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Biomedical subjects

W Müller-Ruchholtz

Publications and source records attributed to W Müller-Ruchholtz.

At least 19 recordsLinked to original sources
PubMed1997

Fractionation and bioassay of human odor types.

Human urine samples were fractionated to examine the contribution of volatiles to the individual body odor. The samples were obtained from 4 male donors and fractionated using a vacuum technique. The volatiles from the chemical fractions were analyzed using the CLSA technique and gas chromatography. Thereafter, these fractions were tested in a computer-controlled olfactometer by trained rats. Although the rats were able to discriminate the distillation residue, they could not recognize the urine odor in the distilled fraction. The results of gas chromatography indicate a continuous release of volatile constituents in the distillation residue.

Adult↗
PubMed1997

Does cyclophosphamide combined with methylprednisolone affect the expression of leukocyte function associated antigen 1 in refractory rheumatoid arthritis.

In the present study, in order to get a better insight into the mechanism of action of cyclophosphamide (CY) in rheumatoid arthritis (RA), we monitored the changes in lymphocytes' expression of leukocyte function associated antigen 1 (LFA-1). A group of 28 patients with refractory severe RA were treated with CY and methylprednisolone (MO) intravenously. Using flow cytometry we evaluated the changes in LFA-1 molecule expression on peripheral lymphocytes. In the analyzed group of patients the proportion of LFA-1 "dim" cells was reduced. After the treatment the ratio was partly normalized. Twelve months after cessation of the therapy high proportion of LFA-1 "dim" was observed only among CY/MP treated patients. The changes were related to clinical improvement. Based on the obtained data, it seems, that the treatment affecting the expression of LFA-1 may slow down lymphocyte migration and by that limit chronic inflammation within the synovium.

Adult↗
PubMed1996

Cross-matches on donor cadaver retinal pigment epithelial cells in corneal risk patients.

BACKGROUND: Allografts can be rejected either through the antibody-mediated or cellular pathways. The objective of this study was to look at the extent of antibody formation in patients awaiting re-keratoplasty using cross-matches on cadaver retinal pigment epithelial (RPE) cells. METHODS: Cadaver RPE cells were derived by trypsin digestion from donor eyes (n = 1200). After 3 days of cell cultivation, the cells were adherent and began to lose their pigment. By day 7 most cells were clear and grew as a polygonal monolayer. MHC class I expression by RPE cells was studied by the W6/32 (anti-HLA-A, B, C) monoclonal antibody (MoAb) and that of class II (HLA-DR) by the 136 MoAb. Normal RPE cells express few class I and no detectable class II antigens. For the induction of MHC expression, cells were subsequently stimulated with 250 U/ml of recombinant gamma-interferon for 5 days. Cells were used for tissue typing and also for cross-matches with recipient serum. Cross-matches were subsequently performed and measured by flow cytometry. RESULTS: Both class I and class II antigens were strongly enhanced, as could be shown by immunohistochemical staining. Some 20% of those patients awaiting rekeratoplasty (n = 60) were positive for anti-HLA antibodies. In one case anti-DR3 antibodies were detected in a recipient who had had several rejection episodes after keratoplasty. CONCLUSIONS: RPE cells are not only useful for cadaver post-mortem HLA typing but also for donor-specific cross-matches. The degree of antibody formation after keratoplasty in rejecting patients was, however, low. This may imply that anti-HLA antibodies are not the major cause of corneal graft loss after keratoplasty.

Antibodies, Monoclonal↗
PubMed1996

MHC-associated and MHC-independent urinary chemosignals in mice.

The chemosensory identity of mice, rats, and humans is determined partly by polymorphic genes of the major histocompatibility complex (MHC). In inbred strains of mice as well as in seminatural populations MHC-associated mating preferences selectively influence reproductive success. To explore MHC-associated chemosignals in relation to otherwise genetically determined chemosignals a first study was conducted on seven trained rats' responses to the odors of inbred strains of mice. Results of the first study confirmed that neither the MHC nor genes in the genetic background dominate in determining urine odor specificity of mice and that specific olfactory cues associated with either the MHC or the genetic background can be identified by olfaction. In a second study, these specific olfactory cues were analyzed by means of gas chromatography. The results indicate that specific volatile components associated with either the MHC or the genetic background can be found in mouse urine odor, and that profiles of ubiquitous volatile components show some association with either the MHC or the genetic background. Furthermore, results show that a small number of specific compounds as well as a profile of some few ubiquitous volatiles constitute MHC-associated odor cues and that influences of the MHC and genes in the genetic background interact in constituting urine odor specificity in mice.

