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Biomedical subjects

W Müller-Ruchholtz

Publications and source records attributed to W Müller-Ruchholtz.

At least 91 records · Page 5Linked to original sources
PubMed1991

The influence of MHC class II antigen blockade by perfusion with a monoclonal antibody on rat renal graft survival.

To decrease immunogenicity of the rat kidney, grafts were perfused with an anti-MHC class II monoclonal antibody (mAb). How effectively this procedure blocked class II-positive cells, which were mainly dendritic in appearance, was checked by immunostaining renal sections after perfusion and comparing them with in vitro stained sections. Optimum conditions were applied for graft pretreatment before transplantation. This procedure prolonged graft survival, though not satisfactorily from the biological point of view (9.6 +/- 0.8 versus 7.7 +/- 0.5 days in the control group; P less than 0.02). The dendritic cells were not killed but blocked. Several hours after transplantation, the mAb dissociated from these class II-positive cells. It was also shown that donor cells migrate into the recipient's spleen early after transplantation. The number of these cells was smaller when the transplanted organ was perfused with the mAb. Further studies are suggested to deplete the graft of donor dendritic cells more adequately. They should also combine graft perfusion with anti-class II mAb and recipient immunosuppression at reduced doses.

Animals↗
PubMed1991

[Immunological reactions against preserved tracheal transplants? Studies on inbred rat strains].

To determine whether chemical (cialit, merthiolate, alcohol, formaldehyde solution) preserved tracheal segments maintain immunogenicity we performed systematic transplantation-immunological investigations using in vivo-, in vitro- and immunohistological tests. In contrast to other authors no indication of residual antigenicity was found. The survival rates after orthotopic grafting of preserved tracheal segments were shortened. This seems due to the loss of the mucociliary clearance, caused by chemical induced damages in the tracheal mucosa.

Animals↗
PubMed1991

Inhibition of anti-HLA-B7 alloreactive CTL by affinity-purified soluble HLA.

The objective of this study was to elucidate the interaction of naturally occurring soluble MHC class I molecules with alloreactive CTL and to discuss its possible relevance to graft acceptance. An anti-HLA-B7 specific CTL-line, BV.B7, was generated in vitro. On phenotyping the cells after 6 weeks, 80% were found to be CD8+, 14% CD4+ and 6% CD8+CD4+. CD4+ CTL were depleted using immunomagnetic beads precoated with an anti-CD4 antibody. Of the recovered CTL greater than 96% were CD8+. A total of 12 HLA-B7 target cell lines and PHA blasts tested were specifically lysed in a 51Cr-release assay. Soluble HLA class I molecules were isolated on affinity chromatography columns using the anti-HLA-B7 ME 1 and the anti-heavy chain W6/32 monoclonal antibodies. Antigen purity was confirmed by analysis on SDS-PAGE gels. CTL were preincubated with 0.1-1.8 micrograms/ml soluble HLA for 30 min at 37 degrees C and subsequently tested for cytotoxicity in the 51Cr-release assay; 1.1 micrograms/ml HLA-B7 molecules reduced CTL cytotoxicity by 50% whereas non-B7 HLA had no effect. Further, CTL cytotoxicity was reduced by preincubation with anti-CD8, anti-TcR, and anti-CD3 antibodies. We anticipate a possible down-regulatory role of soluble HLA on CTL in allogeneic transplantation.

Antibodies↗
PubMed1991

Expression of MHC class I and II molecules by cadaver retinal pigment epithelium cells: optimization of post-mortem HLA typing.

The objective of this study was to investigate the expression of MHC antigens by retinal pigment epithelium cells (RPE) after stimulation with interferon-gamma (IFN-gamma) and to improve the currently practised technique of cadaver HLA typing. A concentration of 100 U/ml IFN-gamma induced expression of class I molecules up to greater than 90% 3 days after stimulation, whereas 50 U/ml were required for the expression of HLA-DR to greater than 90%. A concentration of 750 U/ml induced 35-45% expression of HLA-DP and less than 25% HLA-DQ after 3 days. Cells were serologically typed using the standard lymphocytotoxicity assay 3 days after stimulation with 250 U/ml IFN-gamma. Typing of class I specificities was complemented by one-dimensional isoelectric focusing (1D-IEF). We observed high concordance between the results of the RPE typing and the lymphocytotoxicity test on the same donors. Our results show complete typing of class I and II antigens post-mortem, which, in particular, enables graft matching and improvement of graft survival in recipients of organs removed many hours after death such as the cornea.

Adolescent↗
PubMed1991

MHC allograft immunogenicity--its role in bone marrow transplantation and its constant downregulation.

