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Biomedical subjects

W Müller-Ruchholtz

Publications and source records attributed to W Müller-Ruchholtz.

At least 37 records · Page 2Linked to original sources
PubMed1995

Leukemia-specific allogeneic donor T cells: quantification by limiting dilution assay.

Allogeneic T cells are capable of discriminating between leukemia cells and non-malignant hematopoetic cells. This has been concluded from clinical BMT data and demonstrated by in vitro experiments. We analyzed the frequency and specificity of leukemia-reactive T cells from syngeneic and allogeneic blood donors by limiting dilution assays for interleukin-2-producing (TH1) and cytotoxic (CTLp) T cells. Target cells were leukemia blasts obtained from a patient with common ALL. Control targets were generated by EBV transformation. Effector cells were generated from the peripheral blood of the patient in remission, from his syngeneic brother and from eight healthy, HLA-mismatched volunteers. The effector cells were stimulated with leukemia cells and interleukin-2. Neither the patient nor his brother were able to generate anti-leukemic CTLp or TH1. The HLA-mismatched allogeneic donors displayed anti-leukemic CTLp frequencies with a range from 0 to 68 million. TH1 cells with anti-leukemic specificity were not detectable. We conclude that there are great inter-individual differences in the GVL potential of fully allogeneic peripheral T lymphocytes. It is possible to identify T cell lines with reactivity against leukemia blasts and non-reactivity against normal hematological cells from the same individual. These cell lines are potential effector cells for immunotherapy of human leukemia.

Blood Donors↗
PubMed1995

Allogeneic mouse T cell lines distinguish three Balb/c leukemias from each other and from non-leukemic lymphocytes.

In order to separate graft-versus-leukemia (GVL) effect from graft-versus-host (GVH) reactions in allogeneic cell therapy, we established cytotoxic T cell lines (CTL) against irradiated A20, WEHI-3 and PU cells, ie cells from 3 hematopoietic tumors of Balb/c origin. Immunization with these cell lines did not lead to prolonged survival in Balb/c mice. The cytotoxic activity of the CTL was tested against the 3 leukemias. In all 9 combinations the highest specific lysis was achieved against the stimulating tumor. Cold target inhibition experiments showed that the activity against 51Cr-labelled leukemia cells could be almost completely inhibited by adding a sufficient amount of unlabelled target cells. On the other hand, when unlabelled concanavalin A-induced Balb/c blast cells were added, the inhibition was incomplete. By means of flow cytometry it was excluded that the different susceptibilities and inhibitory potentials of tumor cells and nonmalignant blasts are caused solely by differences in the MHC expression of the target cells. These findings confirm that allogeneic CTL are capable of discriminating malignant from nonmalignant cells, even if the tumor is nonimmunogenic in syngeneic animals.

Animals↗
PubMed1995

Influence of a rat kidney perfusion using the anti-MHC class II monoclonal antibody and reduced dose of 15-deoxyspergualine on the allogeneic graft survival.

One of the main factors inducing rejection of the allogenic graft are the donor MHC-class II antigens. In this study, the allogenic rat renal graft survival after the blockage of MHC-class II positive cells was analyzed and compared with the effectiveness of the recipient treatment with 15-deoxyspergualine (15-DOS). It was found that the DA (RT1 a) rat kidney perfusion with the anti-MHC class II monoclonal antibody (MoAb 29A1--Kiel) allowed to prolong survival of the graft in the LEW (RT1 1) recipient (9.6 +/- 0.8 vs. 7.7 +/- 0.5 days). Our another study demonstrated that the 14 day treatment of the LEW recipient with 15-DOS at the dose of 0.5 mg/kg body weight can induce tolerance to the grafted kidney from the DA strain. The dose of 0.2 mg/kg body weight of 15-DOS prolonged the graft survival only to a small extend (16.5 +/- 0.5 days). In contrast, the combination of the graft pretreatment with MoAb 29A1 with the application of the reduced dose of 15-DOS to the LEW recipient allowed to further prolong the graft survival (97.4 +/- 59.0 days, n = 5). In 3 cases, the long-time (close to 150 days) graft survival was obtained. The above presented results suggest that the blockade of the MHC-class II antigens can reduce the immunogenicity of the graft. Although this procedure was not sufficient to induce tolerance, it allowed to minimize the immunosuppressive treatment.

