[Use of photodynamic therapy for elimination of residual leukemic cells in autologous transplants of hematopoietic progenitor cells]. / Vyuzití fotodynamické terapie k odstranování reziduálních leukemických bunek z autologních transplantátu hematopoetických progenitorových bunek.

BACKGROUND: The increasing use of autologous hematopoietic cell support in various malignancies including leukemia and lymphoma currently bears the problem of tumor contamination of the graft with tumor cells which after re-infusion contribute to the disease relapses. It is therefore desirable to eradicate the cancer cell fraction of the graft without causing damage to the normal stem cell fraction. The purging processes based on photodynamic treatments appear to be perspective means for this purpose. METHODS AND RESULTS: We investigated the effects of 5-aminolevulinic acid (ALA)--based photodynamic treatment (ALA-PDT) on the proliferation of human leukemia cell lines HL60 (promyelocytic leukemia), ML2 (myelomonocytic leukemia) and HEL (erythroleukemia) by 3H-thymidine incorporation into the cell DNA, on the viability of cell lines HL60, HEL, DAUDI (B-cell leukemia) and JURKAT (T-cell lymphoma) as well as of blast cells of acute myeloid leukemia (AML) patients by flow cytometry-propidium iodide assay, and on the clonogenic activities of normal human bone marrow cells by in vitro cloning assays. Under the conditions used (treatment with 1 mM ALA for 4 h at 37 degrees C followed by exposure to blue light dose of 18 J/cm2) the number of proliferating HL60 cells was reduced by 2.4 logs, of ML2 cells by 3.2 logs and of HEL cells by 1 log. From the mononuclear cell preparations of AML patients the blast cells were substantially reduced in eight out of ten patients. The clonogenic activities of normal bone marrow progenitor cells were largely spared: 52.5 +/- 8.9% of colony-forming units--granulocytes macrophages (CFU-GM), and 48.6 +/- 9.7% burst forming units--erythrocytes (BFU-E). CONCLUSIONS: ALA-PDT appears to be usable principle for the depletion of residual leukemic cells from autologous transplants.

Genes involved in the destruction of leukaemic cells by induced photosensitivity.

Gene expression changes were observed in the HEL and HL-60 cell lines after the stimulation of protoporphyrin IX synthesis by ALA administration and photodynamic process induction. Isolated ribonucleic acids were radiolabelled by reverse transcription, and the cDNA obtained was hybridized to membrane macroarrays (Clontech 7742-1) containing 588 gene probes. Besides changes in the activity of genes supposed to be involved in the programmed cell death and DNA reparation processes, increased or diminished transcription activity was also observed in several other genes; the reason for this phenomenon was not clear. The activation of programmed-cell-death genes appeared after the ALA load application, indicating the toxic effect of ALA. The gene expression changes observed in the two cell lines differed substantially, only a few of them were common for both cell lines.

[Selective photodynamic destruction of leukemic cells]. / Selektivní fotodynamická destrukce leukemických bunek.

BACKGROUND: Residual leukemic cells if present in autologous bone marrow grafts or CD34+ concentrates obtained from peripheral blood may increase the risk of relapse after autotransplantation. We are presenting the employment of a new method which was introduced into the photodynamic therapy, namely enhancement of synthesis of the photosensitizing compound, protoporphyrin IX, in cancer cells, following application of its metabolic precursor, 5-aminolevulinic acid, for the specific destruction of leukemic cells. METHODS AND RESULTS: By determining cell viability using tetrazolium salt reduction (MTT), by flow cytometrypropidium iodide assay and by determining cell proliferation using bromodeoxyuridine incorporation we studied the effect of photodynamic therapy based on the application of 5-aminolevulinic acid on the cells of leukemic cell lines HL60 (human promyelocytic leukemia), HEL (erythroleukemia), DAUDI (B-cell leukemia), JURKAT (T-cell lymphoma), blast cells of patients with acute myelogenous leukemia as well as on normal lymphocytes and normal human bone marrow progenitors. In in vitro experiments photodynamic therapy based on an administration of 5-aminolevulinic acid (1 mM, 4 h, 18 J/cm2) lowered the number of viable leukemic cells by over 2 orders (with the exception of HEL cells) and eliminated blast cells in mononuclear cell preparations of six out of seven patients with acute myelogenous leukemia. On the other hand the viability of normal resting lymphocytes was little affected by photodynamic therapy (number of necrotic cells increased from 6 to 11%) and also the clonogenic activity of the progenitor cells of normal bone marrows did not decrease substantially (CFU-GM to 60% and BFU-E to 55% of the original activity). CONCLUSIONS: Photodynamic therapy based on the application of 5-aminolevulinic acid is a perspective method for the specific destruction of leukemic cells in autologous transplants.

The 5-aminolaevulinic acid-based photodynamic effects on nuclei and nucleoli of HL-60 leukemic granulocytic precursors.

