Search

MHC molecules present peptides in their binding groove to T-cell receptors inducing proliferation or cytotoxicity of alloreactive T cells. A previously generated human monoclonal antibody (mAb) UL-5 A1, recognizing a conformational epitope formed by HLA DR1/DRB1 * 0101 molecules and HLA-A2 derived peptides, demonstrates T-cell-like recognition of the peptide/MHC complex (PMC). To study the genes of the antigen binding region, the nucleotide sequences of the rearranged genes in the variable regions of UL-5 A1 were determined and the V-gene usage (VH3, Vλ2) was identified by comparison with published germlines. The genes encoding heavy (Fd) and light (L) chains of UL-5 A1 were linked and expressed in a bacterial system. Specificity of the recombinant Fab-S A1 was determined with HLA-typed LCLs by flow cytometric analysis. As demonstrated in competitive inhibition assays, UL-5 A1 and Fab-5 A1 recognize the same PMC epitope on HLA-A2 + , -DR1/DRB1 * 0101 + typed LCLs. Additionally, mAb UL-5 Al and Fab-5 A1 both recognize HLA-A2 - , -DR1/DRB1 * 0101 + LCLs exogenously loaded with HLA-A2 peptides (105-117, 103-117). UL-5 Al-like antibodies against peptide/MHC complexes could prove valuable tools for research on T-cell recognition and MHC function.

Circulating maternal T lymphocytes were noted in the peripheral blood of six patients with severe combined immunodeficiency. Phenotypical analyses revealed the presence of both CD4 and CD8 subsets in some but not all cases. The maternal T cells could be stimulated by anti-TCR/CD3 mAb +/- rIL-2, but were virtually silent in the MLR and against the recall Ag purified protein derivative of tuberculin and tetanus toxoid, even in immunized patients engrafted with T cells from a responding mother. Using a panel of mAb against TCR V region gene encoded epitopes including V beta 5, V beta 6, V beta 8, V beta 12, and V alpha 2, we show that maternal T cells displayed a profoundly reduced TCR diversity, characterized by a lack of one or even several TCR V subsets in all six cases and a dramatic (5- to 25-fold) expansion of other TCR V subsets in three cases. In one patient analyzed, limited TCR diversity was also seen in T cells cultured from bone marrow and skin; restimulation experiments of these cells against cells expressing host MHC Ag were unsuccessful, as were attempts to exclusively allocate anti-host proliferative responses of maternal control T cells to the TCR V subsets that had undergone expansion in vivo. We conclude that a severely reduced TCR diversity is a common feature of maternal T cells engrafted in severe combined immunodeficiency patients. These novel findings provide a structural basis to understand the failure of these cells to protect the host from infections and may also help to understand their relative inefficiency to induce lethal, multi-organ, graft vs host disease. Moreover, as an experiment of nature, the reported phenomenon clearly illustrates the functional consequences in vivo of an insufficient TCR diversity.

The identification of the novel allele HLA-B*1546, which has serological B50 and B72 reactivity, was found in two members of a family of Turkish origin. In the sequence analysis the new allele differs from B*1501 by four nucleotides in exon 2. Its structure suggests that it may have originated by gene conversion with B*40, B*41, B*44, B*4501, B*47, B*4901 or B*50. At the protein level, the new allele has three amino acid differences compared to B*1501. Sequence alignment demonstrates that amino acid residue 46 is crucial for serological B72 reactivity. Due to substantial differences with other B*15 variants a possible mismatch may impair clinical outcome of bone marrow transplantation.

We describe the results of a comparative evaluation of a gel test (ID Micro Typing) and a micro-column affinity test (MCAT, Cellbind Screen) for red cell antibody screening and identification under routine conditions.3,000 serum samples of patients from the Mannheim University Hospital were tested in parallel by means of the gel test and the MCAT, using the low-ionic-strength-saline indirect antiglobulin test and the protein G affinity technique, respectively. Test cells used were the same in all tests. In addition, we performed titration studies with all detected antibodies as well as with 59 frozen sera containing antibodies of known specificity.A total of 154 antibodies (5.1%) were detected, 149 by gel test and 147 by MCAT. The overall sensitivity and specificity of the gel test was 96.8 and 96.5% and of the MCAT 95.5 and 97.2%. No significant differences between the gel test and MCAT were found when the titer scores of all 213 (fresh and frozen) antibodies were used to check the results. The mean scores for the gel test and the MCAT were 26.8 and 28.5, respectively. For anti-Fy(a) and anti-Kell, a significantly higher titration score could be obtained in the MCAT, whereas anti-Lu(a) showed a significantly higher score with the gel test.For the screening of unexpected red blood cell antibodies, the MCAT is as sensitive as the gel indirect antiglobulin test. The sensitivity and specificity of the two systems are more or less the same although it seems that IgM antibodies are better detected by the gel test.

