Kurt Wonigeit

A point mutation in PTPRC is associated with the development of multiple sclerosis

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is widely accepted that a dysregulated immune response against brain resident antigens is central to its yet unknown pathogenesis. Although there is evidence that the development of MS has a genetic component, specific genetic factors are largely unknown. Here we investigated the role of a point mutation in the gene (PTPRC) encoding protein-tyrosine phosphatase, receptor-type C (also known as CD45) in the heterozygous state in the development of MS. The nucleotide transition in exon 4 of the gene locus interferes with mRNA splicing and results in altered expression of CD45 isoforms on immune cells. In three of four independent case-control studies, we demonstrated an association of the mutation with MS. We found the PTPRC mutation to be linked to and associated with the disease in three MS nuclear families. In one additional family, we found the same variant CD45 phenotype, with an as-yet-unknown origin, among the members affected with MS. Our findings suggest an association of the mutation in PTPRC with the development of MS in some families.

Patterns of donor-type microchimerism after heart transplantation

Allogeneic microchimerism of donor-type has been demonstrated in stable patients in the long-term after organ transplantation. We have analysed microchimerism in skin and blood of 47 heart-transplanted patients after transplantation with polymerase-chain-reaction amplification specific for donor HLA-DRB1. Microchimerism was detectable in 50% of the patients in the first 6 months, in 100% between 6 months and 2 years, and in 58% in the third postoperative year or later. The state of chimerism was not related to acute or chronic rejections. Patterns of microchimerism after heart transplantation may be dynamic, but any association with clinical and immunological variables remains to be elucidated.

The functional relevance of passenger leukocytes and microchimerism for heart allograft acceptance in the rat.

With an organ transplant, hematopoietic donor cells are transferred to the recipient. To study the relevance of the resulting microchimerism for allograft acceptance, we analyzed a rat model of cyclosporine-induced tolerance for strongly incompatible heart allografts. Using a monoclonal antibody that detects a donor-specific CD45 allotype (RT7a), we selectively depleted donor leukocytes at different times after transplantation (days 0 or 18). Depletion was similarly effective at both times. However, only depletion on day 0 prevented tolerance induction and was associated with severe acute or chronic graft rejection. This indicates that passenger leukocytes have an essential immunomodulatory effect on the induction phase of allograft acceptance.

Persistence Of Donor Lymphocytes In Liver Allograft Recipients

Occasional cases of graft-versus-host disease after liver transplantation indicate a transfer of donor lymphocytes by human liver grafts. However, little is known about the usual fate and potential function of passenger lymphocytes in clinical liver transplantation. In this study, we have analyzed liver graft recipients for the presence of donor lymphocytes in the early course after transplantation. The presence of such cells in blood, the graft, and, occasionally, the skin was studied by the use of mAb to polymorphic HLA class I determinants and double-staining techniques in flow cytometry and immunocytology. The findings were compared with the clinical courses and with the results of routine graft biopsies. Within the first week after transplantation, in all 16 patients, between 1% and 24% donor lymphocytes (T, NK, and B cells) were detectable in blood, and in 14 of 22 patients (64%), between 2% and 23% donor T cells were found in the graft. After more than 2 weeks, donor cells were still present in blood in 2 of 14 patients at very low numbers. The presence of donor lymphocytes in the graft was associated with intragraft immune activation in 5 of 15 patients, but no clinical rejection occurred in these cases; mild graft-versus-host disease was observed in one patient. These findings demonstrate that donor lymphocytes regularly persist in liver-grafted patients for some time; this transient mixed lymphoid chimerism is only rarely associated with clinical graft-versus-host disease and some evidence even suggests that these donor-derived lymphocytes may exert beneficial immunomodulatory properties.

Analysis of sequential changes in major histocompatibility complex expression in human liver grafts after transplantation.

The expression of class I and class II major histocompatibility complex (MHC) antigens in human liver grafts was analyzed in 88 liver biopsies from 22 patients. For the staining of MHC antigens, a panel of monoclonal antibodies directed against monomorphic determinants of class I and class II molecules and an indirect immunoperoxidase method were used. In the reference biopsies, class I antigens were expressed on all cell types but only weakly on hepatocytes; class II was only expressed on Kupffer cells, interstitial cells, and endothelia. After transplantation, this pattern of MHC expression was markedly modified. Increased class I expression on hepatocytes and HLA-DR expression on bile ducts occurred in the absence of clinical rejection. During acute rejection, class I was strongly expressed and HLA-DR weakly expressed on hepatocytes; on bile ducts, HLA-DR expression was further increased. Cytomegalovirus hepatitis caused class I and HLA-DR induction on hepatocytes; strong induction of HLA-DR on bile ducts was also found in cholangitis. These findings have a number of implications for the pathophysiology of rejection of the transplanted liver.

Passenger lymphocytes in human liver allografts and their potential role after transplantation.

