Search PubMed⌕ Search

Biomedical subjects

P Wernet

Publications and source records attributed to P Wernet.

At least 55 records · Page 3Linked to original sources
PubMed1995

Rapid identification of gene defects in protein C deficiency by temperature gradient gel electrophoresis.

Protein C deficiency is an autosomally inherited disorder that is associated with a high risk of recurrent venous thrombosis. The authors have shown that temperature gradient gel electrophoresis (TGGE) is a simple and rapid screening method for the detection of mutations in the protein C gene. Samples from eleven patients with sequence defined point mutations in the promoter region of exon I, and in exons II, III, VII, VIII and IX were analysed by TGGE. In all cases the mutations were readily detected. The exons IV, V and VI were not submissive to TGGE analysis due to amplification difficulties. However, specific computer calculations predict a more general applicability of TGGE for the detection of any mutation in the protein C gene. The presented data establish the usefulness of TGGE as a simple and rapid screening method for the detection of hereditary mutations in the protein C gene.

Base Sequence↗
PubMed1995

Stereoselective suppression of neutrophil function by ketamine?

The effects of the commercially available ketamine preparation (Ketanest), the ketamine racemate and of the two enantiomers, the R(-)-racemate and the S(+)-racemate, as well as its drug-free solvent were examined by N-formyl-methionyl-leucyl-phenylalanine-(FMLP)- and zymosan-induced oxygen radical production of polymorphonuclear cells (PMN). The racemate and the two enantiomers of ketamine suppressed FMLP- and zymosan-induced chemiluminescence of PMN in a dose-dependent fashion to the same extent. Therefore suppression of chemiluminescence of PMN by ketamine does not result from a specific receptor interaction.

Cell-Free System↗
PubMed1995

Detection of mixed chimerism and leukemic relapse after allogeneic bone marrow transplantation in subpopulations of leucocytes by fluorescent in situ hybridization in combination with the simultaneous immunophenotypic analysis of interphase cells.

Serial blood and marrow specimens from eight adult recipients of sex-mismatched transplants (BMT) for chronic myeloid leukemia (CML, n = 3), Ewing sarcoma (n = 1), acute myeloid leukemia (AML) in second remission (n = 1), acute lymphatic leukemia (ALL, n = 1) and multiple myeloma (n = 2) were analyzed by the simultaneous immunophenotypic CD3, CD4, CD8, CD20, CD34, CD10 and genotypic analysis (for X and Y chromosomes). This combined technique of moAb/APAAP staining for cell surface and cytoplasmic antigens and fluorescence in situ hybridization (FISH) for the detection of sex chromosomes allowed the qualitative and quantitative evaluation of mixed chimerism and/or relapse. Using the same slides for moAb/APAAP and FISH allowed the simultaneous identification of the cell lineage, the lymphocyte subpopulation and the genotype (XX or YX) in every blood or BM specimen analyzed. A mixed chimerism in the T cell (CD4, CD8+: median 26% host cells, range 5-44%) and in the myelomonocytic cell population (CD14+ median 16% host cells, range 5-50%) was observed at day +7 after BMT. By days +14 to +18 this mixed chimerism was reduced to 18% host T cells (range 5-50%) and 7% host myelomonocytic cells (range 0-20%). Beyond days +21 to +28 a stable donor chimerism for T cells, myelomonocytic cells and granulocytes was observed in seven of eight patients. Still 0.5-1% host cells of different lineages were detectable in five from the eight patients at later time points (> day + 100). In three patients with CML these cells were CD13 or CD13, CD34 positive and in one was CD4, CD8 positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Do barbiturates impair zymosan-induced granulocyte function?

PURPOSE: The dose-response relationship of commercially available preparations of methohexital, pentobarbital, phenobarbital, and thiopental and their respective drug-free solutions on granulocyte function was investigated to evaluate whether suppression of neutrophil chemiluminescence is mediated by the barbiturates themselves or by their drug-free solutions. Furthermore, it was assessed whether suppression of chemiluminescence is due to an interaction mainly with neutrophils or to free radical scavenging. METHODS: The dose-response effects of the four barbiturates on granulocyte function were tested by zymosan-induced neutrophil chemiluminescence and, in addition, in a cell-free chemiluminescence system. RESULTS: Methohexital and pentobarbital did not influence zymosan-induced neutrophil chemiluminescence, whereas phenobarbital and thiopental decreased neutrophil chemiluminescence in a dose-dependent fashion. Nonphysiological osmolality (531 mosmol/kg) caused this impaired neutrophil chemiluminescence at the greatest concentration of phenobarbital. Thiopental solely suppressed neutrophil chemiluminescence drug specifically. Because thiopental also reduced chemiluminescence generated in a cell-free system, free radical scavenging might contribute to the impaired neutrophil chemiluminescence observed with thiopental. CONCLUSIONS: With the exception of thiopental, barbiturates do not impair oxygen radical production during phagocytosis of neutrophils.