Since 1972 our laboratory has performed a large variety of studies in the rat and mouse models. These have shown that it is possible to achieve MHC fully allogeneic hematopoietic chimerism with complete restitution of immunological reactivity but lasting immunotolerance of donor type cells following transplantation of lymphocyte-purged bone marrow into lethally irradiated recipients. The clinical application of this obvious immunobiological potential is, however, still in its infancy. This is largely due to the belief that graft-versus-host reactions and host-versus-graft reactions balance each other out--a view that cannot be upheld. A decisive element in the network of immunoregulation following BMT is the immunogenic strength of the BM. In this context the following bits of information are given: (1) Bone marrow that has been completely purged of lymphocytes loses its allograft immunogenicity to a great extent and may even allow induction of tolerance, whereas unpurged BM is strongly immunogenic. (2) This reduction of alloimmunogenicity is only partially determined by lymphocytes and other MHC class II-expressing cells. (3) By incubating bone marrow with our new cytotoxic monoclonal antibody K31, directed against an epitope located an all human lymphocytes and defined cells of the monocytic lineage, both the alloimmunogenicity and the accessory cell potential of the bone marrow can be greatly reduced. (4) Recent analyses with macrophage cell clones rather than heterogeneous mixtures of BM cells indicate that oncogene transfections may allow us to completely down-regulate their alloimmunogenicity, in spite of unaltered high MHC expression, by genetically downregulating Il-1. In conclusion, it is suggested that MHC allogeneic BMT will become feasible in the human, as it is well-established in preclinical models.

Animals↗
PubMed1991

[Postmortem serologic and biochemical HLA typing with cultivated retinal pigment epithelium cells].

A quick and reliable method for HLA typing of human cadavers has been established. Retinal pigment epithelial cells (RPE) were obtained from cadaver bulbi 8-36 h post mortem (n = 24). After 24 hours, cell growth correlated inversely with the time lapse between the donor's death and bulbus explantation. Cultivated human RPE were stimulated by different Y-interferon (IFN) concentrations and examined by FACS analysis for the expression of MHC class I and II molecules. A concentration of 100 units/ml human Y-IFN was sufficient to induce maximal class I antigen expression by about 90% of cells after 3 days. At 50 units/ml Y-IFN, the cells showed maximal class II antigen expression for HLA-DR, whereas 100 units/ml I-IFN was required for maximal HLA-DQ expression. Higher concentrations of Y-IFN induced a further HLA-DP expression. However, serological HLA typing was performed after cell stimulation with 250 units/ml Y-IFN for 3 days. A concentration of 500 units/ml Y-IFN yielded equivalent results, but 750 units/ml Y-IFN led to poor identification of class I MHC specificities. Unclear serological results on class I specificities were clarified by 1D-isoelectric focusing (1D-IEF), thus allowing full HLA typing of all donor bulbi examined. In summary, successful HLA typing of cultivated RPE is dependent on both the Y-IFN concentration and the time lapse between stimulation and typing of the RPE. The method provides a much improved basis for cadaver cornea grafting.

Adolescent↗
PubMed1990

Measurement of soluble MHC class I molecules in renal graft patients: a noninvasive allograft monitor.

We investigated the biochemistry of naturally occurring major histocompatibility complex (MHC) class I molecules in human serum and established a quantitative enzyme-linked immunoassay (ELISA) to determine soluble human leukocyte antigen (HLA) concentration. Peptides of 46, 40, 37, 35, and 12 kDa were isolated on affinity chromatography columns using two monoclonal antibodies (MoAbs), the anti-heavy chain W6/32 and the anti-beta 2-microglobulin BBM. 1. These peptides were confirmed on Western blots by the HC-10 MoAb, which binds a monomorphic epitope on denatured heavy chains. In detergent-binding experiments, only the 46-kDa peptide could be isolated from solubilized cell membranes. No quantitative differences between serum and plasma HLAs of the same individual were measured. Soluble HLA expression in 12 renal graft recipients was measured over 1-3 mo posttransplantation. A highly significant increase of 50%-100% was noted during rejection episodes. Clinical signs of rejection were accompanied by poor renal function, including elevated creatinine values. After the crises had been managed, class I levels normalized to 0.3-1.5 micrograms/ml, which is the range in healthy persons. Patients who showed no rejection crises maintained constant levels within the period of study. We anticipate the application of soluble HLA measurement in clinical practice as a noninvasive graft monitor.

Enzyme-Linked Immunosorbent Assay↗
PubMed1990

Evidence that reduction of immunogenicity of T-depleted bone marrow depends on additional depletion of accessory cells. Implications for prevention of graft rejection.

