Inhibitory effects of 5-azapyrimidine nucleosides on cellular immunity.

The effect of 5-azacytidine (5-AzCR) and 5-aza-deoxycytidine (5-AzCdR) on the survival of skin grafts in mice and rats, the action of these drugs on regional GVH reaction, as well as the formation of haemopoietic colonies (CFU-5) in the spleen were studied. Both drugs prolonged the life span of skin grafts when administered 24 hr before transplantation, or on the 4th post-transplantation day. However, they were little effective when injected 24 hr after skin grafting, or after induction of the regional GVHR. Following intraperitoneal administration, they inhibited CFU-5 formation. Two-hour incubation in vitro of cells with 5-AzCR significantly reduced their GVH reactivity and capacity to form CFU-5; 5-AzCdR under the same conditions was ineffective.

Transplantation of allogeneic peripheral blood progenitor cells--a single centre experience.

13 patients have been transplanted at Institute of Hematology and Blood Transfusion since 1995 using allogeneic PBPC either alone or with bone marrow as a source of progenitor cells. All donors were G-CSF mobilised HLA identical family members. PBPC harvests were performed on D 4,5, (6) of G-CSF administration. The medium content of TNC, CD34+, CD3+, CD4+and CD8+cells/kg b.w. of the recipients in the grafts were: 13,1x10(8)(TNC), 11,4x10(6)(CD34+), 393x10(6)(CD3+) 243x10(6)(CD4+), 125x10(6)(CD8+) The patients received either BuCy2 or CyTBI preparative regimen and Cyclosporin A + short course of Methotrexate for GVHD prophylaxis. Engraftment of ANC >500 was achieved by D+16 and PLT >20.000 by D+19. Three of ten evaluable patients developed acute and three of nine chronic GVHD. 8 patients survive with the longest follow up 776 days.

The influence of methylpropionic acid and pyridazinone-3 derivatives on some immunologic and hemopoietic functions.

There was studied the influence on the cell-mediated and humoral response in vivo manifested by selected methylpropionic acid and pyridazinone-3 derivatives which had been found to possess strong immunotropic effects in the in vitro screening previously. It was shown that the compounds were generally poorly tolerated by animals, and they exerted only weak suppressive effects on antibody production, the contact hypersensitivity and survival of skin grafts. This immunosuppressive activity was accompanied by a slight decrease in the number of spleen colony forming cells (CFU-s). Only limited correlation between the biological activity of the preparations and their chemical structure was found.

Induction of NK and LAK activities in human lymphocyte culture by a cytosol fraction from leukemic myeloblasts and by monoclonal antibody CD 3.

The stimulating effect of cytosol fraction (F3) isolated from human myeloblasts (m.w. ranging from 30 to 100 kDa) and monoclonal antibody CD 3 (MEM 57) was tested on NK and LAK cell activities in peripheral mononuclear cells (PMNC) of normal donors and leukemic patients. The F3 fraction added in 10% of the total volume of RPMI 1640 medium induced significantly increased lytic activity of normal lymphocytes or IL-2 activated lymphocytes against K 562 cells in 3-day culture. Similarly, the proliferation of both cell cultures was enhanced by F3. The effect of F3 on NK and LAK cell activities in culture of PMNC from leukemic patients was less pronounced and synergic action of F3 did not occur, whereas the proliferation of leukemic cells was significantly enhanced. MEM 57 increased the cytotoxicity and cell proliferation in 3-day culture of normal lymphocytes at concentrations ranging from 10 to 250 ng/ml. MEM 57 stimulated also NK cytotoxicity estimated in freshly isolated normal lymphocytes. The deficient NK cell activity observed in PMNC of leukemic patients was induced by MEM 57 or its combination with IL-2 in 3-day culture. These observations indicate that combination of more immunomodulating agents can lead to shortening of the incubation time necessary for correction of the NK cell defective activity in PMNC of leukemic patients.

