Gustav Steinhoff

Adenine nucleotide metabolism and its relation to organ viability in human liver transplantation

The relationship between adenine nucleotide metabolism and ischemic damage was studied in human liver. Thirty transplanted grafts were divided into two groups assording to their functional outcome. Cellular adenine nucleotide levels were assayed by high-performance liquid chromatography. During cold ischema, the adenosine triphosphate (ATP) level was not correlated with graft function, but two grafts with low total adenine nucleotides (TAN) levels showed poor function after transplantation. After recirculation, the ATP level showed good recovery in grafts that functioned satisfactorily (n=24), 5.47±1.51 mUmol/g dry weight), but remained low in poorly functioning grafts (n=6), 3.30pL 1.68 mUmol/g dry weight) (P<0.01). Bile production, used as a parameter of initial function, was observed shortly after implantation in 17 of 24 grafts that functioned satisfactorily, but in only 1 of 6 poorly functioning grafts. It is concluded that loss of ademine nucleotides and lack of bile production during transplantation are good markers of damaged grafts in human liver transplantation.

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.

Analysis of potential porcine endogenous retrovirus (PERV) transmission in a whole‐organ xenotransplantation model without interfering microchimerism

Abstract The question whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for the evaluation of the safety of pig-to-man xenotransplantation. Unfortunately, polymerase chain reaction (PCR)-based analysis of potential PERV infections in nonhuman-primate whole-organ xenotransplantation models is hampered by false positive results due to chimeric porcine cells. To avoid the inherent analytical problem of xenomicrochimerism, we developed a non-life-supporting pig-to-primate kidney xenotransplantation model: porcine kidneys were transplanted, whereas the functioning recipient kidneys remained in situ. Subsequent to rejection (after 2 hours to 15 days), xenografts were removed, and recipients remained alive for up to 287 days. Immunosuppressive therapy based on cyclophosphamide, cyclosporine, and steroids was maintained for 28 days after transplantation. Using appropriate PCR assays, xenochimerism was found in tissue samples and partly even in peripheral blood leukocytes (PBLs) while the porcine kidneys were in situ. After graft removal, xenochimerism was no longer detectable, thus allowing analysis for possible PERV transmission.

Transmission of donor lymphocytes in clinical lung transplantation

Abstract Passenger mononuclear cells in organ grafts are known to influence the alloimmune response to the graft. To assess their relevance in clinical lung transplantation, we studied the amount, distribution, cell types, and surface marker expression of mononuclear cells in human donor lungs. Two major compartments of mononuclear cells could be differentiated: lymph nodes containing resting Tand B lymphocytes, and the lung tissue itself, containing mainly activated lymphocytes as well as monocytes/macrophages. Tissue‐associated mononuclear cells make up 20–40 times 109 cells per lung, about 30–50% of which are lymphocytes. Tissue‐associated lymphocytes are predominantly Tand NK cells; most of the T cells are CD8+ CD45R0+ and express HLA‐DR. Strong expression of the adhesion molecules LFA‐1 and ICAM‐1 is present on infiltrating cells as well as on resident cells of the organ. Moreover, the lymphocytes inside the lung tissue are functionally highly active, with a strong stimulatory as well as alloreactive potency. Thus, large numbers of allogeneic mononuclear cells and particularly large numbers of functionally active lymphocytes are obviously transmitted by human lung allografts. The immunological in vivo relevance of these cells after lung transplantation may include allostimulation and graft‐versus‐host activity, but also beneficial immunomodulatory effects.

Major histocompatibility complex antigens in human liver transplants

Liver transplantation is performed successfully across major HLA differences between donor and recipient. This may be influenced by the organ specific expression of major histocompatibility complex (MHC) molecules which determine the local immune reactivity and rejection response. The tissue expression of MHC molecules on parenchymal and infiltrating cells has been studied in transplanted human liver using monoclonal antibodies and immunohistological methods. A strong induction of class I (HLA-A,B,C; beta 2-microglobulin) and class II (HLA-DR,DQ,DP) MHC antigens was demonstrated on hepatocytes, bile duct epithelium and endothelial cells during rejection episodes and viral and bacterial infections. The massive induction of donor antigens on hepatocytes, bile ducts and endothelia forms part of, and may also augment, the rejection response. During quiescent states without infection or rejection after transplantation, however, a rather restricted expression of class I and class II donor MHC antigens is present. In addition, the donor Kupffer cells and interstitial dendritic cells are gradually replaced by recipient accessory cells expressing self-MHC molecules. The changes in antigen density and distribution of donor MHC alloantigens as the replacement of accessory cells capable of presenting antigens to T-lymphocytes may influence the course of immune reactivity and the rejection response in the liver. This may partly explain the favourable clinical course long after transplantation. Preliminary clinical investigations of the effect of HLA matching have shown a dualistic effect of the matching of class I or class II HLA antigens. The role of HLA matching in liver transplants in large clinical studies, with specific immunological testing however, remains to be investigated. This may lead to prospective HLA matching with wider organ availability and improved preservation time in the future.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver transplantation for hepatocellular carcinoma: clinical results and future aspects

SummaryThe treatment of unresectable hepatocellular carcinoma (HCC) by liver transplantation remains controversial. In our series, the 5-year survival value for 87 patients who underwent transplantations between 1972 and 1990 was 19.6%. There was no difference in the longterm survival of patients who had underlying cirrhosis and those who did not. In patients with early-stage tumours the long-term prognosis was improved, the 5-year survival in stage II disease being 55.6% according to UICC criteria. Even in some cases of more advanced tumour stage, good long-term results were obtained. In a review of the recent literature, we evaluated prognostic factors to work out criteria for a more differentiated indication for liver transplantation. Resection of increased radicality-which will keep its place as the therapy of choice — and transplantation should be performed complementarily. Further developments will reveal the value of multimodal therapeutic strategies, including chemo-embolisation, chemotherapy and immunotherapy.

Expression of adhesion molecules on lymphocytes/monocytes and hepatocytes in human liver grafts

The regulation of expression of adhesion molecules in human liver grafts in the course of rejection and inflammatory reactions was studied. The tissue distribution of adhesion receptor molecules and ligand molecules in graft biopsies taken during complications was compared to that in normal liver and reference biopsies taken at the time of transplantation.