Biphasic effects of selegiline on striatal dopamine: lack of effect on methamphetamine-induced dopamine depletion.

We tested the hypothesis that selegiline can attenuate dopamine depletion if administered following high doses of methamphetamine that cause neurotoxicity in the striatum. Methamphetamine produced decreases of 50% or greater in both striatal concentrations of dopamine and combined concentrations of homovanillic acid and DOPAC in mice. For animals not exposed to methamphetamine, chronic treatment with selegiline over 18 days caused biphasic effects on striatal dopamine content, with decreases, no effect, or increases observed for mice receiving treatment with 0.02, 0.2, and 2.0 mg/kg, respectively. Selegiline failed to modify methamphetamine-induced reductions in striatal dopamine content or combined concentrations of homovanillic acid and DOPAC. Significant increases in mortality following the onset of selegiline treatment (24 hours after the initial dose of methamphetamine) occurred in methamphetamine-treated mice that received saline or 2.0 mg/kg of selegiline, but not for mice treated with 0.02 or 0.2 mg/kg of selegiline. These results indicate that selegiline fails to attenuate dopamine depletion when administered chronically following exposure to methamphetamine, but may attenuate methamphetamine-induced mortality. In control animals that did not receive methamphetamine, low doses of selegiline produced decreases the concentration of striatal dopamine, while high dose treatment caused increases in striatal dopamine content.

The clinical importance of choosing the right assay for detection of HLA-specific donor-reactive antibodies.

BACKGROUND: We found earlier that there is a close clinical correlation between the presence of histocompatibility leukocyte antigens (HLA) class I-specific donor-reactive antibodies in cross-match serum and a significantly higher frequency of early acute rejection episodes and graft loss during the first year after the transplant. METHODS: Specificity determinations of donor-reactive antibodies present in the cross-match serum before allogeneic kidney transplants were performed. In the present study, we compared the suitability and efficiency of (a) platelet absorptions, (b) blocking with anti-HLA monoclonal antibodies in the microcytotoxicity assay, and (c) donor-specific HLA antigen-coated magnetic microbeads in flow cytometric assays for the definition of clinically relevant HLA antibodies and their correlation with early acute rejections and early graft loss. RESULTS: We found that the microlymphocytoxicity test using donor splenic B lymphocytes often gave positive reactions in the absence of class I or class II antibodies; in other words, a high frequency of false positive reactions was observed. Flow cytometric tests are more sensitive than microlymphocytotoxicity, not only because they are more sensitive, but also because noncomplement-binding antibodies are detected. Platelet absorptions, which detect only reactivity against HLA class I antigens, is insufficient for use in specificity determinations of donor-reactive antibodies. We found that a positive test for HLA antibodies using paramagnetic beads coated with solubilized donor-derived HLA antigens (class I or class II) correlates with early immunological complications after a transplant (P<0.001). CONCLUSION: Assays to determine the specificity of donor-reactive antibodies are now available for use during an acute transplant situation. The introduction of such methods is expected to enhance graft survival and to reduce significantly the frequencies of acute rejections episodes after a transplant.

Role of alloimmunity in clinical transplantation.

HLA-specific humoral immunity, as a result of recipient allosensitization, induces hyperacute rejection of allogeneic kidney grafts. Cross-match tests are performed to avoid this complication. However, present techniques do not allow determination of HLA specificity of donor-reactive antibodies in the acute necro-donor situation. New methods are described and discussed in this communication as well as the alloantibody specificities which are of clinical importance. Alloantibodies not only mediate hyperacute rejection, but may also participate in the acute rejection of organ grafts. Clinical associations between early immunological complications, such as acute rejection, in heart, liver and kidney allografted patients and pre-transplantation humoral alloimmunity emphasize the need for proper determination of donor-specific humoral immunity prior to transplantation. Acute rejection episodes are associated with an increased risk of subsequent chronic rejection. Cytomegalovirus (CMV) infection is also an important risk factor for chronic organ graft rejection as well as for chronic graft-versus-host (GvH) disease in bone marrow transplanted patients. CMV infection triggers autoantibody formation against CD13 in transplanted patients which, in turn, has been shown to be associated with the development of chronic GvH disease. Recently, alloactivation of immune reactivity in vitro was found to induce reactivation of latent CMV infection. We present here a hypothesis to explain the series of clinically associated events: acute rejection--CMV infection--chronic rejection.