Animals↗
PubMed1996

Glass slide smears are a suitable source for RT-PCR-based analysis of chromosomal aberrations in leukaemias.

The polymerase chain reaction (PCR) is a well-established method to detect cytogenetic abnormalities. The handling of fresh specimens is difficult, therefore a method to use smears of blood or bone marrow as a source would be advantageous. Furthermore, such a technique would give the opportunity to investigate retrospectively bone marrow smears in leukaemias without cytogenetic results. The aim of the present study was to investigate the influence of staining procedures and laboratory handling of smears. We chose CML cases as a model. We demonstrated that smears are a suitable source for PCR without loss of information caused by previous routine laboratory handling.

Base Sequence↗
PubMed1996

High lytic activity against human leukemia cells after activation of allogeneic NK cells by IL-12 and IL-2.

IL-12 is a novel cytokine with interesting features regarding its potential usefulness in peripheral blood stem cell transplantation and leukemia immunotherapy. We used cryopreserved leukemia cells of 18 patients with acute myelogenous (n= 14) or lymphocytic (n= 4) leukemia to investigate the effect of IL-12, alone or in combination with IL-2, on the cytolytic activity of NK cells against human leukemia targets. Effector cells were peripheral blood mononuclear cells from healthy donors which were depleted from CD3+ T cells by immunomagnetic separation. CD3-negative effector cells (mainly CD56+ NK cells) were treated for 24 h with various concentrations of IL-2 (100 U/ml to 1000 U/ml) and IL-12 (1 U/ml to 100 U/ml). Cytotoxicity was measured in a 4 h 51Cr-release assay. Whereas a two-fold enhancement of cytotoxic activity was observed after incubation with optimal doses of IL-2 or IL-12, the combination of both cytokines (500 U/ml IL-2, 100 U/ml IL-12) increased the lytic activity more than six-fold. This effect was accompanied by increased expression of cellular adhesion molecules (CD2, CD18) and CD25 on CD56+ effector cells. Of 18 leukemias investigated, five were completely resistant to lysis by effector cells activated with IL-2 or IL-12 alone. In three of these five cases, however, high cytolytic activity was observed after coincubation with IL-2 and IL-12. In comparison to allogeneic NK cells, autologous cells of three patients in remission demonstrated significantly lower cytotoxic activity. No killing of nonmalignant cells (PHA blasts) by allogeneic NK cells was observed. Our data demonstrate that IL-12 can enhance or even induce MHC-unrestricted cytotoxicity of IL-2-activated allogeneic natural killer cells. Since IL-12 has also been shown to have stem-cell mobilizing capacities, it could be used for the recruitment of both stem cells and antileukemic effector cells in the context of peripheral blood stem cell transplantation.

Animals↗
PubMed1996

Analysis of tolerance inducing mechanism after application of oncogen-transformed macrophages.

The purpose of this study was to examine the expression of T cell receptors (TCR) and their V beta subclasses under the influence of the parental cell line P388D1 and its clones mos2 and mos3, using a mouse model. It was shown, that v-mos oncogene-transformed cells of this line (mos2) induced selective immunological unresponsiveness in vitro. Because the induction of tolerance is of a central importance for the organ transplantation, this phenomenon, found in vitro, was also studied in vivo. We found that the in vivo injection of mos2 cells into mice induced a state of selective noncreativity. To further analyse these effects, we studied whether specific tolerance is the consequence of a decreased number of essential receptors or receptor families. For this purpose C57BL/6 mice were immunized with cells of the parental line P388D1 or mos2 and mos3 clones. Their spleen and thymus cells were examined phenotypically. The most impressive result of this study was a clearly changed amount of T cells receptors in mos2 immunized mice, in which a state of tolerance was induced. In these mice only the expression of CD3 T receptors as well as that of the V beta 11 chains was reduced. In spleen of these mice the CD3 expression was decreased, compared to D1 or nonimmunized control animals by 54-58% and compared to mos3 mice by 38-40%. Even though the differences in the thymus were not very pronounced, we still saw a decrease in CD3 stained cells selective in mos2 immunized C57B1/6. The expression of V beta 11 chains on the surface of spleen cells of mos2 animals was reduced by 33.3%, on the thymocytes even by 50% comparing to that in nonimmunized mice. Whether the reduced expression of T receptor V beta families is due to changes in the genetic material (cDNA), has to be studied.

