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Directed immunosuppression therapy in tracheal transplantation
488
Novel immunosuppressives
P.5.23.04
Prevention and therapy of allograft rejection reactions and induction of long-term altograft survival by MNA 279 and MNA 715
J.K. Lindner ‘, N. Zantl 2, H.U. Schorlemmerl. ’ Hoechst Marion Roussel, DG-Rheumatology/immunology, Matburg, Germany 2Depattment of Surgery and Medicine, Technische U&emit& Mtinchen, Germany Introduction: The malononitrilamides (MNA) 279 and 715 are denvatives of A77 1726, the active metabollte of Leflunomide. MNA 279 and 715 inhibit the pyrimidine synthesis pathway by interfering with the enzyme DHODH. By that way T and B cell proliferation, IgM and IgG synthesis are suppressed. Here we report the efficacy of NINA 279 and MNA 715 in the prevention and therapeutic treatment of allograft rejection reactions. Materialand Methods:Male inbred Lewis (RTl’) rats served as recipients and Brown Norway (BN) (RTl”) (10 to 14 weeks of age) rats were used as donors for hetemtopic cardiac transplantation. Graft rejection was taken as the complete cessation of heart beats and was confirmed by lapratomy. The MNAs 279 and 715 were dissolved in 1% CMC and given orally. Reeutts: MNA 279 and MNA 715 were applied from day 0 to day 20 at dosages from 5 to 20 mg/k@day to test their efficacy on the prevention of allograft rejection reactions. A dose dependent increase of efficacy was seen by the application of both drugs (contml: 6.9 f 03,279: 5 rng&g 25.0 f 22.0, 10 ma/kg 42.3 f 16.2, 20 mg/kg 58.2 f 17.0; 715: 5 mgikg 14.6 f 3.0, 10 mg/kg 31 .O f 20.5, 20 mgikg 50.3 f 22.9). Within the 20 mgkg dosage groups more than 50% long term surviving grafts (~70 days) were observed. To investigate the immunological mechanism of cardiac acceptance in this model second-set (BN) and third-party (DA) skin transplantations are performed. Spleen cells from Lewis rats with long term surviving BN hearts were examined for their capacity to transfer donor specific tolerance to BN cardiac allografts in syngeneic Lewis hosts. To test the efficacy of MNA 279 and MNA 715 in suppressing an ongoing ‘immune reaction, the beginning of drug application (15 mg/kg) was delayed to day 3 after heart transplantation (therapeutic treatment). Both substances showed a prolongation of allograft survival from 7.3 f 1.l days in the control group up to 32.3 f 4.0 days in MNA 279 group. Conclusion: MNA 279 and MNA 715 show a dose dependent increase of efficacy within a preventive treatment schedule. Both MNAs are effective in the treatment of ongoing rejection episodes. Long term surviving grafts are observed within the MNA 279 and MNA 715 monotherapy groups.
P.5.23.05
25 June 1997 -Poster presentations
and xerwtransplantation
The effect of kinase inhibitor hyperlcin on normal and tumoral T-cells
C. Ursaciuc ‘, M.Rotaru ‘, P. de Wine *. ‘Dept.Immunology “V Sabes” Institute Bucharest, Romania, 2Dept. Pharmacology; _. Catholic UniversiW Leuven, Belgium
Intmductlon:The naphtodianthmne compound hypedcin (Hy) is known to show intracellular kinase-inhibition activity in tumour cells and consequentty antltumoral effect. Other charactedsttcs consist in antiviral and cytotoxic properties. In order to establish and compare the effects of Hy on normal and malignant T-lymphocytes, we realised two in vitro systems with mouse splenocytes and EL4 thymoma cells modulated with the kinase inhibitor. Yatwlsls and Methods: Splenocytes from C57b/6 mice were tested by blast transformation test in response to in the presence of Hy 0.02 PM. ConA and IL-2 were added to the cultures simultaneously or after 24 hours from Hy addition. The cuttures were illuminated by fluorescence light at 24 hours from Hy addition. The degree of lymphocyte transformation was expressed as 3H-thymidine incorporation index. Different Hy concentrations (between 20-0.002 FM) were added to EL4 cultures 0.1 ml/ml; after 24 h from Hy addition the cultures were illuminated 15 min. by fluorescent light and then m-cultured for another 24 h. After this, the cells were counted and checked for viability by tdpan blue exclusion. Results: The inhibition of normal cells proliferation was dependent on the time of Hy contact with the cell, while the inhibition of EL4 tumour cells proliferation was dose-dependent. Hy 0.02 PM inhibited ConA-stimulated lymphocyte transformation only if the mitogen was added after 24 hours from the Hy priming. An inhibition effect registered also in IL-2 stimulated cells in both situations of cttokine addition from the onset or after 24 hours of culture. In the EL4 cell cultures Hy demonstrated a dose-dependent antiproliferative action, effective at doses between 0.4-0.004 &I. Conofuslons: Kinase-inhibitor Hy showed an antipmliferative effect both in normal stimulated T-lymphocytes and tumoral thymocytes. The inhibition of normal T-cell transformation seems to invotve the signal transduction reactions in case of ConA a&Nation and respectively the membrane receptors in case of IL-2 activation. The antipmiiierative effect in tumour EL4 cells could be due to a dose-dependent intracellular Hy-induced enzyme inhibiiion.
