXCR4 (a receptor for the Cys-X-Cys class of chemokines) is a CD4-associated coreceptor for T-cell-tropic strains of human immunodeficiency virus 1 (HIV-1) and represents a target for antiviral therapy. Infection by T-tropic HIV-1 can be blocked by stromal-cell-derived factor-1 (SDF-1), the natural ligand of CXCR4. The broad variety of cells expressing CXCR4 and the perturbations observed in mice deficient for SDF-1 suggest that antiviral compounds antagonizing the signalling activity of CXCR4 might have severe side effects in vivo. Compounds that interfere selectively with HIV entry and not with SDF-1 signalling would therefore be useful.Results: A series of peptides, each of 13 residues, spanning the whole SDF-1α sequence were tested for their ability to block HIV-1 infection. The antiviral and signalling properties of SDF-1 were retained by a peptide corresponding to its amino terminus. Removal of the first two residues resulted in an antiviral antagonist of the SDF-1–CXCR4 signalling pathway. We prepared 234 single-substitution analogues and identified one antiviral analogue that had drastically reduced agonistic or antagonistic properties. The antiviral peptides competed with the monoclonal antibody 12G5 for CXCR4 binding. Their antiviral activity seems to be due to receptor occupancy rather than induction of receptor endocytosis.Conclusions: The amino terminus of the SDF-1 chemokine is sufficient for signal transduction via CXCR4 and for inhibition of HIV-1 entry, but these activities could be dissociated in a peptide analogue. This peptide represents a lead molecule for the design of low molecular weight antiviral drugs