Positive effect of direct current on cytotoxicity of human lymphocytes.

Normal or leukemic human lymphocytes treated with direct current (DC) showed enhanced antileukemic cytotoxicity. The enhancing effect of DC-treated lymphocytes was dependent on current density and time exposure. A desirable effect was achieved with current densities ranging from 5 to 10 mA/cm2 at a short exposition time (5--10s). Enhanced lymphocyte cytotoxicity occurred after a 48 h cultivation at 37 degrees C in a humidified atmosphere containing 5% CO2 and was proved by the increased number of trypan blue stained target cells, tumor-binding cells, and lymphocytes with activated nucleoli. Lymphocyte cytotoxicity measured immediately after DC-treatment was not enhanced. Furthermore, the cytotoxic effect was potentiated using media conditioned with interleukin-2 (IL-2) or cytosol fraction (F3) isolated from human leukemic cells. Such in vitro stimulated cytotoxic cells displayed reactivity against K 562 cells as well as fresh leukemic cells of allogeneic origin. Of considerable clinical interest is the observation that lymphocytes treated with DC in IL-2 or F3 conditioned media may enhance antileukemic cytotoxicity in peripheral lymphocytes of patients with hematological malignancies.

Derivatives of benzo(c)fluorene. XI. Effect of Benfluron on hemopoietic stem cells.

The effect of Benfluron-- 5-(2-N,N-dimethylamino-ethoxy)-7-oxo-7H-benzo(c)fluorene hydrochloride--on hemopoietic stem cells was tested by the production of spleen cell colonies (CFU-S) in irradiated mice following application of bone marrow cells and by the production of hemopoietic colonies (CFU-C) in semisolid agar. The reduced numbers of CFU-S were found in mice applied Benfluron or Benfluron-treated bone marrow cells. The increased numbers of CFU-S, however, were found in mice receiving bone marrow cells from Benfluron-treated donors. The production of CFU-C was decreased after 2 h incubation of bone marrow cells with 0.5-5 micrograms/ml of Benfluron, and fully inhibited at higher concentrations.

Derivatives of benzo(c)fluorene. X. Inhibitory effect of Benfluron on cellular immunity.

The effect of a cytostatic drug Benfluron-- 5(2-N,N-dimethyl-amino-ethoxy)-7-oxo-7H-benzo(c)fluorene hydrochloride was tested in mice on skin graft survival, graft-versus-host reaction (GVHR) and for mitogenic stimulation of human lymphocytes. Application of Benfluron resulted in a prolonged skin graft survival. The regional and systemic GVHR was potentially inhibited by p. o. administration of Benfluron at a dose of 100 mg/kg of body weight. Allogeneic spleen cells incubated with Benfluron at concentration of 5-20 micrograms/ml showed reduced capability to induce GVHR. PHA or PWM stimulation of lymphocytes was completely inhibited in the presence of 2 micrograms/ml Benfluron. Benfluron as a potent immunosuppressive agent can be considered useful for the prevention of GVHR.

Comparison of two different methods for CD34+ selection and T cell depletion in peripheral blood stem cell grafts--our experiences with CellPro, E rosetting and CliniMACS technique.