An effective prophylaxis of graft-versus-host reaction in clinical bone marrow transplantation can be achieved by T depletion of the graft. However, this kind of graft manipulation is accompanied by a strongly increased incidence of graft rejection. In order to eliminate this hazardous complication we studied the possibility of preventing rejection of a BM graft by reducing its immunogenicity. In this report we demonstrate that the capacity of human BM cells to induce proliferation of allogeneic T cells is strongly reduced after treatment with the antilymphocyte/antimonocyte monoclonal antibody K31+ rabbit complement (C), indicating a reduced immunogenicity of the BM cells. On the other hand, the allostimulatory capacity of BM treated with clinically established T-depleting MoAbs, including the more broadly reactive CAMPATH-1, was not or only slightly reduced. MHC class II induction on BM cells with human recombinant gamma-interferon did not restore the allostimulatory capacity reduced by K31+C. Exogenous IL-1 partially inhibited the K31-mediated reduction of allostimulatory capacity. Reduction of the allostimulatory capacity of BM cells with K31+C correlated with a reduction of their capacity to support mitogen-induced proliferation of purified autologous T cells, indicating the absence of accessory functions. Addition of autologous peripheral adherent cells to K31+C treated BM restored its accessory function as well as its allostimulatory capacity. Labeling studies confirmed that accessory cells are critical for the described K31-mediated effects. We conclude that depletion of accessory cells in addition to lymphocytes with K31+C strongly reduces the immunogenicity of a BM graft, may help prevent graft rejection, and may serve as a model for preconditioning other organ grafts.

Antibodies, Monoclonal↗
PubMed1990

Characterization of soluble HLA molecules in sweat and quantitative HLA differences in serum of healthy individuals.

Soluble class I molecules were immunoprecipitated from human sweat and serum using the BB7.7 monoclonal antibody (mAb) coupled to immunomagnetic beads. Molecules were analysed biochemically on SDS-PAGE gels and finally by ID-isoelectric-focusing (IEF). Serum- and sweat-HLA IEF-band patterns of the same individual were fully identical, showing that HLA excreted in sweat possess polymorphic structures like those in serum. Quantitatively, we used a highly sensitive competitive enzyme-linked immunosorbent (ELISA) assay to determine soluble class I concentrations. The first group was that of non-HLA-A9 and -Bw62 sera, which were found to contain HLA levels with a mean concentration of 0.82 +/- 0.63 microgram/ml (n = 44). However, sera that were HLA-A23 or -24 (splits of HLA-A9) contained higher levels, with a mean of 3.2 +/- 0.94 microgram/ml (n = 20). Similarly, HLA-Bw62 individuals had a higher mean of 2.05 +/- 0.65 micrograms/ml (n = 10). The difference of the HLA-A9 group to the first group was statistically highly significant, P less than 0.0001, and that of the HLA-Bw62 to the first was also significant, P less than 0.004. Individuals who were both HLA-A9 and -Bw62 (n = 5) did not express significantly higher levels than those who only had one of these specificities. Sweat HLA levels had a mean of 0.42 +/- 0.4 microgram/ml (n = 10). These results show for the first time that soluble class I peptides are excreted in relatively high concentrations in sweat and possess polymorphic structures identical to those of serum HLA and that serum HLA levels are allotype dependent.

Female↗
PubMed1990

Immunogenicity testing of food proteins: in vitro and in vivo trials in rats.

The immunogenicity of an industrially produced bacterial food protein (single cell protein, SCP) was analyzed in a comparative study. SCP, casein and ovalbumin were injected into or fed to rats. The systemic response was tested in vitro with a lymphocyte transformation test and in vivo with a footpad swelling assay. Feeding of casein induced cell-mediated reactivity against casein, while feeding of SCP or ovalbumin had no effect. One injection of casein led to sensitization compared with three injections of SCP. Prior feeding of SCP did not abrogate the response to injected SCP. The footpad swelling test is suggested as a particularly effective model for helping to determine the immunological risk of foodstuffs to man.

Animals↗
PubMed1990

Human monoclonal antibody Ha6D3, a candidate for treatment of leukaemia? In vitro reactivity of Ha6D3 with leukaemic cells and in vivo applications in a chimpanzee.

The human monoclonal antibody Ha6D3 of the IgM type was used to stain malignant lymphoma cells from peripheral blood in flow cytometry and from cryosections of lymph nodes using the immunoperoxidase technique. It was found to react with peripheral white blood cells of all 12 cases of leukaemia and with lymph node cells of seven out of 11 B cell lymphomas and with the one T cell lymphoma tested so far. For in vivo experiments a batch of 70 mg Ha6D3 was purified and 6 mg Ha6D3 was injected intravenously into a chimpanzee with time intervals of 10 months and 1 month. The side effects observed were shivering, some muscular spasms and variations in the heart frequency. A decrease of lymphocytes of more than 50% was documented by haematogram analysis. The flow cytometry data showed that the Ha6D3 antigen does not modulate. Even after three repeated injections applied in a time interval of several months no immune response to Ha6D3 could be detected in vivo or in vitro. Based on these data we suggest that Ha6D3 may become a candidate for the treatment of certain leukaemias in vivo.

Animals↗