To provide more information on the 5-aminolaevulinic acid (ALA)-based photodynamic effect (PDE) on nuclei and nucleoli of individual leukemic cells, these structures were studied in cultured HL-60 cells which originated from leukemic highly immature and less differentiated precursors of granulocytes. The nuclear morphology was visualized by panoptic May-Grünwald/Giemsa staining and cytochemical method for DNA, nucleoli were visualized by cytochemical methods for the demonstration of RNA and silver stainable proteins including those of interphase silver stained nucleolus organizer regions (AgNORs). In most cells ALA-based photodynamic treatment (PDT) produced marked alterations such as formation of apoptotic bodies, and large condensation of nuclear chromatin structure but without nuclear segmentation. Such changes are in harmony with the apoptotic process induced in these cells but without previous terminal differentiation. In nucleoli ALA-based PDT produced the reduction and disappearance of nucleolar silver stainable particles (SSPs) representing AgNORs which apparently reflected the alteration of the nucleolar biosynthetic activity and cell proliferation. The latter is also reflected by the disappearance of mitotic divisions. On the other hand, a small subpopulation of cells was less sensitive or resistant to the ALA-based PDE since they did not show mentioned nuclear and nucleolar alterations.

Selective destruction of leukaemic cells by photo-activation of 5-aminolaevulinic acid-induced protoporphyrin-IX.

The effect of 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and proliferation of leukaemia/lymphoma cells as well as normal human lymphocytes has been investigated by flow cytometry-propidium iodide assay (FC-PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) incorporation and on clonogenic activity of normal human bone marrow progenitor cells by clonogenic methods. ALA-PDT (1 mM 5-ALA, 4 h, 18 J cm-2) reduces the number and/or suppressed proliferation of leukaemic cells of promyelocytic (HL60), B-cell-derived (DAUDI) and T-cell-derived (JURKAT) cell lines by 2 logs and that of the HEL erythroleukaemia cells by 77%. The effect of ALA-PDT on quiescent human lymphocytes is small (85% viable cells after ALA-PDT). The proliferation of lymphocytes subjected to ALA-PDT and induced with phytohaemagglutinin (PHA) decreases by 75% as compared to the untreated control. For normal human bone marrow progenitors, 58% of colony-forming units-granulocytes-macrophages (CFU-GM) and 55% burst-forming units-erythrocytes (BFU-E) activities are preserved.

Photodynamic effects of meso-tetra (4-sulfonatophenyl)porphine on human leukemia cells HEL and HL60, human lymphocytes and bone marrow progenitor cells.

Meso-tetra(4-sulfonatophenyl)porphine (TPPS4), in combination with a light dose of 14 J cm-2, has a profound negative effect on the proliferation and viability of leukemia cells HL60 (human promyelocytic leukemia) and HEL (human erythroleukemia), the viability of normal lymphocytes and the colony-forming activity of human bone marrow progenitor cells. However, normal leukocytes (monocytes, granulocytes) are, to a large extent, resistant to photodynamic treatment (PDT). Whilst DNA fragmentation suggesting apoptosis is induced in HL60 cells, accumulation in the interphase of the cell cycle (G0/G1, G2/M) is mainly operative in the TPPS4-mediated PDT of HEL cells. The "dark" effect of TPPS4 on the cell viability is below 15% up to a concentration of 40 microM.

Comparison of several mouse and rat monoclonal antibodies against human fibrinogen.

Six monoclonal antibodies raised against human fibrinogen have been characterized. Mouse monoclonal antibodies were targeted against sequential epitopes on the immunodominant D-domain of fibrinogen and they crossreacted with all molecules containing the D-domain [fibrin, fibrin(ogen)-degradation products]. Their behavior was not influenced by proteolytic degradation of fibrinogen with plasmin. Rat MoAbs were specific for the conformational epitopes on intact fibrinogen. Their reactivities were substantially lower with fibrin(ogen)-degradation products. Degradation of structures on intact fibrinogen was concomitant with the decay of rat MoAbs reactivity. Those structures were presumably on the C-terminal end of fibrinogen alpha chain and/or on the N-terminal end of fibrinogen beta chain.

Monoclonal antibodies against human hemopexin and their application.

Six monoclonal antibodies raised against human serum hemopexin have been characterized. The antibodies reacted with serum hemopexin as well as with the isolated protein in apo-form and with heme-protein complexes on immunoblots obtained following both PAGE and SDS-PAGE. In the case of PAGE blots 1 ng of hemopexin could be detected using a streptavidin-AP detection system. ELISA procedures employing two different pairs of monoclonal antibodies gave working ranges of 0.3-3 mg/l and 10-100 micrograms/l respectively.

[Fibrinogen seen as an independent cardiovascular factor from the molecular aspect]. / Fibrinogen jako nezávislý kardiovaskulární faktor z molekulárního hlediska.