Differential motile response of human malignant mesothelioma cells to fibronectin, laminin and collagen type IV: the role of beta1 integrins.

Beta1 integrins are widely expressed in human tissues but their presence and function on malignant mesothelioma cells have not been examined. In this study, we have investigated the expression and function of beta1 integrins in 7 human malignant mesothelioma cell lines. Immunofluorescence staining and FACS analysis showed similar expression of beta1 integrins with strongest expression of alpha3beta1 in all investigated mesothelioma cell lines. Using the Boyden chamber assay, we found that mesothelioma cell lines migrated to soluble (chemotaxis) and substrate-bound (haptotaxis) fibronectin, laminin and collagen type IV. In order to investigate the biological function of integrins in mesothelioma cells, we pre-incubated the cells with blocking anti-integrin monoclonal antibodies (MAbs) prior to the adhesion and migration assays. Anti-beta1 antibodies inhibited cell adhesion, chemotaxis and haptotaxis in all cell lines. Generally, anti-alpha2 integrin antibodies inhibited cell adhesion, chemotactic and haptotactic migration to collagen type IV, whereas antibodies to the alpha5 and alpha6 subunits inhibited cell adhesion and migration to fibronectin and laminin, respectively. Preincubation of mesothelioma cells with anti-alpha3 antibodies inhibited the migration to either collagen type IV, laminin or fibronectin in all cell lines. Interestingly, in 3 cell lines anti-alpha3 antibodies inhibited cell migration to laminin and collagen type IV without affecting the ability of the cells to adhere to these proteins. Furthermore, in 2 cell lines, antibodies to the alpha3 chain inhibited chemotaxis but not haptotaxis to collagen type IV, indicating the presence of distinct signalling pathways.

Hyperacute rejections of two consecutive renal allografts and early loss of the third transplant caused by non-HLA antibodies specific for endothelial cells.

The immunological rejection of HLA-identical kidney transplants indicates that non-HLA antigens may also be targets for transplant rejection. Interest in the possible role of endothelial specific antigens has grown steadily over the years. Most of the studies published, regarding the association of such antibodies with rejection, have demonstrated the reactivity of endothelial antibodies also with monocytes and keratinocytes, but not with lymphocytes. Such antibodies escape detection in conventional crossmatch tests. In this paper, we present a case report of a 10-year-old girl, whose two consecutive kidney allografts, (one living and one cadaveric donor) were hyperacutely rejected in spite of the fact that she had neither been alloimmunized, nor had any HLA-specific antibodies. Endothelial cell specific antibodies were detected in vivo and in vitro after transplantation only 11 days apart, which were considered to be responsible for rejection. The third cadaveric kidney was lost within 1 week post-transplant. Immunopathological investigation of the three rejected grafts revealed deposition of IgM in the endothelium of arteries and in some glomeruli. No deposition of IgG antibodies was found. Antibodies from this patient did not react with lymphocytes, monocytes or keratinocytes. Patient serum had IgM antibodies that were specifically reactive with cultured endothelial cells, demonstrated by binding in vitro and by complement-dependent cytotoxicity of IL-beta stimulated endothelial cells. No HLA antibodies were found following the first two transplantations, but were demonstrated 1 week after the third transplantation, at the time of an acute irreversible rejection. Western blots of proteins solubilized from endothelial cell membranes, indicated that the antibodies reacted with a 97-110 kD protein. Endothelial cell antigen preparations were made from several different umbilical cord veins. Some primary cell cultures, but not all, reacted with the patient's serum. Therefore, we suggest that the target determinant might be polymorphic. These findings imply that the non-HLA endothelial cell specific molecules may function as target(s) for hyperacute antibody-mediated destruction of kidney allografts.

Acid treatment of lymphocytes selectively decreases the expression of HLA class I antigens: a method to confirm that a positive clinical crossmatch test was due to class I antibodies.

