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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
PubMed1992

New parental cell lines for generating human hybridomas.

In contrast to the success achieved with the production of hybridomas in the mouse system, creating human hybridomas is problematic. The reason is believed to be a lack of suitable malignant human cell lines. The work presented here demonstrates the establishment of three human parent cell lines--two of which are of T cell origin--by installing hypoxanthine guanosine phosphoribosyl transferase (HGPRT) and/or thimidine kinase (TK) deficiencies into the leukemic cell lines REH, 1301 and SKW-3. In order to isolate true hybridomas, selection procedures must guarantee complete death of the enzyme-deficient malignant parent cells. In this respect sublines with a combined HGPRT and TK deficiency proved to be superior to those with only one enzyme deficiency, especially in combination with the newly developed hypoxanthine/aminopterine/thymidine/azaserine (HATA) selection medium. However, selection media should be of low nonspecific toxicity. This was shown to be a particular property of the thymidine-free azaserine/hypoxanthine (AH) selection medium. Preliminary data show an extraordinary ability of one subclone of the hypoxanthine guanosine phosphoribosyl transferase-negative T cell line SKW-3 to generate human T-T hybridomas. They are of a stable, nearly tetraploid karyotype and express new surface antigens, thus providing new possibilities for the investigation of human T lymphocyte function by means of hybridoma technology.

Cell Line↗
PubMed1992

Specific down-regulation of allograft reactivity at the cellular level: graft cells and responder T cells.

Local immune reactivity in an allograft is largely determined by (1) the immunogenicity of the grafted cells and (2) the presence of recipient allospecific T lymphocytes and, much more importantly, their capacity to react and initiate damage to the graft. Ad (1): It is still widely believed that allograft immunogenicity is sufficiently described by the cell surface expression of transplantation antigens, in particular MHC class I and II molecules. In extension of earlier, more complex models it will be demonstrated that transfected subclones of a macrophage cell line show unaltered, strong MHC expression but have become non-immunogenic and, most importantly, have acquired the capacity to render mature naive T cells anergic in a specific fashion. Ad (2): Starting from the above and from other observations, we have found in human organ graft recipients that cytotoxic T lymphocyte (CTL) frequencies may be specifically down-regulated but may regain their reactivity after cultivation under ex vivo conditions. In keeping with the latter we can now show quantitatively the variable presence and the functional potential of cell-released (soluble) human MHC (HLA class I) molecules: they are able to specifically inhibit human CTL reactivity in a dose-dependent fashion. Taken together, it may be concluded that studies at the cellular level, in particular the level of human cells, will increasingly help us to better understand and manipulate the immunological interplay between the local graft cell and its most important antagonist, the T lymphocyte.

Animals↗
PubMed1992

Post-mortem HLA tissue typing of retinal pigment epithelial cells.

Retinal pigment epithelial cells (RPE) were derived from bulbi of cornea donors and maintained in culture. The time-span between donor's death and cell cultivation ranged from 2 to 122 h. The mean numbers of hours was 25.6 (n = 130). After IFN-gamma stimulation, cells were serologically typed for class I and class II antigens. Unclear class I allospecificities were verified by one-dimensional isoelectric focusing. Data from the serological typing of lymphocytes and those of the serological and biochemical typing of RPE from the same donors were compared in 22 cases. There was a discrepancy of less than 5%, whereby either the typing on lymphocytes could not identify some specificities declared as blanks or the RPE typing had failed to clearly define a specificity. Our data show that the strategy adopted here is very successful for tissue typing post mortem, thus increasing the number of available HLA-matched corneas and consequently reducing the number of corneal graft rejections.

Adolescent↗
PubMed1992

Tissue distribution of MHC class II--positive cells, their down-manipulation by monoclonal antibodies and potential role in organ allograft immunogenicity.

This review article presents current state of knowledge of tissue distribution of MHC class II antigens in rat kidney. Our attention focuses particularly on down manipulation of these antigens by monoclonal antibodies. The results of organ perfusion procedure on prolongation of graft survival and perspectives of further studies are also discussed.

Animals↗
PubMed1992

[Characteristics of changes of personal odor after experimental bone marrow transplantation].

In an operant conditioning paradigm 4 Balb/c inbred strain mice were conditioned to discriminate between urine samples of two other inbred strains. The purpose was to examine whether or not the known stimulus configuration during training had any effect upon the rate of recognition of other stimuli. On the basis of this procedure, we subsequently investigated the extent to which an allogeneic bone marrow transplantation interferes with the olfactory identity of an individual. For the transplants a strain was inbred which had no contact to the animals tested, a procedure used for the first time here. Furthermore, also for the first time, it was possible to demonstrate in a direct comparison, the role played by donor versus recipient characteristics on the olfactory composition of the recipients odor.

Animals↗
PubMed1991

Freezing of cells--replacement of serum by oxypolygelatine.

A commercially available plasma expander (Gelifundol) containing 5.5% oxypolygelatine in buffered saline was used instead of serum as a supplement for freezing several types of human cell and a mouse myeloma cell line in liquid nitrogen. Viability, recovery, proliferation and cytotoxic activity were compared after freezing with a plasma expander and after conventional freezing with human AB serum or fetal calf serum. The plasma expander proved to be equivalent or superior to AB serum by all parameters tested and acceptable even when compared with fetal calf serum. Furthermore, this preparation is cheap, sterile, free of BSE, viral and mycoplasmal contamination or antibodies and foreign serum proteins. We therefore recommend it for freezing of cells for culture of HLA typing.

Animals↗
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↗