Biomedical subjects
Publications and source records attributed to M Kawalec.
The thymocytes were analyzed on the 7th day after i.p. inoculation of 10(6) leukemia L 1210 cells to syngeneic DBA/2 Wf mice. A three-fold decrease of the total number of thymocytes was found as well as 1.7-fold decrease of the per cent of thymocytes with Lyt 2+ phenotype, while the per cent of cells with phenotypes Thy 1.2+ and Lyt 1+ was unchanged.
We have tested the immunologic status and hematologic parameters of mice 2 months (short-term survivors) or 18 months (long-term survivors) after lethal total body irradiation and syngeneic bone marrow transplantation (SBMT), and of normal mice of corresponding age. Long-term SBMT survivors showed significantly lowered bone marrow and spleen cellularities, decreased numbers of CFU-S in hemopoietic organs and severe impairment in the formation of CFU-F colonies compared with short-term SBMT survivors and normal mice. The peripheral blood parameters (hematocrit, erythrocytes, reticulocytes, platelets, white blood cells and granulocyte counts), however, remained unaltered. In long-term SBMT survivors we also observed a relative increase of Lyt-2+ lymphocytes (CD8+, cytotoxic/suppressor) and Mac-1+ cells among splenocytes. At the same time the L3T4+/Lyt-2+ ratio (CD4+/CD8+) was decreased. Relative contents of Ig+, Thy-1+ and L3T4+ cells were unchanged. The ability of splenocytes to generate IL-2R+ cells after in vitro stimulation with concanavalin A was greatly diminished. In summary, the immunohematopoietic status after initial normalization is again impaired in long-term SBMT survivors.
BALB/c x DBA/2 F1 (CD2F1) mice were lethally irradiated and reconstituted with syngeneic bone marrow cells (SBMC) obtained from normal or previously immunized (against L1210 lymphatic leukemia) donors. These recipient mice are called TBI + SBMT or TBI + Imm-SBMT mice, respectively. TBI + Imm-SBMT, but not TBI + SBMT mice, were able to develop strong immune resistance against L1210 leukemia, but not against MOPC 104E plasmacytoma, if the immunization procedure (four i.p. injections at weekly intervals of immunogenic L1210 cells) was started as early as 7 days posttransplantation. Incubation of Imm-SBMC with mafosfamide (ASTA Z7654) before grafting abrogated the ability of the recipient mice to develop early resistance against the leukemia. Treatment of Imm-SBMC with monoclonal or polyclonal antibodies plus complement showed that two or three subpopulations of Imm-SBMC were necessary for the transfer of immune information against leukemia: T lymphocytes with phenotype Thy 1.2+, Lyt 1+2-, I-Ad-, macrophages with phenotype Mac-1+, I-Ad-, and probably asialo-GM 1+ cells. Recipient mice immunized against L1210 leukemia before TBI + SBMT do not develop early resistance to the leukemia.
Mouse bone marrow cells (BMC) were frozen by the simple two step method and stored in liquid nitrogen. The reconstitutive potency of fresh and frozen BMC were compared. BMC-fresh and BMC-frozen had a similar potential of colony formation in spleen and reconstitution of lethally total body irradiated (TBI) mice.
Balb/c x DBA/2 F1 mice (CD2F1 mice) bearing L1210 lymphatic (10 L1210 cells i.p. injected on day 0) were subjected to chemoimmunotherapy. They received 100 mg/kg of cyclophosphamide i.p. on day +8 and 10(6) or 10(7) immunogenic L1210 cells treated in vitro with mafosfamide - ASTA Z7654 (L1210-Maf cells) i.p. or i.p. + s.c. on days 0, +3, +6, +9, +12 after the leukemia implantation. About 30% of leukemia-bearing mice receiving cyclophosphamide and L1210-Maf cells after L1210 inoculation were able to reject the leukemia (as compared with 0% after injection of L1210-Maf cells only or 5% after cyclophosphamide administration). Better results (54% of cured mice) were obtained if 10(7) L1210-Maf cells were injected i.p. +s.c. beside cyclophosphamide. Biological response modifiers (BRM's): levamisole, BCG, bestatin did not improve these results in the doses used in the experiment. Augmentation of anti-L1210 therapeutic response is dependent on the administration of cyclophosphamide and L1210-Maf cels. Cyclophosphamide not only reduces the tumor burden but probably can potentiate the L1210-Maf dependent antitumor immunity as well.
