Cryopreservation of marrow, purging and autologous bone marrow transplantation in childhood.

Since 1984 bone marrow from 42 children with acute lymphoblastic leukaemia, non Hodgkin's lymphoma and neuroblastoma was cryopreserved. In 5 cases (c-ALL, NHL and B type) the marrow was purged by using a cocktail of three monoclonal antibodies (VIL A1, VIB C5, VIB-E3). Up to now 13 children (ALL/10, neuroblastoma/3) were autografted (one of them after purging) after supralethal chemoradiotherapy. Except one child with early death all patients had engraftment: a level of 1.0.10(9)/l leukocytes was reached at days 10-33 (median, 19); platelet level over 60.10(9)/l at days 32-60 (median, 41). 2 children died on treatment related complications, one on infection after full haematological restitution, 2 patients alive with relapse, 8/13 alive in CCR and well.

[Lysis of leukemia cells with a monoclonal antibody-cocktail (e.g. VIP-pool) and human complement]. / Untersuchungen zur Lyse von Leukämiezellen mit einem monoklonalen Antikörpercocktail (sog. VIB-pool) und Humankomplement.

During the lysis of leukemic cells with a monoclonal antibody cocktail (the so-called VIB pool) and complement the attempt was made to replace rabbit serum as a complement source by human serum. For identifying the lysis of leukemic cells the complement-dependent in vitro cytotoxicity test was used and for excluding stem cell toxicity the CFU-c test according to PIKE and ROBINSON. In combination with the applied monoclonal antibody pool against B and c-ALL the human complement could be shown to be suitable to produce a lysis in the same manner as rabbit complement. Similarly to the pretested rabbit serum the treatment with the human complement had no impact on stem cell recovery. An optimal cytotoxic activity (95% against ALL blasts of patients, 100% against NALM) could be identified up to an antibody dilution of 1:32 with a volume percentage of 50% of human complement, an incubation temperature of at least 37 degrees C and an incubation time of 30 mins. With proved high reactivity against leukemic cells and lacking impairment of the haemopoietic power of the bone-marrow, this method can be recommended for "purging" protocol with the possibility of using human serum as a source of complement having advantages as far as clinical application is concerned.

[Detection of glucocorticoid receptors in leukemia cells by dexamethasone-induced cytolysis and [3H]-dexamethasone binding]. / Nachweis von Glukokortikoidrezeptoren auf Leukämiezellen durch Dexamethason-induzierte Zytolyse und [3H]-Dexamathasonbindung.

The presence of glucocorticoid receptors on the leukemic cells of 33 patients affected with acute lymphatic leukemia (ALL) and 6 patients affected with acute myeloic leukemia (AML) was investigated by dexamethasone-induced cytolysis and [3H] dexamethasone binding. The tests undertaken proved that after 20 hours of incubation 9 of 26 non-T-non-B-ALL (c-ALL and unclassified ALL) and 2 of AML were lysed with dexamethasone; blood lymphocytes and bone marrow leukocytes of healthy donors, however, were not affected. Non-T-non-B-ALL and AML were able to bind essentially more [3H] dexamethasone than T-ALL. There existed no correlation between dexamethasone binding and dexamethasone-induced cytolysis.

Different E-rosetting properties of human peripheral blood NK- and K-cells.

Mononuclear, non-adherent blood leukocytes were separated into the spontaneously E-rosetting (E+) and non-E-rosetting (E-) fraction. NK- and K-cell activity was determined simultaneously in a 4 h assay against 51Cr-labeled K 562 cell line cells and rabbit antiserum coated mouse leukemia cells (Gr/E) respectively. In each case E- exhibited a significantly higher K-cell activity than E+ [M16 donors E-:E+ = 46 +/- 11:10 +/- 6 (% specific cytotoxicity)]. With regard to NK-cell activity E- of only 7 donors was significantly more active than E+ [M7 donors E-:E+ = 49 +/- 15:20 +/- 10]. Six times the activities of the two fractions were not significantly different [M6 donors E-:E+ = 46 +/- 15:39 +/- 14]. On the other hand E+ of 3 donors displayed a significantly stronger activity than E-[M3 donors E-:E+ = 13 +/- 10:36 +/- 9]. These results confirm the heterogeneity of NK-cells with respect to E-rosetting properties and indicate that NK- and K-cells of at least 3 donors may belong to different cellular subsets [NK:E- less than E+; K:E- greater than E+].