The aim of this study was to establish a suitable method for in vitro T cell depletion in peripheral blood stem cell grafts for mismatched/haploidentical transplantation in children and adults with severe hematological disorders and for autologous transplantation in patients with autoimmune diseases refractory to conventional immunosuppressive treatment. Two different selection techniques have been used: CD34+ selection using immunoaffinity columns (CellPro Ceprate) followed by T cell depletion by E-rosetting or CD34+ selection using submicroscopic paramagnetic beads (CliniMACS device) with T cell depletion in a one step procedure. The mean purity and recovery of CD34+ cells and efficiency of T cell removal in the final product were compared. From March 1995 to December 1998 we prepared twelve allografts using Cell Pro system for eight children with high-risk hematological malignancies and six autografts for six patients with severe autoimmune diseases. From January 1999 to October 2000 we prepared fifteen allografts using CliniMACS system for ten children with high-risk hematological diseases and inborn metabolic disorders or primary immunodeficiences, five allografts for three adult patients with high-risk hematological malignancies and two autografts for two patients with autoimmune diseases. In allogeneic transplantation the median purity of CD34+ cells in the final products after CellPro and E-rosetting was 85.6% (55.3%-95.7%); median recovery was 24.8% (17%-35%), median transplanted doses of T cells per kilogram of body weight were 0.66x10(4) (0-2.8); in autologous transplantation the median purity of CD34+ was 92.6% (55.6%-96%), median recovery was 28% (22%-46.2%), median transplanted doses of T cells per kilogram of body weight were 0.39x10(4) (0.0-3.6). After CliniMACS technique the median purity of CD34+ cells was 94.87% (69.15%-99%),medianrecoverywas 58% (30%-79.6%), median transplanted doses of T cells per kg of body weight were 0.254x10(4) (0-14.15); in autologous transplantation the median purity of CD34+ was 94% (94%-94%, median recovery was 97.4% (95%-99.8%), median transplanted doses of T cells per kilogram of body weight were 0.87x10(4) (0.49-1.24). We consider both methods of CD34+ selection and T cell depletion suitable for peripheral blood stem cell processing before mismatched hemopoietic stem cell transplantation in patients without identical donor or before autologous transplantation for severe autoimmune diseases. However, magnetic separation using CliniMACS system results in higher levels of purity and recovery with efficient T cell depletion.

Influence of the incubation of cells with zinc and lithium ions on GVH reactivity of cells and on their ability to form haemopoietic colonies.

Mouse spleen and bone marrow cells were incubated for 2 h with ZnCl2 and Li2SO4 at different concentrations and tested for the ability to evoke the graft-versus-host reactions (GVHR) and to form pluripotent haemopoietic colonies. ZnCl2 at concentrations 5 X 10(-6) to 5 X 10(-4) M inhibited the regional GVHR. At a concentration of 5 X 10(-6) ZnCl2 also inhibited the ability to elicit the systemic GVHR in irradiated mice. Haemopoiesis was stimulated in cells incubated with ZnCl2 at concentration 5 X 10(-6) M, it was inhibited after incubation of A/01a cells with 5 X 10(-4) M ZnCl2, whereas cells from non-inbred ICR mice were stimulated by the latter concentration of ZnCl2 for haemopoiesis. Li2SO4 inhibited the ability to induce the regional GVHR at concentrations of 5 X 10(-3) and 10(-2) M, had no effect on survival of mice during the systemic GVHR and exerted rather an inhibitory effect on haemopoiesis.

Effect of the treatment of parental cells in vitro with anti-T antibody on bone marrow transplantation in F1 hybrid mice.

Anti-T antibody was obtained from xenogeneic antithymocyte serum by adsorption and elution with brain immunosorbent. Bone marrow and spleen cells from the parental A strain were treated in vitro with anti-T antibody and complement or with the original ATS and complement. So treated cells were assayed for the ability to induce a local and a total GVH reaction in (A X C57BL)F1 hybrid mice, to prolong the survival of lethally irradiated, identical F1 hybrid mice and to form haemopoietic colonies in spleens of the syngeneic irradiated recipients. The local GVH reaction induced by anti-T antibody- and ATS-treated spleen cells was somewhat lower. Administration of a mixture of spleen and bone marrow cells treated with anti-T antibody prolonged the survival of lethally irradiated recipients but did not prevent the consequences of late GVH reaction. The colony-forming activity of bone marrow cells treated with anti-T antibody was not reduced in most experiments. It can be concluded that the treatment of cells from mouse haemopoietic organs with anti-T antibody and complement damages mature T lymphocytes and in this way reduces the ability to induce GVH reaction, but does not damage T lymphocyte precursors and haemopoietic stem cells.