The immunohematopoietic reconstitution of mice lethally irradiated (TBI) and reconstituted with syngeneic bone marrow cells untreated or treated with mafosfamide (ASTA Z 7654) [TBI + SBMT or TBI + SBMT-Maf mice, respectively] was examined. The number of CFU-S was greatly reduced in TBI + SBMT-Maf mice compared with those in TBI + SBMT mice. The recovery of blood parameters (hematocrit, reticulocytes, erythrocytes, white blood cells, granulocytes, platelets) and of bone marrow and spleen cells, but not of peritoneal exudate cells, was slightly delayed in TBI + SBMT-Maf mice compared with those in TBI + SBMT mice. The time for immune system regeneration was, however, considerably longer in TBI + SBMT-Maf than in TBI + SBMT mice, as measured by the incidence of Ig+, Thy-1.2+, L3T4+, Lyt-2+, and IL-2R+ cells in the spleens. The appearance of Mac-1+ and asialo-GM 1+ cells was only slightly prolonged or unchanged, respectively.
Lymphatic leukemia L1210-bearing semisyngeneic Balb/c x DBA/2Wf F1 (CD2F1) mice were subjected to chemoradiotherapy (2 x 100 mg/kg of cyclophosphamide i.p. and 1000 cGy of total body irradiation) and reconstitution with 10(7) syngeneic bone marrow cells i.v. The bone marrow obtained from leukemic mice was previously ex vivo purged of the leukemia cells with mafosfamide (ASTA Z7654) and stored in liquid nitrogen. Eight weeks after cytoreductive therapy and bone marrow transplantation we tried to immunize the mice against the lethal dose of the leukemia by i.p. injections of L1210-Maf cells (L1210 cells treated in vitro with mafosfamide for inhibition of their growth). About 75% of such mice were able to reject the subsequent 10(3) L1210 leukemia cell challenge, as compared with 70% of normal immunized mice and 55% of mice reconstituted with bone marrow cells not treated with mafosfamide.
The immunogenicity of cyclophosphamide-treated murine tumor cells was tested on syngeneic or semisyngeneic mice. The majority of mice immunized with modified Ehrlich carcinoma cells, L1210 leukemia cells or AA sarcoma cells survived after challenge with a lethal dose of unmodified tumor cells. Plasmacytomas RPC-5 and MOPC 104E were less immunogenic and L-1 sarcoma cells were not immunogenic.
Lymphatic leukemia L 1210 cells were treated in vitro with various concentrations of Mafosfamide--a stabilized active derivative of cyclophosphamide (4-hydroxycyclophosphamide). L 1210 cells treated with Mafosfamide (L 1210-MAF cells) were used for vaccination of semisyngeneic CD2F1 mice against L 1210 leukemia. These cells do not grow in vivo but are viable in the test with trypan blue. L 1210-MAF cells, obtained by treatment of L 1210 cells two times with 50 micrograms/ml or 100 micrograms/ml of Mafosfamide, and injected into the mice induced resistance against L 1210 leukemia in these animals. L 1210 cells treated two times with higher concentration of Mafosfamide (200 micrograms/ml or 400 micrograms/ml) did not give this effect.
CD2F1 mice were immunized against semisyngeneic L 1210 leukemia. Immunization was achieved by four i.p. injections, in weekly intervals, of L 1210 cells treated in vivo twice with 200 mg/kg of cyclophosphamide. The immunized animals survived i.p. challenge with 1000 untreated L 1210 cells that was lethal for nonimmunized mice. The immunity could be abrogated in vivo with anti-mouse thymocyte serum, carrageenan or reserpine, but not by anti-mouse IgG serum, suggesting participation of T lymphocytes and macrophages in the response. Moreover, lymphocytes and macrophages from the peritoneal cavity of immunized mice were cytotoxic in vitro for L 1210 cells. The immunity, at least partially, could be adoptively transferred with peritoneal exudate cells or splenocytes.