[NK and K cell activity in children with leukemias and aplastic anemias before and after allogeneic bone marrow transplantation]. / NK- und K-Zell-Aktivität bei Kindern mit Leukämien und aplastischen Anämien vor und nach allogener Knochenmarktransplantation.

Mononuclear leukocytes from the peripheral blood and bone-marrow of children affected with aplastic anemia and leukemia were investigated for K-cell activity (antibody-dependent cellular cytotoxicity) and NK-cell activity before and after allogenous bone-marrow transplantation. 51Cr liberation test against murine Graffi erythroblast leukemic cells covered with xenoantibodies and K-562 cells were used for identification. Strongly lowered NK- and K-cell activities could be found in aplastic anemia prior to bone-marrow transplantation. However, NK-cell activity was only lowered significantly in leukemic patients with indication of bone-marrow transplantation. K-cell and NK-cell activities normalised after bone-marrow transplantation. K-cell and NK-cell activities could be observed to be reconstituted very early after bone-marrow transplantation.

[Nonspecific killer cells in premature and mature neonates and infants]. / Unspezifische Killerzellen bei unreifen und reifen Neugeborenen und Säuglingen.

K (killer) and natural killer (NK) cells were investigated in peripheral blood of 76 children, preterm small for date babies (n = 8), preterm babies (n = 15), fullterm small for date babies (n = 6) fullterm babies (n = 7) and infants up to 12 months age (n = 40). The K and NK cell activity of human leukocytes was analysed as compared with those cells of the K 562 cell line and murine cells covered by xenologous antibodies in Graffi erythroblast leukemia by means of the 51Cr release test. K cell activities were significantly lower in preterm small for date babies to infants with 1-12 months of age. In our results it is shown that NK capacity of preterm or term newborns and infants up to 6 months age does not differ significantly from each other. Children who are 6-12 months old will have significantly higher NK cell activities. It can be concluded that K cell activities are fully developed during pregnancy and NK cell activities later when the children are between 6 and 12 months of age.

[Spontaneous lymphocyte-mediated cytotoxicity and antibody-dependent cell cytotoxicity of human effector cells]. / Spontane lymphozytenvermittelte Zytotoxizität und antikörperabhängige zelluläre Zytotoxizität menschlicher Effektorzellen.

Antigen dependent cellular cytotoxity (ADCC) and non-killer cell activities of haematological healthy donors were investigated in the 51Cr release test. Attempts of cell fraction reveal that lymphocytes are active as killer and non-killer cells. Granulocytes were efficient effector cells of antigen dependent cellular cytotoxity (ADCC), however, they had no natural-killer activity. In testing leukocyte fractions of 11 donors, killer cell would only be found in the non-T-fraction. In contrast to that, three types could be observed in the distribution on non-killer cells: Distribution on T-lymphocyte fraction (27.3%), distribution on non-T-lymphocyte fraction (9.1%) and approximately equal distribution on T- and non-T-lymphocyte fraction 63.7%). Without any treatment patients with acute lymphocytic leukemia showed an antigen dependent cellular cytotoxity and non-killer activity only in exceptional cases. Normal activities were reached in remission, with chemotherapy having a depressive effect on non-killer activity.

[Cytotoxic complement-dependent anti-thrombocyte antibodies in rheumatoid arthritis patients]. / Zytotoxische komplementabhängige Anti- Thymozyten-Antikörper bei Rheumatoid-Arthritis-Patienten.

Sera of 38 patients and 10 blood donors were examined for cytotoxicity against human thymocytes. As method for testing the complement-dependent cytotoxicity the lymphocytotoxicity test was adapted for thymocytes. The sera of blood donors and of patients with non-rheumatic diseases showed no or only a very small rate of cytolysis (3.6%). The rate of cytolysis of sera of patients with arthroses (8.5%) and of collagenoses (14.5%) were below 20%, i.e. still within the limits of normal. The sera of seronegative arthritides (30%) and of patients with rheumatoid arthritis (40%) showed an unequivocally pathological cytotoxicity. The complement-dependent thymocytotoxicity might be coordinated to the clinical arthritis.

[Human nonspecific killer cells]. / Unspezifische Killerzellen des Menschen.