Effect of a synthetic poly N-(2-hydroxypropyl)methacrylamide (Duxon) on haemopoiesis and graft-versus-host reaction.

A synthetic polymer, Duxon, was developed and tested as a substitute of blood plasma for transfusion purposes. Tests of this preparation included a test for its influence on the haemopoietic stem cells and the graft-versus-host reaction (GVHR). A single dose or repeated doses of Duxon did not reduce the number of pluripotent haemopoietic colonies (CFU-S) in mice and short-term incubation of cells from haemopoietic organs of mice with Duxon resulted in a slight, yet significant, increase in the number of CFU-S. Injection of Duxon non-significantly diminished the manifestations of the regional GVHR. Incubation of spleen cells with Duxon significantly reduced their GVH reactivity in the regional test. The systemic GVHR was moderately inhibited by repeated doses of Duxon in sublethally irradiated mice. A significant delay in deaths of animals from GVHR was seen in lethally irradiated mice receiving a single dose of Duxon on day 4 after GVHR induction. Survival of lethally and sublethally irradiated mice was prolonged following injection of cells incubated with Duxon. Possible uses of Duxon, a preparation with mild immunosuppressive properties, not impairing haemopoiesis, are discussed.

An attempt at isolation and partial characterization of specific antibodies from antithymocyte serum.

Specific anti-T antibody was isolated from rabbit anti-mouse and anti-rat thymocyte antiserum on immunosorbent prepared by fixation of brain homogenate in glutaraldehyde. Conditions of isolation, purity of obtained antibody and cytotoxic activity were followed. The resulting preparation containing immunoglobulin G is contaminated with beta globulin and albumin, contains non-precipitating proteins originating from brain tissue and is cytotoxic for thymocytes. Cytotoxicity for bone marrow cells was not demonstrated.

Some biological properties of mouse spleen cells fractionated by the adherence of Sephadex G 25 and glass bead columns.

The possibilities of separation of haemopoietic cells from lymphocytes capable of eliciting the graft-versus-host reaction through column chromatography were investigated. Strain-A mouse spleen cells were fractionated into the adherent and non-adherent fraction on Sephadex G-25, glass bead columns and glass beads coated with antibody against mouse globulin. Increased numbers of cells forming haemopoietic colonies were found in the cell fraction which did not adhere to the antibody-coated glass beads and in cells reversibly adhering to glass beads. No significant decrease in local graft-versus-host reaction was found in any fraction obtained, and the prolonged survival of irradiated semiallogeneic recipients was observed in both fractions obtained on Sephadex G-25 columns.

Immunosuppressive effects of a synthetic polymer poly N-(2-hydroxypropyl)methacrylamide (Duxon).

Poly N-(2-hydroxypropyl)methacrylamide (Duxon) was studied as a substitute of blood plasma and was found to prolong significantly the survival of semiallogeneic skin grafts in mice and rats and to reduce MLC reactions of human lymphocytes. It did not inhibit the growth of lymphocytes and the growth of the fibroblast-like (diploid) or epithelioid (heteroploid) cell lines in tissue culture.

Immunosuppressive effects of bovine seminal fluid fractions with ribonuclease activity.

Using different isolation procedures (after acidification and saturation with 3 M ammonium sulphate) three fractions were isolated from bull seminal vesicle fluid and assayed for their effects on cell immunity in vitro and in vivo. Two of these preparations (ZS RNase and AS RNase) possessing a high level of ribonuclease activity at concentrations of 50 micrograms/ml showed inhibitory effects (up to 80%) on 3H-thymidine incorporation into the DNA of mitogen-or antigen-stimulated human lymphocytes. The third preparation (3M-P) possessing low ribonuclease activity showed lesser inhibitory effects. The potency of mouse spleen cells to cause regional GVH reaction was significantly decreased after preincubation of spleen cells to cause of 1 mg per AS RNase or ZS RNase whereas 3M-P was ineffective in this test. A single dose of 1 mg per mouse of ZS RNase or 3M-P administered i.p. on day 4 after skin transplantation significantly prolonged graft rejection. Both preparations at this dose potentiated the effect of cyclophosphamide on skin graft survival. All tested preparations preincubated with mouse bone marrow cells had no adverse effects on their colony-forming activity (in the spleens of irradiated mice). The possibility of utilizing the preparations with ribonuclease activity isolated from vesicle fluid in clinical bone marrow transplantation is discussed.