Cell-Free System↗
PubMed1994

Selection of unrelated bone marrow donors by PCR-SSP typing and subsequent nonradioactive sequence-based typing for HLA DRB1/3/4/5, DQB1, and DPB1 alleles.

HLA incompatibility between bone marrow recipients and unrelated donors is one of the main obstacles in bone marrow transplantation. HLA class I and generic class II DR and DQ typing is generally performed by serology. Precise subtyping of HLA class II genes, however, can only be achieved by molecular genetic methods. Here, the final selection of serologically pretyped unrelated bone marrow donors by confirmatory PCR-SSP (PCR-sequence-specific primers) typing and subsequent nucleic acid sequence analysis of the second exon of DRB1, DRB3, DRB4, DRB5, DQB1, and DPB1 alleles is presented. Serologically identical potential marrow donors and their corresponding recipients were analyzed for HLA-DRB identity by PCR-SSP analysis. After solid-phase single-strand separation, direct sequencing of the allele- or group-specific DRB amplified products was performed by applying fluorophorlabelled sequencing primers. Electrophoretically separated sequencing products were detected by means of an automated DNA sequencer. Group-specific amplification and sequencing of DQB1 alleles was carried out for all potential bone marrow donors and recipients, while only the final donor-recipient pair was analyzed for DPB1 alleles. Thus, the presented amplification strategy in combination with direct sequencing of PCR products allows matching of bone marrow transplant pairs with the highest degree of reliability for the assessment of HLA class II identity.

Base Sequence↗
PubMed1994

Rapid DNA crossmatch analysis of HLA class II genotypic polymorphisms by temperature gradient gel electrophoresis in unrelated bone marrow donor selection.

The present paper demonstrates the usefulness of the temperature gradient gel electrophoresis (TGGE) as a precise method for the rapid identification of HLA-DR full matched donors by molecular crossmatch analysis. The resulting TGGE fingerprints are highly diverse for different HLA-DR genotypes and identical for samples with identical HLA-DR type, as shown for a panel of 25 randomly selected PCR-SSO pretyped DNA samples. In addition, TGGE analysis of a panel of homozygous typing cell lines (HTCs) established, that even subtle differences in the DR4 subtypes are detectable by TGGE crossmatch analysis. Furthermore, TGGE crossmatch analysis of unrelated donor/patient pairs demonstrated, that only HLA-DR identical patient/donor combinations showed identical TGGE patterns. Thus crossmatch analysis by TGGE presents a novel basis for a rapid and safe unrelated bone marrow donor selection strategy. Only HLA class I identical donors which also show HLA-DR identity in the TGGE crossmatch test are selected for the final confirmation of the HLA class II genes by a suitable DNA subtyping method.

Alleles↗
PubMed1994

The rapid detection of the quantity (genotype) and cell lineage (immunophenotype) of contaminating maternal white cells in cord blood samples by fluorescence in situ hybridization combined with confocal laser scanning microscopy.

The role of maternal blood cell contamination in cord blood as hematopoietic stem cell transplant could be an important factor driving the graft vs. host reactivity. Therefore, the efficiacy of fluorescence-labeled chromosome probes for the X and Y chromosomes to study contaminating maternal cells (XX+, Y-) in nucleated cells of male cord blood specimen (X+, Y+) by fluorescence in situ hybridization (FISH) was evaluated here. Unfortunately, the FISH technique evaluated by standard indirect immunofluorescence was not sufficiently sensitive to safely detect maternal contamination below 2-3%. Thus, the analysis of FISH had to be adapted with confocal laser scanning microscopy, which then gave no false-negative reading values. A Bio-Rad MRC-600 laser scanning microscope with a very high sensitivity for low-intensity and hidden signals was used for the observation of cord blood cells. Sixteen samples from the whole cord blood (CB), 10 analyses of mononuclear cells, 6 of the granulocytic fraction, 6 of picked erythroid burst-forming unit (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) colonies and 5 of enriched CD34+ cells were performed. Only in 1 of 16 cases was a contamination by maternal cells detected: 10% of all nucleated cells from the whole CB, 5% of the CD3+ T-cell fraction, 15% of the myelomonocytic cells from mononuclear cells (MNCs), and 15% of picked BFU-E and CFU-GM colonies were of maternal origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1994

Do barbiturates and their solutions suppress FMLP-induced neutrophil chemiluminescence?