A rapid and reliable method for eliminating HLA class I antigens from the surface of lymphocytes without damaging the cells is described. Lymphocytes were exposed to an acid solution (pH 3.0) which selectively destroys the antigenicity of HLA class I antigens. Alloantisera containing multispecific HLA class I antibodies reacted with phosphate-buffered saline (PBS)-treated, but not with acid-treated, lymphocytes. Specificity controls included: antibodies against HLA class II antigens and CD3, CD4, CD8; markers expressed on T cells and CD19, CD23; markers expressed on B cells. No change in lymphocyte reactivity to any of these surface antigens or to autoantibodies was observed. The viability of acid-treated lymphocytes was regularly around 90%. We propose that acid-treated lymphocytes are suitable targets for determination of the sole presence of class I antibodies in crossmatch sera of patients awaiting organ transplants.

The use of magnetic beads coated with soluble HLA class I or class II proteins in antibody screening and for specificity determinations of donor-reactive antibodies.

An adequate method that will permit rapid specificity determination of donor reactive antibodies is urgently needed. Such a method could also be used for monitoring the presence of HLA antibodies in panel scanning. We here describe a method using magnetic beads coated with soluble HLA antigens that can be directly added to patient serum for efficient absorption of HLA antibodies. The entire procedure takes 45 min, and can therefore be easily adopted for use in acute crossmatching situations. Furthermore since no live cells are required for identification of alloantibodies in the screening for HLA specific panel-reactive antibodies, it can be used as an ideal complement for screening of panel-reactive antibodies. Binding of antibodies to the antigen-coated beads is easily visualized using flow cytometry. A new method for quick purification of soluble MA antigens from a pool of lymphocytes or thrombocytes is also presented.

CD13-specific autoimmunity in cytomegalovirus-infected immunocompromised patients.

Cytomegalovirus (CMV) infection has been suggested to be associated with various autoimmune manifestations, such as hemolytic anemia, granulocytopenia, and the formation of autoantibodies. Earlier we found that CMV is associated with a human protein, CD13 (aminopeptidase N), emanating from CMV-infected cells and serving an important function during CMV infection of susceptible cells. We hypothesized that CD13 might become immunogenic if presented to the immune system as a part of the CMV virion. The presence of CD13-specific antibodies was tested using a microcytotoxicity assay against CD13-positive human monocytes, or by flow cytometric assays against mouse cells transfected with human CD13; specificity was assessed by specific blocking with monoclonal antibodies. CD13 reactivity was also demonstrated in immunoprecipitation experiments. CD13-specific antibodies were identified in 15 of 33 bone marrow transplant patients, but exclusively in patients who had experienced either CMV disease (9/10) or CMV viremia (6/9), and appeared at the time of CMV detection. None of the remaining 14 patients without signs of CMV infection were positive for CD13 antibodies (P<0.0001). No antibody of this specificity was found in any of the control individuals (0/24), including patients with various autoimmune diseases, CMV-seropositive or -seronegative healthy individuals, and patients with acute EBV or HSV-1 infections. Thus, the CMV-associated autoantigen CD13 is immunogenic during CMV infection in bone marrow transplant patients. A specific response against autoantigens associated with infectious virus particles is suggested as a new and general mechanism to explain virus-induced autoimmune manifestations in man.

Cytomegalovirus-induced CD13-specific autoimmunity--a possible cause of chronic graft-vs-host disease.

Cytomegalovirus (CMV) infection has been suggested to be associated with certain autoimmune phenomena as well as with the development of chronic graft-versus-host disease (cGVHD) following allogeneic bone marrow transplantation. Earlier we found that the CMV-associated host protein CD13 is immunogenic during CMV infection in allogeneic bone marrow transplant patients, resulting in production of CD13-specific antibodies (7). Here we found a close correlation between CD13-specific immunity and later development of cGVHD in 26 of 33 patients who could be evaluated for this disease. Of seven patients with CMV disease, six developed extensive cGVHD, all of whom had CD13 specific antibodies (P=0.0002). All 14 patients who were CD13-immune later developed either limited or extensive cGVHD (P=0.0008). Antibodies in sera from the CD13-immune patients suffering from cGVHD recognized normal structures in cryosectioned skin biopsies from control individuals, producing a staining pattern similar to that of CD13-specific monoclonal antibodies. The antibody binding could be specifically blocked by preincubation of the skin sections with a mixture of monoclonal antibodies against CD13, and was also abolished after preabsorption of sera to mouse cells expressing human CD13. No other common autoantibodies were identified in more than single patients. Furthermore, in vivo binding of IgM antibodies in a CD13-like fashion was preferentially demonstrated in skin and oral mucosa biopsies from the CD13-immune patients suffering from cGVHD. Thus, we suggest that CMV-induced CD13-specific autoimmunity contribute to tissue damage in chronic graft-versus-host reactions.