The NK and K-cell activity of human leukocytes was investigated as compared with those cells of the K 562 cell line and murine cells covered by xenoantibodies in Graffi erythroblast leukaemia by means of the 51Cr release test. NK and K-cells could be identified in the blood and bone-marrow. However, they could not be identified in the thymus, lymph-nodes, and tonsils. Attempts of cell fraction with the blood of healthy donors revealed that the K-cells must be attributed to non-T-lymphocytes. NK-cells may be found in the fraction of non-T-lymphocytes as well as in that of T-lymphocytes. Killer cell activity tests in children with acute leukaemia resulted in leukaemia cells having NK and K-cell activity only in very rare cases. ALL patients in remission had strongly lowered NK-cell values under chemotherapy. In comparison to that, chemotherapy had no influence on K-cell activity. On the one hand, NK-cell activities were induced in mixed cultures of allogenous lymphocytes of the blood and, on the other hand, in cells of lymph-nodes. Attempts of fractionation, investigations for determining the influence of chemotherapy and attempts of inducing killer cell activity in vitro lead to the conclusion that NK and K-cells may be regarded as similar cell populations, being, however, not identical.

[Antibody-dependent cellular cytotoxicity (AAZZ) against leukemia cells--K-cell activity of human leukocytes]. / Antikörper-abhängige zelluläre Zytotoxizität (AAZZ) gegen Leukämiezellen--K-Zell-Aktivität menschlicher Leukozyten.

The activity of human leukocytes from the peripheral blood (PB) and bone-marrow (BM) to take the function as K-cells in the antibody dependent cellular cytotoxicity (ADCC) was tested in a 51Cr-test against mouse leukaemic cells and ALL cells covered with specific heterologous antibodies. Mononuclear PB-leukocytes and granulocytes of healthy donors and patients with leukemia and lymphoma in remission lysed murine and human leukaemic cells in the presence of specific antibodies. There was no lowering of K-cell activity of mononuclear PB-leukocytes of patients with leukaemia and lymphoma in remission under chemotherapy as compared with healthy donors and patients in remission without chemotherapy. There was a good correlation between the percentage of K-cell active mononuclear leukocytes in PB and BM. Attempts of fractionation with peripheral blood leukocytes of healthy donors resulted in the non-adherent mononuclear PB-leukocytes (lymphocytes) and granulocytes being effector cells in ADCC. To a high degree K-cell active lymphocytes could be identified in the non-B-fraction and only slightly in the fraction forming E rosettes.

[Subtypification of acute lymphocytic leukaemia (ALL) in childhood by characterization of immunological surface membrane markers (author's transl)]. / Subtypisierung der akuten lymphatischen Leukämie (ALL) im Kindesalter durch Bestimmung immunologischer Oberflächenmaker.

Leukaemic blast cells isolated from bone marrow or blood of 42 children with ALL were investigated for presence of immunological surface membrane markers. By characterization of 5 surface markers (reaction with an anti-ALL serum for demonstration of a leukaemia-associated antigen, reaction with an anti-thymocyte serum and formation of E-rosettes for demonstration of T-lymphozytes, as well as reaction with an anti-Ig serum and formation of EAC-rosettes for demonstration of B-lymphocytes) the ALL cells of the 42 patients could be divided into 5 subtypes: I. 18 patients (42,7%( O-ALL with common ALL antigen II. 13 patients (31%) O-ALL without common ALL antigen III. 7 patients (16,7%) T-ALL with E-rosette formation IV. 3 patients (7,2) T-ALL without E-rosette formation V. 1 patients (2,4%) B-ALL.

Human leukaemia-associated antigens expressed by acute myelocytic leukaemia cells and their detection by heterologous antisera.

Antisera against human acute myelocytic leukaemias were tested in complement-dependent in-vitro cytotocity tests against leukaemia cells and normal cells as targets. After absorption with erythrocytes and spleen cells from allogeneous donors the antisera reacted with leukaemia cells, but not with leukocytes from bone marrow and the peripheral blood of children in remission, lymphocytes from healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-induced blasts and cord blood lymphocytes. Extensive cross reactions were obtained in the tests against leukaemia cells. The antisera reacted not only with AML cells, but also with ALL, CLL, and CML cells. It was possible to remove the cross-reactivity with ALL cells through absorption with ALL cells or with fetal tissue, and to remove the cross reactivity with CLL cells through absorption with CLL. A complete absorption of the anti-AML sera was possible with AML and CML cells. After absorption with fetal tissue and CLL cells the antisera showed exclusively specificity for myelocytic leukaemias. Thus, AML cells contain three leukaemia-associated membrane antigen components: an antigen of fetal origin, a "CLL-specific" antigen, and an antigen that occurs on myelocytic leukaemias.