P.5.23.08
Directed immunosuppression therapy in tracheal transplantation
A. Karpitzky ‘, S. Panko, V. Anytchkin, A. Oladko, G. Sapko, A. Lyzikov, V. Taller. Medical Institute of Vitebsk, Belams, ’ Research Institute for Radiation Medicine of Vitebsk, Selarus
Intmductlon:Existing methods of suppressing transplant rejection reactions based on general immunosuppression, are sufficiently effective in the transplantation of organs and tissues into the internal medium of the organism. Materialsand Methods:We have developed a method of allotransplantation of the trachea with omentopexia. followed by a special method of immunosuppression therapy with specially developed prolonged action forms of Cyclospodn A. The experiments were carried out on 56 mongrel dogs in 3 series (1) replacement of circular tracheal defects by unconsewed allografts 28 dogs (2) replacement of circular tracheal defects by unconserved allografts, along with omentopexia and the use of prolonged action forms of Cyclospodn A - 23 dogs; (3) replacement of circular tracheal defects by unconserved transplants with a specially developed protective covering along with omentopexia and directed transport of Cyclosporin A -15 dogs. Results: The given method consists of allotransplantation of the trachea, followed by the insertion of a specially developed prolonged action microcapsulated form of Cyclospodn A into the transferred omentum (series 2). This significantly reduces (upto 8.7%) the number of transplantation complications (lysis of carttlage rings, stenosis), which caused the death of all operated animals from series 1 within 25 days of surgery. Likewise, the doses and frequency of insertion of the immunosuppressant are also reduced. The pmtectiie covering of the tracheal allotransplant @ties 3) increases the selectivity of drugs of this group because of its impermeability to immunopasittve cells and prevents the rejection of the tracheal tube allotissue. Endcecopic, radiological and macroscopic control of the tmcheobronchial tree in animals from series 2 and 3 shows that the transplants are viable, their carcass rlgidt and lumen are maintained. Conclusion:Allotmnsplantatlon of the trachea in combination wtth omentopexia and immunosuppression therapy with prolonged action forms of Cyclosponn A re-establishes the blood supply to the transplanted airway segment, prevents insufficiency of the tmcheo-transplant joints, causes long term inhibition of rejection and maintains the viability of the allogmft. P.5.23.07
Selenium influence on immunological reactions
RR. Tuhvatshin, V.A. Nasymv, R.A. Zulkameyev, G.B. Usenkulova. Central Research Laboratory Bishkek. Kjqyzstan The aim of research work is to study selenatum nattium glutaminant (SNGA) and selenitum natrium glutaminant (SNGI) influence on indices of humoml and cells, immunity. Subjects were 3 groups of white mouses. The first group accept selenatum natrtum glutaminant, the second - selenitum natrium glutaminant, and the 3rd control. 1st and 2nd group of white mouse use SNGA and SNGI 7 and 15 days in dose of 0.4 mg/kg. Immunologic reaction was examined on the fifth day after peritoneal immunisation of sheep erythrocytes(SE) 2 x 10s cells by counting; (1) antitelgenemtion cells in spleen (AGC), (haemagglutininum serum (HS), (3) msettforming cells in spleen (RHS). Resuh Group with selenit natrium glutaminant during 15 days the number of AGC increase to 467 f 58.3 (as control 180 f 44.9 (p = 0.01) and during 7 days - no changes, titre HS was excluded control in 2.3 times; (2) The selenat natrium glutaminant use dudng 15 days was increased AHC in 3 times (p = 0.01). and tttre of HS in 3.4 times. We conclude that SNGA and SNGI in dose of 0.4 mg/kg activated anttbodygeneration, by increasing the AHC number in spleen, and had poor influence on T activity of cell immunity.