MHC molecules present peptides in their binding groove to T-cell receptors inducing proliferation or cytotoxicity of alloreactive T cells. A previously generated human monoclonal antibody (mAb) UL-5 A1, recognizing a conformational epitope formed by HLA DR1/DRB1*0101 molecules and HLA-A2 derived peptides, demonstrates T-cell-like recognition of the peptide/MHC complex (PMC). To study the genes of the antigen binding region, the nucleotide sequences of the rearranged genes in the variable regions of UL-5 A1 were determined and the V-gene usage (VH3, V lambda 2) was identified by comparison with published germlines. The genes encoding heavy (Fd) and light (L) chains of UL-5 A1 were linked and expressed in a bacterial system. Specificity of the recombinant Fab-5 A1 was determined with HLA-typed LCLs by flow cytometric analysis. As demonstrated in competitive inhibition assays, UL-5 A1 and Fab-5 A1 recognize the same PMC epitope on HLA-A2+, -DR1/DRB1*0101+ typed LCLs. Additionally, mAb UL-5 A1 and Fab-5 A1 both recognize HLA-A2-, -DR1/DRB1*0101+ LCLs exogenously loaded with HLA-A2 peptides (105-117, 103-117). UL-5 A1-like antibodies against peptide/MHC complexes could prove valuable tools for research on T-cell recognition and MHC function.

On the basis of historical findings, 10% dimethylsulfoxide is still used to cryopreserve stem cells. We studied a final concentration of 5% dimethylsulfoxide in cryopreservation of cord blood-derived stem cells in autologous plasma. In our opinion, a final concentration of 5% dimethylsulfoxide in autologous plasma without further additives is sufficient for cryopreservation of cord blood stem cells in a banking routine.

The very sensitive solid-phase antiglobulin test is used widely in blood group serology. However, a disadvantage of the test is the sedimentation variability of indicator cells during centrifugation, which causes errors in the photometric evaluation.To solve this problem a technique was developed which depends on the 'length measurement of the absorption curve' of the sedimented indicator cells.9,075 sera were tested for the presence of alloantibodies on the basis of the visual type of measurement and the newly developed technique. The agreement between the two evaluation systems was 99.94%. No false-negative results were observed.The new method allows the complete automation of the solid-phase antiglobulin test, enabling the analysis and evaluation of the solid-phase microtiter plate assay to be more discriminating and safe.

BACKGROUND: The passive latex agglutination test is commonly used for the identification of cytomegalovirus IgG and IgM antibodies. This test is used because of its sensitivity, specificity, speed, and ease of performance, but it is unsuitable for large numbers of samples or for batch processing. STUDY DESIGN AND METHODS: To solve this problem, comparative studies to assess the cytomegalovirus passive latex agglutination test on microtiter plates were done with a new photometric particle agglutination method (PPAM) and an enzyme-linked immunosorbent assay as a control. RESULTS: A total of 3430 sera were tested using both the PPAM and enzyme-linked immunosorbent assay. A high degree (97.6%) of correspondence between the results of the tests was observed. The new PPAM was easier and faster to use (96 wells in 25 min). CONCLUSION: These results, as well as the possibility of adapting this method to a fully automated system, suggest that the PPAM could be an important contribution to the field of infection serology.

Comparing HLA data from 851 patients with serological HLA-A, -B, -DR and/or PCR-SSO HLA-DRB, -DQB reevaluated HLA types produced 107 (12.5%) different results. The high discrepancy of HLA data from patients confirm the demand for a serological HLA-A, -B and a DNA HLA-DRB, -DQB reevaluation before starting an unrelated donor search for bone marrow transplantation. The reliability of serological HLA-DRB, -DQB typing was evaluated as 97.8%, whereas DNA HLA-DRB, -DQB typing was 99.5%.