The dose-response relationship of four commercially available barbiturates (methohexitone, pentobarbitone, phenobarbitone and thiopentone) and of their drug-free solutions on the production of oxygen radicals by neutrophils were tested by N-formylmethionyl-leucyl-phenylalanine (FMLP)-induced granulocyte chemiluminescence and in a cell-free chemiluminescence system. Methohexitone had no effect on neutrophil chemiluminescence. Pentobarbitone, phenobarbitone and thiopentone dose-dependently decreased FMLP-induced chemiluminescence and cell-free chemiluminescence. Suppression of neutrophil chemiluminescence by pentobarbitone and phenobarbitone was due to effects of the drug-free solutions and an osmolality greater than 360 mosmol kg-1. Only thiopentone suppressed granulocyte chemiluminescence drug-specifically. The physicochemical properties of commercially available barbiturate preparations and their solutions, as well as free radical scavenging capacity, have to be considered if these preparations are used to evaluate drug-specific effects on the production of oxygen radicals by neutrophils.

Barbiturates↗
PubMed1993

Transfer of insulin-dependent diabetes between HLA-identical siblings by bone marrow transplantation.

Insulin-dependent diabetes was observed in a woman, aged 29, 4 years after transplantation of bone marrow from her HLA-identical brother with insulin-dependent diabetes. Both had classic symptoms and insulin dependency from onset. At diagnosis of diabetes the recipient was positive for high-titre islet cell antibodies (ICA) whereas she had been ICA negative before transplantation. Chromosomal analyses verified that all circulating leucocytes were of male donor type. These findings suggest transfer of insulin-dependent diabetes by bone marrow cells and confirm the immune nature of the disease.

Adult↗
PubMed1993

Instant therapy of acquired agranulocytosis and sepsis by recombinant granulocyte-macrophage colony-stimulating factor in a polytrauma patient.

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was given to an intensive-care patient with polytrauma in a life-threatening situation with acquired agranulocytosis and sepsis. Mature granulocytes reappeared in the blood 2 days after initiation of rhGM-CSF therapy; granulocyte precursors peaked at 43% after 5 days. Bone marrow examination performed 7 days after the beginning of rhGM-CSF therapy revealed complete regeneration of granulopoiesis. The functional analysis of these blood leukocytes in vitro showed regular production of reactive oxygen radicals. Clinically, the patient recovered without any serious side effects due to the rhGM-CSF therapy. These results suggest that rhGM-CSF accelerates granulocyte recovery from acquired agranulocytosis with the presence of their functional activity.

Adult↗
PubMed1993

Simultaneous genotypic and immunophenotypic analysis of interphase cells for the detection of contaminating maternal cells in cord blood and their respective CFU-GM and BFU-E.

Contamination of cord blood (CB) specimens by maternal blood provides a source of cells that may be capable of graft-versus-host reactivity. To confirm the genetic purity of collected CB samples (six samples, volume 110 +/- 21 ml, total nucleated cells 1.22 +/- 0.36 x 10(9)) the HLA-DR beta exon 2 for the noninherited material allele was examined by polymerase chain reaction amplification. No maternal cell contamination was detected in samples of 1 x 10(5) cells. In the case where the mother was homozygous for DR and DQ, the purity of the sample could not be tested by PCR and therefore in situ hybridization on interphase cells with a fluorescein labeled Y- and X-probe in male CB specimens was performed. This sensitive method also revealed no contamination of the CB by maternal white cells. In addition, picked CB-CFU-GM and BFU-E colonies (cultured in hu-SLF, GM-CSF, and Epo) were analyzed by simultaneous genotypic (for Y and X) and immunophenotypic analysis (monoclonal antibodies [MAbs] CD13, CD14, CD2, CD8, CD4, and glycophorin A). This approach permits simultaneous visualization of both the immunophenotype (MAbs, APAAP, red fluorescence) and the genotype (chromosomes, fluorescein isothiocyanate, green fluorescence) within the same cell. In contrast to PCR and restriction fragment length polymorphism, this method has the advantage that the donor-recipient origin of each lymphohematopoietic lineage (i.e., BFU-E, CFU-GM, T cells, B cells) can be determined in sex-mismatched transplantations. Thus confocal scanning laser microscopy is most suitable not only for the detection of contaminating maternal cells, but also for the detection of mixed hematopoietic chimerism after transplantation.

Adult↗
PubMed1993

Benzodiazepines and their solvents influence neutrophil granulocyte function.