Animals↗
PubMed1995

Cell migration between graft and host--an analysis with monoclonal antibodies after allogeneic rat kidney transplantation.

In order to analyse migration patterns of donor MHC class II cells out of transplanted kidney and accumulation of host cells within the graft, immunomorphological studies were performed using monoclonal antibodies in rat allogeneic kidney transplantation model. To answer the question of how many donor cells migrate out of the renal cortex MRC 0 x 3 monoclonal antibody (MoAb) against LEW MHC class II antigens was used. In the grafts explanted after 4,24 48 and 73 h, a slow reduction of donor class II cells was observed and some areas in cortex showed only very few, if any, donor cells. At the same time, starting from day 2 after transplantation accumulation of donor cells was found in perivascular spaces. Spleen sections stained at 24, 48, 72 and 96 h after transplantation revealed donor cells present in recipient's spleen. They were detected up to day 3 after surgery. Their numbers, however, decreased after day 2. After 2 and 3 days, accumulations of recipient's cells between tubules were detected. It was found that many cells in infiltrations were stained with anti-T lymphocyte MoAb. Expression of class II antigen on rat kidney cells increases significantly from the day 4 after transplantation.

Animals↗
PubMed1994

Failure of in vitro T-cell assays to predict clinical outcome after human kidney transplantation.

Allotransplant rejection is a T-cell-dependent reaction. Functional in vitro T-cell assays are being used widely for donor-recipient matching in bone marrow transplantation and have recently been used in some centres for transplant monitoring. In order to assess tolerance induction after clinical transplantation, we measured the T-cell response of the host against donor spleen cells of 33 kidney transplant patients before and every 3 months after transplantation over a period of 18 months. The T-cell reactivity before transplantation was not significantly different in any of the assays in rejecting and non-rejecting patients. In the classical mixed lymphocyte culture (MLC), a donor-specific loss of reactivity was seen only in a patient with a CMV-associated irreversible transplant rejection. One patient with chronic rejection acquired a very high MLC response against donor spleen cells and a high response against third-party cells. Little or nonspecific changes were seen in the MLCs of all other patients. Using the method of limiting dilution analysis (LDA), we found a significant reduction of donor-specific cytotoxic T-cell precursors (CTL-p) within the first 3 months after transplantation in most patients with high antidonor CTL-p frequencies before transplantation. The reduction of donor-specific CTL-p was seen in patients with rejection episodes as well as in patients without. Thus we conclude, in contrast to others, that MLC and CTL-p LDA have no predictive value on the outcome of clinical transplantation.

Clonal Deletion↗
PubMed1994

Magnetophoresis: I. Detection of magnetically labeled cells.

A new method for the detection of magnetically labeled cells in suspension using an optical microscope is presented. The movement of individual cells in a nonuniform magnetic field (magnetophoresis) can be studied and the direction and velocity of their movement can be determined. Cell-associated magnetic particles (e.g., attached magnetic immunomicrospheres) influence these parameters. We developed a magnetophoresis chamber for measuring the movement of single cells in a high-gradient magnetic field. Paramagnetic erythrocytes were used to monitor the level of magnetic force in the chamber. Bw6 antigen-positive REH cells were used as a biological model. Magnetic microspheres coupled with anti-Bw6 antibody were used as magnetic labels. The movement of magnetically labeled REH cells was investigated versus the intensity of magnetic field. The magnetization of microspheres was experimentally determined by the Faraday balance method. Experimental and theoretical results substantiate the feasibility of detecting a single cell associated with a single magnetic microsphere. Thus, magnetophoresis provides a sensitive biometrical system for magnetically labeled entities.

Humans↗
PubMed1994

Isolation of porcine pancreatic islets: low trypsin activity during the isolation procedure guarantees reproducible high islet yields.

During the past few years, interest in xenotransplantation of porcine islets of Langerhans for the future therapy of type I diabetes has increased markedly. Therefore, we established a semiautomated digestion method for isolating islets from the porcine pancreas. However, although the isolation technique was standardized and collagenase of controlled quality was used, we were unable to attain high islet yields with a satisfactory degree of reproducibility. One hypothesis was that varying degrees of interference by donor pancreatic enzymes were responsible for this failure. The aim of this study was to examine the kinetics of four types of enzymatic activity during the isolation procedure, as well as their effects on islet yield: collagenase, trypsin, neutral protease, and clostripain. Our results indicate that while exogenous collagenase activity decreases slightly during the isolation procedure, the activity of the pancreas enzymes neutral protease and trypsin increases. In some cases, trypsin activity increases very strongly. A strong increase in trypsin activity correlates with poor islet yield, whereas low trypsin activity always correlates with high islet yield. Addition of the protease inhibitor Pefabloc to the isolation medium results in low trypsin activity and reproducible high islet yields.