Animals↗
PubMed1996

Induction of specific tolerance by 15-deoxyspergualin (DOS) treatment after rat skin and kidney transplantation in rats. Analysis of effectivity of various protocols of DOS application.

The purpose of this study was to analyse the effect of various protocols of 15-deoxyspergualin (DOS) application on skin or kidney graft survival. Following rat strain combinations were used: AS-->LEW (MHC identical/non-MHC-different) and DA-->LEW (MHC-different/non-MHC-different). Reference DOS dose was 2.5 mg/kg, i.p. It was shown that the effect of DOS depended on multiple factors, such as: type of tissue or organ, onset of treatment, drug dose and length of drug application. In skin transplantation graft survival was 32-34 days in AS-->LEW and 24-26 days in DA-->LEW. Kidney graft survived more than 150 days. DOS prolonged skin survival when the application was started earlier than day 8, whereas kidney graft survived only when DOS treatment was started not later than 3-4 days after transplantation. In skin transplantation a dose of 0.3 mg/kg had a small effect-prolongation graft survival up to 4 days. Higher doses induced longer graft survival, however, maximal survival of allogeneic skin was 22 days. In kidney transplantation a dose of 0.3 mg/kg led to prolonged graft survival-up to 150 days. Doses of 2.0-2.5 mg/kg were able to induce specific tolerance. The optimal skin or kidney graft survival was obtained when DOS was applied for 14 days. Shorter than 12-day treatment with DOS led to a shorter graft survival. When donor was pretreated with DOS prolongation of non-allogeneic graft survival was observed. Our results showed that short-term application of DOS is safe and effective. To obtain optimal DOS effect the drug application must be started directly after transplantation.

Animals↗
PubMed1996

In vitro analysis of the mode of action of the immuno-suppressive drug 15-deoxyspergualin.

The purpose of the study was to analyse the action of the immunosuppressive drug 15-deoxyspergualin (DOS) in vitro. We studied: a) the influence of DOS alone and DOS in combination with various monoclonal antibodies on alloantigen stimulation in the mixed lymphocyte culture (MLC), b) the influence of DOS treatment on the MHC class I and II expression of splenocytes, lymph node cells and peritoneal macrophages, c) the influence of DOS treatment on a suppressor cell population. Our study showed that: a) DOS inhibits interleukin 1 (IL-1) secretion by macrophages, leading to reduction of immune response to alloantigens. This effect was neutralized by addition of IL-1; b) DOS treatment has no influence on MHC class II antigen expression, but induces changes of MHC class I expression. After DOS application in a population of spleen macrophages a subpopulation of cells with reduced MHC class I antigen expression appeared. Down-regulation of these molecules was also observed in immunomorphological studies of kidney graft sections of rats treated with DOS after transplantation; c) after DOS treatment suppressor cells were detected in "suppressor" MLC, 16-33 days after kidney transplantation. Their activity was confirmed 137 days after treatment with DOS, but were inactive in the case of third party cells. These results suggest that DOS action is based on a blockade of antigen presentation by reducing IL-1 production, down-regulation of MHC class I antigen and by inducing suppressor cell population.

Animals↗
PubMed1995

Magnetophoresis: II. Quantification of iron and hemoglobin content at the single erythrocyte level.

The iron and hemoglobin content of individual erythrocytes was determined using a method based on parallel velocity measurements during magnetophoresis and gravitational sedimentation of individual erythrocytes in suspension. In previous publications we have suggested employing cell magnetophoresis, a biophysical phenomenon characterized by cell movement in a fluid under magnetic field influence, for cytometry. The paramagnetic ferric iron in methemoglobin is used as a magnetic label. The iron content is estimated from the magnetophoresis velocity, and hemoglobin content from the gravitational sedimentation velocity of erythrocytes. Blood samples are also analyzed in a Coulter counter to determine their mean corpuscular hemoglobin. The time course of the reaction of methemoglobin reduction is quantified at the single erythrocyte level. The methemoglobin content in individual erythrocytes is determined following the oxidation reaction. Erythrocytes from patients with normo-, hypo-, or hyperchromic anemia exhibit magnetophoresis and gravitational sedimentation velocities that correlate closely with mean corpuscular hemoglobin. We propose the utilization of magnetophoretic cytometry for detailed diagnostic studies at the single erythrocyte level. Furthermore, the magnetophoresis velocity to gravitational sedimentation velocity ratio is proposed as a standard value for comparative study of magnetically labeled cells in future investigations, as it was found to be constant and independent of hemoglobin content.

Anemia↗
PubMed1995

Short-course cyclosporin A therapy for definite allograft valve survival immunosuppression in allograft valve operations.