Rare cases of graft-versus-host disease after liver transplantation indicate that donor lymphocytes may be transferred to the recipient by human liver grafts. In this study, we have analyzed the number and subpopulations of donor lymphocytes transferred by liver grafts in order to evaluate the potential relevance of these cells after transplantation. Therefore, mononuclear cells were isolated from the tissue of perfused human donor livers and from the associated lymph nodes. The number of lymphocytes, their location, and surface marker expression were determined by immunostaining. The majority of lymphocytes transferred by the grafts were found within the liver tissue (5.3 ± 2.9 × 109 cells). These lymphocytes are mainly T and NK cells, predominantly CD8+, are partially activated (28% HLA-DR+), and show strong adhesion molecule expression (88% LFA-13+). In addition, 20–500×106 of resting lymphocytes, predominantly T and B cells, are transmitted by lymph nodes. These findings demonstrate that considerable numbers of donor lymphocytes of distinct phenotype are regularly transmitted to the recipient by human liver grafts and may be of functional relevance after transplantation.

Production of cytokines (TNF-alpha, IL-1-beta) and endothelial cell activation in human liver allograft rejection.

Intragraft production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) was determined in rejecting human liver grafts during acute rejection and in chronic graft dysfunction. The localization of cytokine-producing cells was then correlated with the distribution of monocytes and macrophages as their main producers, as well as with effector functions such as endothelial cell activation. In selected patients collateral TNF-alpha plasma levels were measured. In normal liver and biopsies taken during an uncomplicated course, few TNF-alpha and even fewer IL-1-beta positive macrophages were found. During acute rejection episodes of all degrees of severity liver grafts were infiltrated by large numbers of TNF-alpha-positive monocytes, and concomitant TNF-alpha plasma levels were elevated compared with uncomplicated controls. In marked contrast IL-1-beta production by macrophages and vascular and sinus endothelial cells was restricted to the most severe, irreversible rejection episodes. The localization of cytokine-positive cells coincided with areas of maximum induction of ICAM-1 and von Willebrand Factor. In chronic graft dysfunction increased numbers of mature macrophages were found. A large proportion of these were positive for TNF-alpha as well as IL-1-beta. Distinct from acute rejection episodes, however, parallel TNF-alpha plasma levels were not elevated, suggesting cytokine storage rather than secretion. The present results indicate an important local role of TNF-alpha and IL-1-beta in the early phase of the rejection process. They presumably activate endothelial cells to upregulate the expression of adhesion molecules, thereby facilitating mononuclear cell adhesion and extravasation. Therefore, specific inactivation of cyto-kines or of their actions may prove to be a powerful tool in the prevention and treatment of allograft rejection in the future.

Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single‐step transduction to introduce an autoregulative tetracycline‐responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue‐independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.—Kues, W.A., Schwinzer, R., Wirth, D., Verhoeyen, E., Lemme, E., Hermann, D., Barg‐Kues, B., Hauser, H., Wonigeit, K., and Niemann, H. Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double‐transgenic pigs. FASEBJ. 20, E357–E366 (2006)

Phenotype and histological distribution of interstitial dendritic cells in the rat pancreas, liver, heart, and kidney

The phenotype and the histological distribution of interstitial dendritic cells was investigated in the rat pancreas, liver, heart, and kidney by immunoperoxidase techniques. Monoclonal antibodies, directed against lymphocyte differentiation antigens (W3/25, W3/13, and Ox8) or against Ia antigens, revealed distinct phenotypes of these cells in all organs investigated—namely W3/25+Ia- dendritic cells, W3/25+Ia+ cells, and a small W3/25-Ia+ population. In the kidney cortex a W3/25+W3/13+ population was additionally shown. Interestingly, the phenotypically distinct cell populations also differed in their topographical distribution: W3/25+Ia+ cells were evenly scattered in the interstitium of the endocrine and exocrine pancreas, heart, and kidney. In contrast, W3/25+Ia- cells showed an additional predilection for connective tissue septa in the exocrine pancreas and for the kidney medulla. In the liver, phagocytic Kupffer cells were W3/25+Ia-, whereas the W3/ 25+Ia+ nonphagocytic dendritic cell population resided periportally and around central veins. These results show a marked heterogeneity of interstitial dendritic cells in the rat, which is discussed in terms of different cell lines or different activation or maturation stages of one single cell type.

Positive and negative MHC class I recognition by rat NK cells.

Summary: The prompt rejection of transplanted allogeneic lymphocytes by rat NK cells in non‐sensitized recipients (allogeneic lymphocyte cyto‐toxicity or ALC) is determined by MHC genes as well as by genes located in the NK complex. The same genetic control is found when NK alloreactivity is measured by an in vitro assay, and we have employed this assay to delineate the specificity of NK cells for the MHC. The MHC of the rat, RT1, contains class 1 genes situated on either side of the class Il/class III region. The majority of these class 1 genes are located in the RT1.C region and expressed class I products usually behave as non ‐classical (class Ib) molecules. They do not serve as restriction elements for the vast majority of conventional a/p T‐cells, in contrast to those class molecules encoded by one or more loci in the classical (doss la) region, RT1. A. However, NK cells appear to recognize the products of either class 1 region. Immunogenetic studies suggest that NK cells are inhibited by RT1.A molecules, whereas RT1.C region molecules may have a dual role in regulating NK cytolytic activity, i.e. they either inhibit or activate natural killing. Based on THESE premises, a model is proposed in which identification of a target as self or non‐self depends on different receptors for class 1 in single NK cells, interpreting coincident positive and negative signals from the various target class I molecules. The putative role of peptides presented by class I, the biological implications, and the evolution of the NK receptors and ether ligands are discussed.