The blood protein fibrinogen is one of the main independent cardiovascular risk factors and it is very likely that an increased fibrinogen level is not the consequence of cardiovascular disease but its direct cause. Fibrinogen participates actively in many processes in the organism and undergoes various changes of the molecule but it is not known what is the molecular background, why even a slight increase of the fibrinogen level is so dangerous as regards increased risk of cardiovascular attacks. In the present work the authors compared, using monoclonal antibodies, the rate of release of fibrinopeptides A and B (FpA, FpB) by the action of thrombin from fibrinogen in solution and from fibrinogen adsorbed to a solid surface. The authors revealed that the rate of FpB breakdown from sorbed fibrinogen, contrary to fibrinogen in solution is comparable to the release rate of FpA and does not depend on the release of FpA (except for competitive inhibition). The release of FpB from sorbed fibrinogen thus takes place, contrary to fibrinogen in solution, at a significant recordable rate from the very onset of thrombin action. Fibrinogen adsorption to a solid surface is associated with conformation changes which cause this effect and which lead to the formation of a two-dimensional formation formed by fibrinogen after its interaction with the surface of activated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies against human antithrombin III.

Three monoclonal antibodies identified as D8, B11 and C5 of different specificities have been produced against human antithrombin III (AT). The apparent dissociation constants (Kd app) of the AT-antibody interaction were determined by ELISA method: Kd app (D8) = 2.4 nmole, Kd app (B11) = 13 nmole, Kd app (C5) = 24 nmole. All three antibodies reacted with isolated AT on immunoblots obtained with "native" PAGE. The D8 antibody also reacted with plasma and serum AT while B11 antibody reacted with serum thrombin-antithrombin (TAT) complexes as well.

[Monoclonal antibodies against fibrinogen and fibrin and against their proteolytic degradation products]. / Monoklonální protilátky proti fibrinogenu a fibrinu a proti jejich proteolytickým degradacním produktum.

The level of fibrinogen (FDP) and fibrin (fDP) degradation products is one of the surprisingly few unequivocal indicators of the activity of the coagulation and fibrino (geno) lytic system. Today it is beyond doubt that, similarly as the FDP/fDP level is of unequivocal importance, traditional methods of their assessment are very equivocal. Fibrin and FDP/fDP are molecules derived from fibrinogen, which are formed from the latter as a result of a series of reactions during which structures are formed or are made available which are not present in the fibrinogen molecule. New antigenic determinants are formed, in relation to the original molecule so-called neoepitopes, neoantigens. The great predominance of monoclonal antibodies prepared in the meantime reacts with fibrinogen as well as with fibrin and their degradation products. By means of specific approaches and original methods it proved, however, possible to prepare some specific monoclonal antibodies which were successfully used in diagnostic tests in vitro; their use for detection of thrombi in vivo is tested as well as their use for an increasingly effective thrombolytic therapy after their conjugation with plasminogen activators. It may be expected that this problem will be intensely developed in the near future.

Experiences with evaluation of immunosuppressive activity of alpha-globulin fraction of human blood plasma.

Presented are experiences with the determination of immunosuppressive activity of alpha globulin fraction of human blood plasma, crude (Cohn fraction IV), partially purified and so-called low-molecular isolates of these substances. For testing the following techniques were used: the lymphocytotoxic and lymphoagglutination test, rosette-inhibition tests with human lymphocytes and splenocytes of mice immunized with sheep erythrocytes, lymphocyte blastic transformation inhibition test using PHA stimulated cells as well as in the mixed culture, inhibition of haemolytic plaque formation tests, determination of GVH reactivity using regional tests in the popliteal nodes, determination of HVG reactivity by skin graft survival test, determination of the effect on formation of precipitins against soluble antigens. The best results were obtained with the determination of survival times for skin grafts and the regional test in the popliteal nodes Fairly reproducible results were obtained in experiments on influencing the humoral antibody response and inhibition of formation of precipitating antibodies. Of in vitro methods tests of inhibition of lymphocyte blastic transformation both after PHA stimulation and in the mixed culture are applicable. Fully unsuitable proved the rosette, lymphocytotoxic and lymphoagglutination tests. In vitro modification of haemolytic plaque formation tests. In vitro modification of haemolytic plaque formation test is unfit for testing the crude fractions or partially purified drugs, its in vivo modification seems more feasible provided a large panel of animals is examined.

Study on immunosuppressive activity of alpha-globulin fraction of human blood plasma.

Immunosuppressive activity of alpha-globulin fraction of human blood plasma (Cohn fraction IV) was studied to determine the possible clinical utilization of this fraction as such, purified, or as the starting material for isolation of the active factor. The ethanol fractions IV, isolated and waste in the production of blood derivatives were found to be directly inapplicable because of relatively low activity and poor solubility. Therefore purification experiments were done to remove the denatured proteins and lipids and to increase the specific activity, they were performed on laboratory scale using DEAE ionex chromatography, and on semipilot scale utilizing the principles of ethanol fractionation of plasma. From the viewpoint of technology, the latter procedure seems more feasible; partial purification yielded good solubility and somewhat higher specific activity. Under study are further techniques capable of eliminating the contaminating proteins with do not carry the immunosuppressively active component, as well as the effectivity of pasteurization at 60 degrees C/10 hrs., which seems indispensable with respect to inactivation of infectious hepatitis viruses and the potential clinical application. Experimental isolations of the active component have led immunosuppressively active low-molecular part, which, however, is chemically nonhomogeneous. Its subsequent subfractionation will be necessary.