[Manipulation of hematopoietic stem cell grafts and their use in clinical practice]. / Manipulace se stepy hematopoetických progenitorových bunek a jejich pouzití v klinické praxi.

Autologous stem cell transplantation has been successfully used in treatment of various hematological malignancies and solid tumors in children and adults. Published data have confirmed that bone marrow harvests and peripheral blood stem cell collections frequently contain a significant number of tumor cells. Contaminating tumor cells can contribute to the disease relapse in posttransplant period, so attempts are made to eliminate contaminating tumor cells from autografts. In the case of allogenic transplantation, T-lymphocytes depletion from graft decreases the risk of the graft versus host disease after transplantation. In this article we comment techniques available and method used for elimination of tumor cells. Commentary is aimed on potential benefits and risks of every method.

[Separation of hematopoietic progenitor cells from peripheral blood in patients with hemato-oncologic malignancy]. / Separace hemopoetických progenitorových bunek z periferní krve (PBPC) u pacientu s hematoonkologickými malignitami.

BACKGROUND: Transplantations of haematopoietic progenitor cells from peripheral blood (PBPC) are able to ensure haematopoietic and immunological reconstitution as well as stable long term engraftment. Autologous PBPC are administered after previous myeloablative chemotherapy to patients with haematological and non-haematological malignancies. The objective of the submitted study was to follow-up the results of autologous separations of PBPC in patients with a good effect of mobilisation therapy. The authors evaluated in PBPC concentrates the content of cell parameters needed for transplantation. In the subsequent part of the trial they mention the times of engraftment after autologous transplantation. METHODS AND RESULTS: The authors evaluated parameters of 26 separations of PBPC in 11 haematooncological patients with a good effect of mobilisation therapy and with concentration of CD 34+ cells higher than 20 x 10(3)/ml of peripheral blood. The separations of PBPC were implemented on the Cobe Spectra and Baxter CS 3000 Plus equipment under a standard regime with processing of 12 l blood, i.e. 2.7 total blood volumes of the patients. In the mentioned group of patients already from one separation an adequate amount of CD 34+ cells for their transplantation was obtained. Transplantation doses were prepared on average from two separations and amounted as regards MNC/kg, CD 34+ cells/kg and CFU-GM/kg to 4.3 x 10(8), 17.1 x 10(6) and 2.5 x 10(4). The assembled parameters correspond to, or in some parameters exceed, recommended amounts for their transplantation. CONCLUSIONS: In well mobilised patients under the regime of standard separations adequate amounts of progenitor cells for their autologous transplantation were obtained. Transplantation doses were prepared from two collections. Investigation of pre-separation concentration of CD 34+ cells in the peripheral blood is a reliable indicator for starting PBPC separation. Early post-transplantation results indicate the time of engraftment 10 days on average and minor need of substitution therapy with blood products.

[Results of bone marrow transplantation at the Institute of Hematology and Blood Transfusion in Prague]. / Výsledky transplantace kostní drene v Ustavu hematologie a krevní transfúze v Praze.