Specific inhibition of HLA class I and II antibodies by soluble antigens--a method for the identification of antibody specificity in sera from alloimmunized individuals.

HLA class I and II antigens were purified to be used for the determination of the specificity of lymphocyte reactive antibodies in renal transplant patients. Purification of HLA antigens was achieved by affinity chromatography using rabbit antibodies directed to human beta 2 microglobulin, W6/32 antibodies that recognize a nonpolymorphic determinant on HLA class I molecules, and BU25 monoclonal antibodies directed to a monomorphic determinant on HLA class II molecules. Pooled platelets and spleen lymphocytes from a large number of donors were used as a source of class I and II antigens. HLA antigen preparations, highly enriched as shown in Western blot experiments, were obtained that caused a dose-dependent inhibition of the binding as well as of the cytotoxicity of W6/32 and BU25 antibodies. The HLA class I preparation did not inhibit the binding of monoclonal antibodies to T or B cell-specific surface markers or to HLA class II antigens. Similar kinds of results were obtained with the HLA class II antigen preparation, which only specifically blocked the class II antibody binding. The antigen preparations were tested for their ability to block binding of antibodies in sera from alloimmunized patients. In order to avoid complications by soluble HLA antigens, all sera were absorbed in ELISA plates coated with anti-class I and anti-class II monoclonal antibodies. The HLA class I and class II antigen preparations were found to be HLA-specific. All data were compared with the conventional method to determine HLA specificity--i.e., specific blocking of class I and class II reactivity using monoclonal antibodies and unabsorbed sera. Soluble HLA antigens, added directly to mixtures of patient sera and lymphocytes used in the cytotoxicity or binding tests, can therefore be used for determination of the presence of HLA antibodies in sera of alloimmunized patients.

Antibodies in alloimmunized uraemic patients treated with recombinant erythropoietin.

We have retrospectively analysed sera from 52 already sensitized uraemic patients collected over 1 year and compared erythropoietin (EPO)-treated with non-EPO-treated patients. Significantly fewer (P<0.01) patients (33%) on dialysis because of the rejection of their kidney grafts received EPO than patients on dialysis because of underlying kidney disease (71%). EPO treatment reduced the number of additional blood transfusions, since 3/28 EPO-treated but 12/24 non-EPO-treated patients were given blood (P<0.05). Among the EPO-treated patients, 64% showed a loss of panel-reactive antibodies (PRA), as measured by the micro-lymphocytotoxic technique, while only 12.5% of the non-treated patients showed a loss of PRA (P<0.01). In the subgroup of transplanted patients, PRA loss was only found among the EPO-treated patients, but their number was small (P<0.05). The class, subclass and specificities of the antibodies, as determined by FACS (flow cytometry) analyses, showed no distinct differences between EPO- and non-EPO-treated patients. The differences were significant between transfused and previously transplanted patients.

The occurrence of cytotoxic and non-complement-fixing antibodies in the crossmatch serum of patients with early acute rejection episodes.

In an earlier study we found a strong correlation between the presence of donor-reactive HLA-specific antibodies in the crossmatch serum and early acute rejection episodes. Our experience was also that some of these antibodies were not cytotoxic and could therefore not be detected using the microcytotoxicity assays. In the present study, 11 patients from the earlier study who had weakly positive B cell reactive cytotoxic antibodies of the IgG class were further characterized. In addition, 14 new patients were selected who experienced early acute rejections but had a completely negative donor-B cell cytotoxicity crossmatch. A group of 12 controls without immunological complications was added, as well as 5 patients with early graft losses due to nonimmunological causes. Using the flow cytometric crossmatch test we confirmed the presence of HLA-specific antibodies in all 11 patients from the earlier study. In addition, positive flow cytometric crossmatches shown to be caused by HLA antibodies were observed in 11 of the 14 patients with acute rejections and negative cytotoxic crossmatch. One of 17 control patients had antibodies that were not HLA-reactive. IgA antibodies as well as IgG subclass determinations were performed in all positive sera. A substantial proportion of patients had HLA-specific antibodies of non-complement-binding classes (IgG2, IgG4, IgA) often of higher titers than IgG1 and IgG3. The subclass distribution pattern was heterogeneous and often included several subclasses. We conclude that non-complement-fixing antibodies can also contribute to the risk for development of early acute rejections after necrokidney transplantation. Immunological mechanisms for these findings are discussed.