Human leukaemia-associated antigens expressed by acute lymphocytic leukaemias and their detection with heterologous antisera to T, B-, and non-T-non-B subtype AL blasts.

Antisera from rabbits and goats against subtypes of acute lymphocytic leukaemia (ALL with T-cell markers, ALL with B-cell markers, Non-T-non-B ALL) were tested for their specificity in complement-dependent in-vitro cytotoxicity testing. After absorption of the fivefold diluted antisera with erythrocytes and spleen cells of allogenous donors they reacted with ALL cells, but not with leukaemias of other types (AML, CLL, CML), lymphocytes of healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-stimulated lymphocytes, cord lymphocytes and bone marrow lymphocytes of patients in remission. In the reactions of the antisera against ALL cells the subtype of ALL is of major importance: Six rabbit antisera and one goat antiserum against T-subtype ALL reacted in all 19 tests with the leukaemia cells of 5 patients with T-cell ALL and in all 9 tests with thymocytes of 3 donors, but only in 14 out of 41 tests with the leukaemia cells of 14 Non-T-non-B ALL patients. One antiserum against a B-subtype ALL lysed B-cell ALL (1/1), but not T-cell ALL (0/3), Non-T-non-B-cell ALL (1/5) and thymocytes (0/2). Four antisera against Non-T-non-B-subtype ALL reacted in 22 out of 46 tests with the Non-T-non-B cells of 17 ALL patients, but did not react with the leukaemia cells of 4 children with T-cell ALL (0/16), one child with B-cell ALL (0/1) thymocytes of 2 donors (0/4). The reactions of the anti-ALL sera with fetal liver cells, complete absorbability of the antileukaemic activity of the antisera with fetal tissue and the reactions of an anti-fetal serum with ALL cells point to the existence of fetal antigen components as leukaemia-associated antigens.

[Serological diagnosis of the antigenic markers of acute lymphatic leukaemias and non-Hodgkin lymphomas in childhood (author's transl)]. / Serologische Bestimmung der antigenen Marker von akuten lymphatischen Leukämien und Non-Hodgkin-Lymphonen im Kindesalter.

Leukaemia cells of 11 children with acute lymphatic leukaemias (ALL) and lymphoma cells of 4 children with lymphosarcoma (LS) were studied for the presence of T- and B-lymphocyte markers on their cell membranes. Spontaneous rosette formation (E-rosettes) of the malignant cells after adding sheep erythrocytes and the reaction with antithymocyteserum in cytotoxic test served as T-cell-markers and the surface immunoglobulins as B-cell-markers. The leukaemia cells of 5 ALL-patients with mediastinal tumours formed E-rosettes and reacted with the anti-thymocyte-serum. Six ALL-patients did not show these reactions. None of the ALL-patients had surface-immunoglobulins. Two of the 4 LS-children were E-rosette-positive, the cells of the other two LS-children contained surface immunoglobulins. There was a good correlation between the formation of E-rosettes and the reaction with the anti-thymocyte-serum. After absorption of the anti-thymocyte-serum with peripheral leukocytes it reacted with some of the E-positive lymphoblasts and thymocytes but not with normal peripheral T-lymphocytes. Anti-leukaemia-sera against the ALL-cells with T-cell markers of two patients after absorption with spleen cells did not react with peripheral lymphocytes but did effect lysis of thymocytes. Consequently, leukaemia cells which are E-rosette-positive possess a T-lymphocyte-associated antigen and a Thymus-associated antigen.

[Foetal antigens on leukaemia cells (author's transl)]. / Fötale Antigene auf Leukämiezellen.

An antiserum against human foetal liver cells reacted in in-vitro-cytotoxic test with the leukaemia cells of 6 out of 10 children with acute lymphocytic leukaemias (ALL) and with the lymphoma cells of 1 out 2 children with lymphosarcoma (LS). No cytotoxic reactions were obtained against leukaemia cells of 5 children with acute myelogenic leukaemia (AML), leukaemia cells of 7 adults with chronic lymphocytic leukaemia (CLL), bone marrow cells of 9 children in clinical remission and lymphocytes of normal donors. The cytotoxic activity of the anti-foetalserum was removed by absorption with foetal liver but not with adult liver. The results suggest that foetal antigens may occur on ALL-cells and LS-cells.