This study was designed to determine the effect of short-course cyclosporin A therapy (10 mg/kg daily for 14 days) on allograft valve survival across the histocompatibility barriers in the following rat models; (1) syngeneic Lewis to Lewis (herein referred to as autografts), (2) weakly allogeneic AS to Lewis (RT1 compatible, non-RT1-incompatible), and (3) strongly allogeneic CAP to Lewis (RT1 and non-RT1-incompatible). Cyclosporin A-treated and untreated recipient animals (Lewis) received allovital and antibiotic-treated viable allografts implanted into the infrarenal aorta. Second-set skin grafting was performed 3 weeks after heterotopic valve implantation to test for immunogenicity and presensitization. The animals (Lewis) were sacrificed serially on days 20, 50, 100, and 150 for immunofluorescence study using mouse monoclonal antibodies (OX6) directed at class II endothelial surface antigens. The allografts in weakly allogenic strains showed no humoral response under a short course of cyclosporin A. The cyclosporin A-untreated allovital grafts and the viable (antibiotic-treated) valves demonstrated fibrocalcification on the 100th and 150th postoperative days, respectively. In conclusion, it seems that a short course of nontoxic immunosuppression could arrest allograft rejection and thus prevent early degeneration of allografts. Furthermore, antibiotic-treated viable allografts seemed to be more durable than allovital grafts.

Animals↗
PubMed1995

Porcine valves are reendothelialized by human recipient endothelium in vivo.

The degeneration of human allogeneic and porcine xenogeneic heart valves has not been clearly understood. The question is whether the observed loss of function and calcification is primarily an immunologic process or a mechanical process or is influenced by both factors. In the current study, we looked at explanted xenogeneic heart valves for the presence of recipient endothelium. Explanted valves were shock frozen and stored at -80 degrees C before use. They were subsequently examined by immunohistochemical staining with a variety of monoclonal antibodies. Xenogeneic valves showed clearly positive results for the human major histocompatibility complex class I and class II antigens and morphologically showed a thin layer of viable endothelium restricted to the annular region of the valve. Additionally, they were also positive for intercellular adhesion molecule-1 and the H-Y antigen. Although the xenogeneic valves were significantly degenerated, the endothelium was clearly defined and could be identified immunohistochemically as being of recipient origin. The grafts remained negative for endothelial cell-leukocyte adhesion molecule-1 and factor VIII. These data allow speculation on whether reendothelialization of valvular grafts with recipient endothelium is a normal repair mechanism in vivo.

Animals↗
PubMed1995

G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: comparison of T cell depletion strategies using different CD34+ selection systems or CAMPATH-1.

Allogeneic transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) appears to be an attractive alternative to allogeneic bone marrow transplantation (BMT). However, because vast amounts of potentially graft-vs.-host-reactive T cells are transfused with PBPC grafts, the use of PBPC in the allogeneic setting may be associated with an increased incidence or severity of graft-vs.-host disease (GVHD). To evaluate strategies for prevention of GVHD after PBPC allografting, we have studied T cell depletion (TCD) of G-CSF-mobilized PBPC samples harvested from six healthy donors and from five patients scheduled for autologous PBPC transplantation. Three approaches (CAMPATH-1 plus autologous complement [C], immunomagnetic CD34+ cell selection, and biotin-avidin-mediated CD34+ cell selection) were compared. TCD of PBPC samples with the monoclonal antibody (MAb) CAMPATH-1 plus autologous C resulted in a median elimination of 2.16 log CD3+ T cells, whereas 39% of CD56+ natural killer (NK) cells and 56% of CD34+ progenitor cells were recovered. TCD by CD34+ cell selection with the Isolex (Baxter, Munich, Germany) or Ceprate (CellPro, Bothell, WA) devices achieved median depletions (Isolex vs. Ceprate) of 4.04 vs. 3.12 log T cells and > 5 vs. 3.27 log NK cells while allowing the recovery of 36 vs. 27% CD34+ cells. The median purity of CD34+ cells in the final product was 1.7 (CAMPATH-1), 94 (Isolex), and 65% (Ceprate). We conclude that all methods tested effectively deplete T cells from PBPC preparations harvested from healthy donors. Whereas immunomagnetic CD34+ selection is most effective in terms of elimination of T cells, the less intensive T and NK cell depletions achieved with CAMPATH-1 might be advantageous with regard to retaining engraftment potential and graft-vs.-leukemia (GVL) activity of PBPC allografts.

Adolescent↗