Bone marrow transplantation from unrelated donors is being used increasingly for the treatment of patients with leukaemia and other disorders of lymphohaemopoiesis. Selection of histocompatible unrelated bone marrow donors currently relies on serological HLA identity and negative mixed lymphocyte cultures (MLC) between potential donor-recipient pairs. Since serological HLA-DP typing is not feasible, we used the HLA-DPB1 oligonucleotide typing method to test whether the current selection procedure can also guarantee identity for HLA-DP. In 40 consecutive patients, one-third (62/193) of the serologically HLA-A, -B, -C, -DR and -DQ identical donors were judged as MLC negative (relative response below 5%) with the presumptive recipient. HLA-DPB1 oligonucleotide typing of the MLC negative donors revealed that only one-third of these (20/62) were also identical for DP. In the majority of the pairs, we found a DPB1 disparity. A difference in the graft-versus-host direction was seen in 25/62 cases in the host-versus-graft direction in 28/62 cases and in both directions in 29/62 cases. These data indicate that, in spite of the strict MLC criteria used, the current procedure did not guarantee complete MHC class II identity. Therefore oligotyping for DPB1 can improve matching for DP and should be introduced for typing of volunteers. We suspect that DP differences may contribute to the higher incidence of graft-versus-host disease or graft rejection in unrelated donor transplants.

Circulating maternal T lymphocytes were noted in the peripheral blood of six patients with severe combined immunodeficiency. Phenotypical analyses revealed the presence of both CD4 and CD8 subsets in some but not all cases. The maternal T cells could be stimulated by anti-TCR/CD3 mAb +/- rIL-2, but were virtually silent in the MLR and against the recall Ag purified protein derivative of tuberculin and tetanus toxoid, even in immunized patients engrafted with T cells from a responding mother. Using a panel of mAb against TCR V region gene encoded epitopes including V beta 5, V beta 6, V beta 8, V beta 12, and V alpha 2, we show that maternal T cells displayed a profoundly reduced TCR diversity, characterized by a lack of one or even several TCR V subsets in all six cases and a dramatic (5- to 25-fold) expansion of other TCR V subsets in three cases. In one patient analyzed, limited TCR diversity was also seen in T cells cultured from bone marrow and skin; restimulation experiments of these cells against cells expressing host MHC Ag were unsuccessful, as were attempts to exclusively allocate anti-host proliferative responses of maternal control T cells to the TCR V subsets that had undergone expansion in vivo. We conclude that a severely reduced TCR diversity is a common feature of maternal T cells engrafted in severe combined immunodeficiency patients. These novel findings provide a structural basis to understand the failure of these cells to protect the host from infections and may also help to understand their relative inefficiency to induce lethal, multi-organ, graft vs host disease. Moreover, as an experiment of nature, the reported phenomenon clearly illustrates the functional consequences in vivo of an insufficient TCR diversity.

Reticular Dysgenesis (RD) basically represents a lymphopenic severe combined immunodeficiency (SCID) in association with congenital agranulocytosis. It is presumed that RD results from a primary defect of pluripotent hematopoietic stem cells (HSC). Alternatively RD might be due to an alloreaction induced by T-cells derived from intrauterine transfusion of maternal cells into an immunoincompetent host. In the past 15 years, among 49 newborns with SCID taken care of in the University Hospital of Ulm, 5 children (4 boys, 1 girl) exhibited the characteristics of RD. In 3 of 4 cases studied, maternal T-cells were detected by HLA-typing. All 3 children showed signs of graft-versus-host-disease (GvHD), confirmed histologically. However, 9 of 45 newborns with SCID without congenital agranulocytosis also disclosed maternal T-cell-engraftment; 5 of the 9 had signs of GvHD. Therefore, it is unlikely that RD is caused by GvHD secondary to maternofetal transfusion. The fact that erythropoiesis, thrombopoiesis and the monocyte-macrophage-system basically are intact argues against a global maturation defect of HSC in RD.

Supportive care with blood component transfusions has greatly improved prognosis in patients with bone marrow failure. This progress has made possible by newly developed techniques for separation of blood cells and by a better understanding of the antigenicity of human blood cells and of immunologic reactions following their transfusion. Transfusion of white cell and platelet-poor red cell preparations prevent alloimmunization to leukocyte and platelet-bound alloantigens, or non-hemolytic transfusion reactions in already alloimmunized patients. Alloimmunization can be circumvened and effective long-term platelet support to thrombocytopenic patients can be provided by matching donor and recipient for HL-A antigens. The place of granulocyte transfusion in clinical therapy has yet to be defined, although their usefulness in infected granulocytopenic patients is suggested by the few studies reported so far.

The presence of lymphocytotoxic antibodies was studied in 22 patients receiving granulocyte transfusions. Due to previous blood transfusions 7 out of 22 patients had lymphocytotoxic antibodies before granulocyte transfusions were started. 6 out of 15 unsensitized patients developed antibodies during granulocyte transfusion therapy, and 9 out of 15 patients had no detectable antibodies at any time. Transfusion reactions occurred in the presence of lymphocytotoxic antibodies not directed against donor cells.