Fewer acute rejection episodes and improved outcome in kidney-transplanted patients with selection criteria based on crossmatching.

Cross-matches performed on sera from presensitized patients have so far failed to differentiate clinically "relevant" from clinically "irrelevant" antibodies due to insufficient specificity characterization. In order to find a basis for such a possibility, 72 consecutive kidney transplanted patients were selected and studied retrospectively. Our policy has been to accept weakly positive (less than 50%) cytotoxicity on donor B splenic lymphocytes in the crossmatch test. Forty-six patients who had early acute rejections and 26 who had no acute rejections were selected for the study. Antibodies in current sera were characterized according to their target cell reactivity, immunoglobulin class, and HLA/non-HLA specificities. Crossmatches were performed using both the cytotoxicity and the flow cytometric method. We found that the factor that differentiates clinically "relevant" from clinically "irrelevant" antibodies is the HLA specificity of the lymphocyte-reactive antibodies. A high proportion of patients with posttransplant complications such as acute rejections had antibodies with specificities for HLA (particularly class I) antigens, while patients without rejections had either no detectable antibodies or antibodies reactive with non-HLA antigens only. In the present study, after elimination of false-positive reactions by ultracentrifugation of current sera, we found that flow cytometric crossmatches may not necessarily be more specific than the ordinary cytotoxicity crossmatch, since a positive flow cytometric crossmatch is often, but not always, associated with a weakly positive B cell cytotoxic crossmatch (10-25% reactivity). Antibodies causing a positive flow cytometric crossmatch could constitute low-titerd complement-fixing antibodies, non-complement-fixing antibodies, or both. Thus our study shows that it is possible to identify, prior to transplantation, patients with a risk of early posttransplant immunological complications such as acute rejection and to differentiate these from those who are more likely to have an uncomplicated posttransplant clinical course. A more careful patient selection based on adequate crossmatch testing, including specificity determination, might reduce the frequency of acute rejections and improve the outcome of transplantation.

Relevance of a positive crossmatch in liver transplantation.

We studied 27 liver transplants in 24 patients performed between November 1984 and January 1988. We investigated retrospectively the importance of donor reactive HLA class I and class II and of non-HLA antibodies for graft survival in these patients. In order to determine the specificity and class of the antibodies, we used monoclonal antibodies to HLA-A, -B, -C and DR and DQ antigens to block cytotoxicity of sera and the reagent dithiothreitol to characterize the immunoglobulin class. We found that humoral immunity to HLA antigens in liver-grafted patients, demonstrable as the presence of cytotoxic antibodies reactive with donor splenic T and/or B cells in the pretransplantation period, is associated with significantly lower graft survival as compared with patients without demonstrable preformed HLA antibodies (P = 0.01). In addition we found that a substantial proportion of patients had donor-reactive cytotoxic antibodies which were not HLA specific. Thus, our study shows that HLA immunity can influence liver allograft survival, and that it is useful to have patient cytotoxic antibodies characterized with regard to HLA reactivity prior to transplantation.

Characterization and significance of donor-reactive B cell antibodies in current sera of kidney transplant patients.

The specificity and class of antibodies resulting in a positive B cell crossmatch were studied in 47 kidney-transplanted patients. The study was performed to determine instances where a positive B cell crossmatch would not be deleterious to the survival of the graft. In order to determine the specificity and class of the antibodies, we used monoclonal antibodies to HLA-A,B,C and DR antigens to block cytotoxicity of sera, and the reagent DTT to characterize the immunoglobulin class. We found that graft survival in patients with DR antibodies was significantly better than in patients with class I antibodies (P less than 0.02). No difference in graft survival in patients with IgM antibodies versus patients with IgG antibodies was observed. The presence of weak HLA class I antibodies in patient's sera only detected as reactivity on B lymphoid cells should be considered a contraindication to transplantation. Thus our study shows that a fraction of patients who have cytotoxic B cell reactive antibodies at the time of transplantation can be successfully transplanted, provided the specificity of each serum is defined prior to transplantation.