The composition of peritoneal exudate and spleen cells of CD2F1 mice after fourfold i.p. administration of L 1210 leukemia cells treated with cyclophosphamide (L 1210-CY cells) were examined. The number of cells in peritoneal cavity did not increase, however, the spleen weight rose after administration of L 1210-CY cells. The per cent of lymphocytes T was increased 2.5 times but the content of macrophages and lymphocytes B was normal in the peritoneal cavity after L 1210-CY cells injections. In the spleen an 1.4 times increase of the per cent of lymphocytes B, but normal level of macrophages and lymphocytes T were observed.
Balb/c x DBA/2 F1 (CD2F1) mice were lethally irradiated (TBI) and reconstituted with syngeneic bone marrow cells (SBMT) untreated or treated with mafosfamide (ASTA Z 7654) for ex vivo purging of semisyngeneic L1210 leukemia (TBI + SBMT or TBI + SBMT-Maf mice, respectively). At various times after irradiation and reconstitution mice were injected intraperitoneally four times at weekly intervals with 10(6) immunogenic L1210-Maf cells (L1210 cells treated in vitro with mafosfamide for inhibition of their growth in vivo). As positive controls we immunized normal (non-irradiated) CD2F1 mice. Full resistance against L1210 leukemia (as compared to normal immunized mice) could be obtained in TBI + SBMT and TBI + SBMT-Maf mice when the immunization procedure was started from day +28 or day +56 after transplantation, respectively. Earlier immunization of TBI + SBMT mice (from day +14) or TBI + SBMT-Maf mice (from day +14 or +28) caused only partial resistance against the leukemia.
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An experimental method of measuring intercellular adhesion based on cell interactions in Poiseuille flow is presented. Hydrodynamic conditions of suspension flow, concentration of the suspension, and the size of cells allows determination of the capture efficiency, which is a measure of cell adhesiveness. The measurements of cell aggregation were performed with thymus cells and lectin from Ricinus communis. On the ground of theoretical description of the process of cell aggregation, it was possible to estimate the diffusion constant of the receptors in the cell membrane, which is equal to 5.6 X 10(-11) cm2/s. This value is in good agreement with the results of direct measurements of the diffusion constant of the lectin receptor. In the case of formalin-treated cells, the diffusion constant of the lectin receptors is equal to 6.8 X 10(-12) cm2/s. The total number of the receptor sites on the thymus cell surface for lectin R. communis and the affinity constant were also estimated.
We have demonstrated that lymphoid leukaemia L 1210 cells treated with cyclophosphamide or ifosfamide were immunogenic for semisyngeneic CD2F1 mice and that they effectively prevented the development of the later inoculated leukaemia L 1210 cells. Non-dividing and immunogenic L 1210 cells were only obtained when an appropriate dose of cyclophosphamide was applied. These cells lost their immunogenicity after killing by repeated freezing and thawing or after fixing with glutaraldehyde. Non-dividing immunogenic L 1210 cells treated with cyclophosphamide possessed the histocompatibility and tumour-associated transplantation antigens and had the ability to synthesize proteins, the RNA and partially the DNA.
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The median survival time of DBA/2Wf or CD2F1 hybrid mice increased after administration of 10(3) cells of lymphoid leukaemia L 1210 when the recipient mice were injected earlier with L 1210 cells pretreated in vivo with cyclophosphamide. To achieve this effect, at least 10(5) cells treated with cyclophosphamide should be used per mouse. This effect was immunologically specific and could not be induced by L-1 cells treated with cyclophosphamide, and was transferable by means of splenocytes. Such immunoprophylaxis was efficient for at least 100 days. Attempts at immunoprevention by means of the same cells treated with mitomycin C or killed by osmotic shock were unsuccessful.
A 3000--6000-fold purified kallikrein was obtained from human serum in 10--25% yield by chromatography on QAE-Sephadex A-50, Molselect CM-50 and on soybean trypsin inhibitor (SBTI)-AH-Sepharose 4-B. The enzyme had a specific activity of 14--23 U, as measured by BAEE hydrolysis. Some properties of highly purified kallikrein are described.