Transplant recipients previously sensitized to leukocytes and platelets by pregnancy and transfusion are subject to hyperacute allograft rejection after transplantation. Current methods for preparing buffy coat-poor blood for transfusion utilize differential centrifugation, filtration of heparinized whole blood, or sedimentation of the red blood cells in the presence of dextran. The average removal of leukocytes with these methods is 70–85%. The procedure described in the present paper increases leukocyte and platelet removal to 96% by passing blood that has been sedimented in the presence of dextran through nylon fibre filters. It is suggested that the technique is an effective method for preparation of buffy coat-poor blood for prospective allograft recipients.

Es wurden Resistenzmodalitaten zwischen Erythromycin, Lincomycin und Staphylomycin mit Hilfe von Bakterienpassagen in flussigem Nahrmilieu untersucht. Die daraus resultierenden Resistenzkoppelungen werden beschrieben und diskutiert.

A method for preparing buffy coat-poor red cell suspensions from stored ACD blood is described. Stored ACD blood is sedimented in presence of dextran and then passed through nylon fibre niters. A removal of 94% of leukocytes and 97% of platelets is achieved with this procedure. Red cell recovery is over 70%.

Es wurden Methoden zur Herstellung leukozyten- und thrombozytenarmer Blutkonserven bei unterschiedlichem Stabilisatorenzusatz untersucht. Als bestes Verfahren erwies sich dabei die Sedimentation der Erythrozyten im ACD-Macrodex-Gemisch mit anschlieseder Filtration des Sedimentes. Die Reduktionsrate der Leukozyten bzw. der Thrombozyten betrug dabei 96% bwz. 97,8%.

The polymorphism of the coagulation factor XIIIA and B subunits was determined before and after bone marrow or liver transplantation. It could be shown that the FXIIIA phenotype of the recipient was replaced by donor phenotype after bone marrow transplantation, whereas the phenotype of FXIIIB remained of recipient origin. In contrast, the FXIIIB phenotype changed to donor type after orthotopic liver transplantation but not the FXIIIA phenotype. The results indicate that the FXIIIA protein is produced in hemopoiesis, most likely in megakaryocytes and FXIIIB protein in the liver. Depending on the site of synthesis, FXIII alleles can be used as a marker in engraftment of FXIIIA-incompatible bone marrow transplantation or as a marker in FXIIIB-incompatible orthotopic liver transplantation.

Data obtained in the Seventh International Histocompatibility Workshop suggested the existence of two alleles subtypic to HLA-B17(1,6). The subdivision of HLA-B17 was confirmed during The Eighth and Ninth International Histocompatibility Workshops, and the two subtypic were designated HLA-Bw57 and HLA-Bw58 (2–5). These two alleles are both included in HLA-Bw4 (1–6).

A patient with severe combined immunodeficiency received three transplants of bone marrow from the HLA-B- and -D-identical mother. The first transplantation led to a severe graft-versus-host reaction followed by immunological reconstitution. A split chimerism was found with engraftment only of the maternal lymphocytes. Five months after the transplantation an autoimmune hemolytic anemia was observed which was due to rhesus incompatibility as well as polyspecific antibodies. At the same time agranulocytosis developed and 9 mth after the first transplantation the child suffered from aplastic anemia. Two further attempts failed to engraft the maternal hematopoiesis. The child died during the treatment with cyclophosphamide as conditioning for a third transplantation.

A bone marrow transplantation (BMT) was successfully carried out in a seven-year-old boy with aplastic anaemia. An HLA and MLC-identical brother served as donor. During the first 16 days after BMT between 0.55 and 7.11X10(10) granulocytes were transfused 14 times (an average of 4.03X10(10) per transfusion). The maximum increment in the peripheral blood after transfusion was over 4000 granulocytes per microliter. The course of local infections was taken as parameter for determining the clinical effectiveness of the granulocyte transfusion. Under the substitution therapy the necrotic ulcer of a fingertip and the mucous membrane ulcers in the oral cavity subsided. The presence of transfused granulocytes in the oral cavity ("orogranulocytes") could be established. There was a statistically significant relationship between the number of granulocytes in the peripheral blood and the number of orogranulocytes. It could be demonstrated that in children a temporary normalisation of the number of granulocytes in the peripheral blood can be attained by means of granulocyte substitution and that the transfused granulocytes are clinically effective.

A semi-micro method for the mixed lymphocyte culture (MLC) test in dogs is described. Optimization of various factors influencing the test were invistigated. Discrimination between allogeneic and isogeneic cell mixtures was possible after 90 h or culture but a culture period of 144 h was found to be optimal. The method measures MLC identity or non-identity and can be used as a tool for histocompatibility testing of lymphocyte defined (LD) Antigens.