Animals↗
PubMed1994

Soluble MHC class I molecules in human body fluids.

Apart from their well-known function of antigen presentation in the form of peptides, major histocompatibility antigens (MHC) have been found to be unique markers of individual body odors in murine experimental models. In the current study we examined the nature and expression of soluble human MHC class I molecules in body fluids. Biochemical analysis of affinity purified serum class I molecules revealed a variety of molecules within the molecular weight region of 45 to 21 kD. SDS-Western blotting of HLA derived from hepatocytes and spleen cells suggested that much of the small molecular mass fraction of sHLA (< 45 kD) found in serum is derived from the liver. sHLA were detected and quantitated in serum, plasma, urine, cerebrospinal fluid, and sweat. No sHLA were detectable in cerebrospinal fluid (n = 20). In addition, sHLA was measured in serum of women during the menstrual cycle. A significant increase in sHLA was observed during the first half of the cycle, suggesting that sexual hormones may increase sHLA concentration. The observed increase was most prominent in women that were HLA-A24.

Adult↗
PubMed1994

In vitro effects of solubilized HLA-DR--role in immunoregulation?

To test the hypothesis that soluble HLA-DR antigens (sHLA-DR), binding to the T-cell receptor (TCR) and/or CD4 structures, compete with and abrogate functions of their cell-bound counterparts, we studied effects of detergent-solubilized, affinity-purified HLA-DR molecules on the DNA synthesis, IL-2, and IL-1 secretion by human peripheral blood mononuclear cells (PBMC). While resting T cells did not show any response, there was a dose-dependent suppression of T-cell responses induced by mitogen (phytohemagglutinin, PHA), recall antigen (purified protein derivative of tuberculin, PPD), or HLA class II alloantigens (Daudi cells). In the PHA system, sHLA-DR affected DR-identical and DR-disparate PBMC with equal efficiency, suggesting a nonspecific interference with accessory functions of cell-bound HLA class II molecules. A competitive ligation of lymphocyte (rather than of monocyte) CD4 is suggested based on the failure of sHLA-DR (i) to potentiate inhibitory effects of anti-CD4 mAb, (ii) to suppress mitogen responses after depletion of CD4+ lymphocytes and also following separate monocyte (vs lymphocyte) pretreatment, and (iii) to induce a reproducible IL-1 secretion inhibition. In the PPD system, suppressive sHLA-DR effects on autologous or DR-identical PBMC significantly exceeded that on DR-disparate PBMC, and in the MLR, third party allogeneic sHLA-DR was exceeded in its suppressive potency both by sHLA-DA pertinent to responder cells and by sHLA-DR pertinent to stimulator cells. These additional specific effects may result from competition (with cell-bound class II restriction and recognition determinants) at the TCR level rather than from peptide competition at the antigen-presenting cell level. Interference by sHLA-DR with the primary and/or accessory signaling may offer new therapeutic strategies in allotransplantation and autoimmunity.

Dose-Response Relationship, Drug↗
PubMed1994

Quantitative and biochemical characterization of soluble HLA class I antigens shed by cultured human cells.

sHLA has been described in human serum and other body fluids. In this study sHLA shed by cultivated human cells and their biochemical nature in solution were studied. EBV-transformed human B-lymphoblastoid cell lines (n = 4), permanent human lymphoblastoid tumor cell lines (n = 4), and PBLs from three donors were cultivated in vitro and sHLA measured in the supernatants. The Daudi cell line was used as a negative control in all experiments. Maximum expression of sHLA was measured after 8 hours, after which the concentrations gradually declined. The allospecificities A2 and B7 were also detectable in the ELISA. sHLA in the supernatants was further characterized by 1D-IEF. All bands representing the allotypes were detected, showing that cell supernatants can be used as antigen sources for biochemical tissue typing. These data show that sHLA expression is a characteristic of viable cells.

Antibodies, Monoclonal↗
PubMed1994

G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: safety, kinetics of mobilization, and composition of the graft.