We have examined the effects of commercially available preparations and drug-free solvents of diazepam (Valium, Diazepam-Lipuro) and midazolam (Dormicum) by N-formylmethionyl-leucylphenylalanine (FMLP)- and zymosan-induced polymorphonuclear cell (PMN) chemiluminescence and in a cell-free chemiluminescence system. In the case of Valium, drug-free solvent and diazepam suppressed PMN chemiluminescence. With Diazepam-Lipuro, the solvent stimulated and diazepam inhibited PMN chemiluminescence. With midazolam (Dormicum), only the active drug depressed PMN chemiluminescence. FMLP-induced PMN chemiluminescence was depressed 10-100 fold more by diazepam and midazolam than zymosan-induced chemiluminescence.

Diazepam↗
PubMed1993

Identification of an alternatively spliced transcript of human interleukin-4 lacking the sequence encoded by exon 2.

The expression of interleukin-4 (IL-4) mRNA of human peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA) for 15 hours was analyzed by reverse transcription and subsequent polymerase chain reaction (RT/PCR). These analyses revealed an additional smaller fragment that hybridizes with an IL-4 cDNA probe and an oligonucleotide that is specific for a fragment lacking the sequence encoded by exon 2. Sequencing of this fragment demonstrates that it is generated from an alternatively spliced transcript of the IL-4 gene with the sequence encoded by exon 2 being skipped. Skipping of exon 2 does not result in a frame shift but would delete part of the mature protein (48 bp coding for amino acid residues 22 to 37), including Cys24 but not a region directly involved in receptor binding. Differential splicing of other exons or exon combinations has not been observed. The data suggest that the alternatively spliced transcript is not generated by a splicing or PCR error and is not detectable solely because of the high sensitivity of RT/PCR, but in contrast, argue for a physiological role of the transcript and its potentially encoded protein.

Alternative Splicing↗
PubMed1992

Human glioblastoma cell line 86HG39 activates T cells in an antigen specific major histocompatibility complex class II-dependent manner.

The capacity of three different human glioblastoma cell lines to activate human T cells was analysed by measuring major histocompatibility complex (MHC) antigen expression, monokine secretion and lectin, mAb OKT3 and antigen-driven T cell proliferation. All glioblastoma cells tested were able to induce PHA and concanavalin A (ConA)-driven T cell proliferation in a dose-dependent fashion, while all failed to induce T cell activation with mAb OKT3. In addition, the glioblastoma cell line 86HG39 was able to induce tetanus toxoid and toxoplasma lysate antigen-specific T cell proliferation. The responding T cell lines originated from only one out of five different donors. This foreign antigen-specific T cell proliferation induced by 86HG39 cells could be inhibited with mAb L243 directed against HLA-DR molecules. The study of monokine secretion by 86HG39 cells showed a strong interleukin (IL)-6 secretion after lipopolysaccharide (LPS) treatment, whilst no IL-1 secretion was observed. Furthermore, only 86HG39 cells were positive for HLA-DR molecules, whereas interferon (IFN) gamma treatment of 87HG28 and 87HG31 cells was necessary for the induction of class II antigen expression. Thus, cell line 86HG39 shows many features of an antigen presenting cell and the interaction of these cells with MHC compatible human T cells might be a useful model to study cellular immune reactions within the central nervous system.

Animals↗
PubMed1992

One-step molecular HLA-DR prescreening employing a set of 14 sequence specific oligonucleotides in a non-radioactive tetramethylammonium chloride hybridization protocol.

A novel non-radioactive protocol for molecular generic HLA-DR typing is introduced, employing sequence specific oligonucleotides (SSOs) enzymatically 3'-labelled with biotin-14-dATP via terminal deoxynucleotidyltransferase in a tetramethylammonium chloride hybridization procedure. The detection reaction is carried out, using streptavidin conjugated horseradish peroxidase which is bound to the SSOs, in combination with a light emitting detection system. Fourteen SSOs and one control SSO are employed for generic HLA-DR typing in a one-step protocol. In order to demonstrate the suitability of this procedure, 5 homozygous typing cell lines and samples of 11 pretyped individuals which include most serologically defined HLA-DR specificities (DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9, 10, 52, and DR53) are analysed with the panel of 14 SSOs. The typing results show that this protocol, which avoids the use of radioisotopes, combines high specificity and easy handling. It also allows typing of poorly amplified samples because even after longer exposition times no false positive signals were observed and is particularly suitable for routine molecular HLA-DR typing on the generic level. In addition it can easily be adapted to DP and DQ typing or DR subtyping.

Base Sequence↗