Bw60 was serologically defined as a split of Bw40 at the Eighth International Histocompatibility Workshop 1980 [1]. It could be clearly recognized with monospecific sera, and separated from Bw61, the other Bw40 split, and from the cross-reacting antigen Bw48.

A panel of eleven HLA-DR5 homozygous lymphoblastoid cell lines was investigated for structural heterogeneity on the product level. HLA class II antigens were isolated by immunoprecipitation with different anti-class II monoclonal antibodies and separated by two-dimensional (2-D) gel electrophoresis. As a result, three distinct DRB1, one commonly expressed DRB3, and two distinct DQ gene products could be identified that combined to four different haplotypes associated with HLA-DR5. A hitherto serologically undetected split of HLA-DRw11 was presented by three cell lines. HLA-DRw11 and HLA-DRw12 were found to be related allospecifities that differ only in their DRB1 locus products, but are closely associated with the supertypic DRB3 allele HLA-DRw52b and with HLA-DQw7. The DRB3 alleles HLA-DRw52a and DRw52c were not detected in our cell line panel, indicating that these supertypic determinants are in negative linkage disequilibrium with HLA-DR5. Our data suggest that intra HLA-DR/DQ crossing-over events contribute to the development of the HLA class II polymorphism. Evidence is presented that the T cell defined HLA-D allospecifities are commonly determined by DRB1 and DQ gene products.

The definition of DRw8 at the last Histocompatibility Workshop was based on the reactions of five sera with r values between 0.40 and 0.58 [1]. For this Workshop, analysis of DRw8 was studied in relation to DR5 and DRw6. More extensive serum reaction patterns are presented in all three reports to permit a better comparison of these cross-reacting specificities (Table 1). The segregation pattern of the sera shown in Table 1 were studied in informative Workshop families. The mode of selection is given in details in the Joint Reports on DR5 (Betuel et al., this volume) and on DRw6 (Schreuder et al., this volume). Examples of such haplotypes are presented in Table 2. Several DRw8-related patterns were observed in which all DRw52 (MT2), DR5 + w8 + w13 (6.6) and DRw8 sera were positive.

In 167 first cadaver kidney recipients and their donors the blood groups ABO, Rhesus, Lewis, MN, Ss, P, Kell and Duffy were determined. The influence of incompatibility in each system as well as of simultaneous presence of several mismatches was analysed. Whereas one-year graft survival of Lewis-compatible grafts was 67 per cent (p = 0.02). The other blood groups showed no significant effect on graft outcome. Cumulative red cell incompatibilities, however, led to decreased survival rates. One-year graft survival in the group with greater than or equal to 4 incompatibilities was only 51 per cent versus 69 per cent in transplants with less than 4 incompatibilities (18% difference, p less than 0.01). When the Lewis system was excluded from analysis, the difference in survival rates was reduced to only six per cent. These data indicate that cumulative incompatibilities of red cell antigens have an unfavourable effect on graft survival. Of the different blood groups, the Lewis system is of major importance.

Patient survival after BMT is directly correlated with the HLA-type of the donor. The survival rate after BMT from an HLA-genotypically identical sibling is 56% in acute leukemia, 55% in combined severe immunodeficiency disease (SCID) and 67/83% in severe aplastic anemia patients. The usage of only HLA-D identical related or unrelated donors in SCID revealed a 37% survival, compared to 18% survival in acute leukemia and 11% in severe aplastic anemia using HLA-phenotypical identical or HLA-D identical related donors. BMT from HLA-phenotypical and MLC identical unrelated donors resulted in death of the grafted patients. Non of the patients grafted with HLA-different marrow survived BMT. Survival of BMT patients depended beside the histocompatibility matching on the clinical treatment and the clinical constellation of the patient: The survival rate decreased in aplastic anemia patients due to sensibilisation caused by pre-BMT blood transfusion and was significantly higher in leukemia when BMT was performed in remission.

C4 polymorphism was investigated in 13 orthotopic liver transplantations. It could be shown that recipient C4 phenotype disappears after transplantation and is replaced by donor phenotype on days 10-19 posttransplantation. This indicates that C4 is mainly produced in the liver. The delayed appearance of donor C4 phenotype compared with other complement components produced in the liver cannot be explained by different rates of synthesis or serum protein levels. The limited number of patients investigated in this study does not permit assessment of the role of C4, C3, Bf, and HLA-A, B, and DR in orthotopic liver transplantation.