The first allogenic bone marrow transplantation (TKD), when for the preparation whole body irradiation was used, was implemented in the Institute of Haematology and Blood Transfusion (UHKT) in Prague in 1986. Before June 1992 36 TKD were performed incl. 28 allogenic, 2 syngenic and 6 autologous. For the first time bone marrow from a non-related donor was transplanted. Of 30 allogenic and syngenic TKD to the present time 17 patients survive, i.e. 56.6% of the whole group. According to individual diagnoses 8 patients with the diagnosis of chronic myeloid leukaemia (CML) survive, 5 of 10 patients with the diagnosis of acute leukaemia (AL) and 3 of 4 patients with the diagnosis of severe aplastic anaemia (SAA) or with Fancon's anaemia (FA) resp. The survival period of the whole group is from 1-62 months since the transplantation. The main cause of death of 8 from 13 patients who died were infections associated with acute or chronic disease of the graft against the host (GVHD). In autologous TKD the bone marrow was treated with etoposide. Of the six transplanted patients with AL five survive 1.5-30 months after transplantation. The authors present some general information of pretransplantation preparation, prevention of GVHD, its incidence and results of TKD.

[Allogenic transplantation of bone marrow in childhood]. / Alogenní transplantace kostní drene v detském veku.

BACKGROUND: Bone marrow transplantation has become the therapeutic method in some forms of malignant haemotopoietic diseases, malignant tumours, inborn errors of metabolism and immunodeficiency states. The objective of the presented work is the analysis of 40 allogenic bone marrow transplantations in children made in 1989-1994. METHODS AND RESULTS: Bone marrow transplantation was made in 40 children (26 boys and 14 girls), mean age 10.5 years (range 1.5-17.5 years). Indications were acute lymphoblastic leukaemia in 11, acute myeloid leukaemia in 10, chronic myeloid leukaemia in 6, myelodysplastic syndrome in 2, aplastic anaemia and Fanconi's anaemia in 7, non-Hodgkin lymphoma in 2 and inborn errors in 2 children. The donor was in 33 patients in HLA identical sibling and in seven instances a monozygotic twin, HLA non-identical sibling or relative or unrelated matched donor. Bone marrow engraftment was achieved in 35 (87.5%) patients, in one instance the bone marrow was rejected (2.5%) and in four patients (10%) early death occurred before the bone marrow engraftment. On Aug. 15, 1995 20 patients (50%) survived, a relapse developed in 7 (17.5%) and 13 patients died in conjunction with the transplantation (32.5%). The most frequent cause of death were infectious complications (9 children) either in conjunction with the development of graft versus host reaction (6x) or without signs of this reaction (3x). As a prophylaxis of graft versus host disease 24x Cyclosporine A with corticosteroids was used, 16x with methotrexate. A chronic graft versus host disease developed in 6 of 28 children surviving 100 days after transplantation. The greatest problem are infectious (bacterial and mycotic) complications in the phase of bone marrow aplasia before engraftment of the transplanted bone marrow or in conjunction with a graft versus host reaction which cannot be completely avoided by preventive measures. CONCLUSIONS: Bone marrow transplantation is also in children an effective therapeutic method of some forms of malignant haematopoietic diseases, malignant tumours and immunodeficiency states. The correct indication, suitable donor, preventive measures against the graft versus host reaction and protection against infectious complications are essential for the success of this pretentious treatment.

[Purging of hemopoietic progenitor cells in autologous transplantation]. / Cistení hemopoetických progenitorových bunek pro autologní transplantace.

The authors describe the results of purification of bone marrow and peripheral progenitor cells (PBPC) for clinical transplantations. Vepeside was used to purify in 1990-1995 a total of 41 bone marrows of adults and children. Of these 23 were transplanted. Maphosphamide was used bone marrow purging in two patients; transplantation was performed in one case. By a combination of Vepeside with methylprednisolone haematopoietic cells of 24 patients were purged, transplantations were performed in 10. Three-day cultivation of haematopoietic cells in the presence of Desferal was used for purging cells of 22 patients with neuroblastoma; transplantations were performed in 10 patients. The authors give the values of nucleated cells, haematopoietic colonies of CFU-GM and CD34 positive cells obtained after purification calculated per kg body weight of the patient and the percentage yields.