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) makes the general anaesthesia of the donor unnecessary and may result in more rapid engraftment and faster recovery of the immune system. We have studied G-CSF-mediated PBPC mobilization in healthy donors and analysed the cellular composition of the resulting PBPC grafts. PBPC grafts were obtained from nine healthy donors (18-67 years old) for allogeneic or syngeneic transplantation. Six donors received 10 micrograms/kg G-CSF per day, the others 5-6 micrograms/kg. Mobilization and harvesting were well tolerated except for moderate bone pain which occurred in all donors primed with 10 micrograms/kg. With 10 micrograms/kg, a 31-fold (9-62) enrichment of circulating CD34+ cells was observed with peak values constantly occurring on day 5 after the start of G-CSF administration. Starting harvest on day 5, one to three collections on consecutive days yielded 5.5 x 10(6)/kg (0.9-10.7) CD34+ cells, 219 x 10(6)/kg (106-314) T cells, and 34 x 10(6)/kg (23-67) NK cells per 10 litres leukapheresis volume. Altogether, PBPC grafts contained 3 times more CD34+ cells, 7 times more T cells, and 20 times more NK cells than five allogeneic marrow grafts that were analysed for comparison. The yield of CD34+ cells per 10 litres apheresis volume as well as the height of the CD34+ peak in peripheral blood were inversely correlated to the age of the donor. In the donors primed with 5-6 micrograms/kg G-CSF the increase of circulating CD34+ cells (4-7-fold enrichment) and the CD34+ cell yield per 10 litres leukapheresis volume (1 x 10(6)/kg [0.8-2.2]) was much smaller compared with the 10 micrograms/kg group. In conclusion, sufficient amounts of PBPC capable of restoring haemopoiesis in allogeneic recipients can be mobilized safely by administration of G-CSF (10 micrograms/kg s.c. for 5 d) in healthy donors, and harvested with one or two leukapheresis procedures. Whether the large numbers of T-cells and NK cells that are contained in the collection products may influence graft-versus-host and graft-versus-leukaemia reactivities of PBPC grafts remains to be determined.

Adolescent↗
PubMed1994

MHC-associated and MHC-independent urinary chemosignals in mice are expressed via the hematopoietic system.

The change in genetically determined urine odors which appears after experimental bone marrow transplantations in mice was examined in order to test whether the HMC is the only group of genes that influences the chemosensory identity via the hematopoietic system. 5 female rats were trained in a computer-controlled olfactometer to discriminate urine odors of two MHC congenic or background congenic inbred strains of mice. Transfer-of-training tests with urine samples of irradiated mice which were restored with bone marrow either from an MHC congenic or a background congenic inbred strain reveal a change of urinary chemosignals after both types of experimental bone marrow transplantation. Thus, both MHC-associated as well as MHC-independent urinary chemosignals are expressed via the hematopoietic system.

Animals↗
PubMed1994

Human monoclonal antibodies recognize early and late viral proteins of human cytomegalovirus.

Human monoclonal antibodies directed against human cytomegalovirus were generated by fusion of in vitro stimulated human spleen lymphocytes from an HCMV-seropositive 53-year-old organ donor with the mouse myeloma cell line Ag8.653. Fourteen human/mouse hybridomas producing anti-cytomegalovirus IgG were screened by an ELISA technique and four selected clones have been established since March 1988, generating about 5-40 micrograms/24 h IgG per ml culture supernatant. Reference and local cytomegalovirus strains were stained by the antibodies without showing cross-reactivity to other herpes viruses. Three monoclonal antibodies, A4B4 (IgG11), A6B3 (IgG1k) and A6A2 (IgG1k), immunoprecipitated a 68 kDa early viral protein which appears during the infectious cycle, first in the nucleus (18-24 h) and then also in the cytoplasm (24-96 h) of infected cells. Inhibition of DNA replication restricted the detection of the 68 kDa viral protein to the nucleus of infected cells. Staining of unfixed infected cells showed that two of the antibodies bound at the surface of a few cells. The fourth monoclonal antibody A3C5 (IgG11) immunoprecipitated a 34/38 kDa late viral protein which appears in the nucleus (48-72 h) of infected cells. These antibodies enable us to study the human host response to human cytomegalovirus and to elucidate the functions of human antibodies especially in their interaction with the T-cell response.

Animals↗