1P.5.23.08 1 Binary sample of platinum drugs and interferon conjugates U. Plotnikov. Virus Leuk. Dept., Lab. of Biomodefs, Tomsk, Russia At present progress on the field of drug antitumor and antivirus therapy is essentially related to the development and use of advanced preparations of new generation. We have worked out a new technology (know-how) of obtaining binary conjugates of the know cytostatic preparation of cisplatinum (cis-DDP) and promising platinum and 6-mercaptopurin complex of the common group of Pt-8-mp. As a carder, in the given case, we used human interferon-gamma. As test systems, we used leukemic K-562, HUT-102 cells, the reaction of lymphocyte blastic transformation. Platinum content in conjugates was established by means of the neutron-activation analysis and chemical methods. The binary immunoconjugates we developed have shown a dose-dependent cytotoxic activity (60-95% of damaged). It should be noted that the above technology allows launching production of new cytostatic preparations on a large scale for scientific and clinic use. We think it expendent to epmloy the above immunological vector
Novel immunosuppressives
P.5.23.04
Prevention and therapy of allograft rejection reactions and induction of long-term altograft survival by MNA 279 and MNA 715
J.K. Lindner ‘, N. Zantl 2, H.U. Schorlemmerl. ’ Hoechst Marion Roussel, DG-Rheumatology/immunology, Matburg, Germany 2Depattment of Surgery and Medicine, Technische U&emit& Mtinchen, Germany Introduction: The malononitrilamides (MNA) 279 and 715 are denvatives of A77 1726, the active metabollte of Leflunomide. MNA 279 and 715 inhibit the pyrimidine synthesis pathway by interfering with the enzyme DHODH. By that way T and B cell proliferation, IgM and IgG synthesis are suppressed. Here we report the efficacy of NINA 279 and MNA 715 in the prevention and therapeutic treatment of allograft rejection reactions. Materialand Methods:Male inbred Lewis (RTl’) rats served as recipients and Brown Norway (BN) (RTl”) (10 to 14 weeks of age) rats were used as donors for hetemtopic cardiac transplantation. Graft rejection was taken as the complete cessation of heart beats and was confirmed by lapratomy. The MNAs 279 and 715 were dissolved in 1% CMC and given orally. Reeutts: MNA 279 and MNA 715 were applied from day 0 to day 20 at dosages from 5 to 20 mg/k@day to test their efficacy on the prevention of allograft rejection reactions. A dose dependent increase of efficacy was seen by the application of both drugs (contml: 6.9 f 03,279: 5 rng&g 25.0 f 22.0, 10 ma/kg 42.3 f 16.2, 20 mg/kg 58.2 f 17.0; 715: 5 mgikg 14.6 f 3.0, 10 mg/kg 31 .O f 20.5, 20 mgikg 50.3 f 22.9). Within the 20 mgkg dosage groups more than 50% long term surviving grafts (~70 days) were observed. To investigate the immunological mechanism of cardiac acceptance in this model second-set (BN) and third-party (DA) skin transplantations are performed. Spleen cells from Lewis rats with long term surviving BN hearts were examined for their capacity to transfer donor specific tolerance to BN cardiac allografts in syngeneic Lewis hosts. To test the efficacy of MNA 279 and MNA 715 in suppressing an ongoing ‘immune reaction, the beginning of drug application (15 mg/kg) was delayed to day 3 after heart transplantation (therapeutic treatment). Both substances showed a prolongation of allograft survival from 7.3 f 1.l days in the control group up to 32.3 f 4.0 days in MNA 279 group. Conclusion: MNA 279 and MNA 715 show a dose dependent increase of efficacy within a preventive treatment schedule. Both MNAs are effective in the treatment of ongoing rejection episodes. Long term surviving grafts are observed within the MNA 279 and MNA 715 monotherapy groups.
P.5.23.05
25 June 1997 -Poster presentations
and xerwtransplantation
The effect of kinase inhibitor hyperlcin on normal and tumoral T-cells
C. Ursaciuc ‘, M.Rotaru ‘, P. de Wine *. ‘Dept.Immunology “V Sabes” Institute Bucharest, Romania, 2Dept. Pharmacology; _. Catholic UniversiW Leuven, Belgium
Intmductlon:The naphtodianthmne compound hypedcin (Hy) is known to show intracellular kinase-inhibition activity in tumour cells and consequentty antltumoral effect. Other charactedsttcs consist in antiviral and cytotoxic properties. In order to establish and compare the effects of Hy on normal and malignant T-lymphocytes, we realised two in vitro systems with mouse splenocytes and EL4 thymoma cells modulated with the kinase inhibitor. Yatwlsls and Methods: Splenocytes from C57b/6 mice were tested by blast transformation test in response to in the presence of Hy 0.02 PM. ConA and IL-2 were added to the cultures simultaneously or after 24 hours from Hy addition. The cuttures were illuminated by fluorescence light at 24 hours from Hy addition. The degree of lymphocyte transformation was expressed as 3H-thymidine incorporation index. Different Hy concentrations (between 20-0.002 FM) were added to EL4 cultures 0.1 ml/ml; after 24 h from Hy addition the cultures were illuminated 15 min. by fluorescent light and then m-cultured for another 24 h. After this, the cells were counted and checked for viability by tdpan blue exclusion. Results: The inhibition of normal cells proliferation was dependent on the time of Hy contact with the cell, while the inhibition of EL4 tumour cells proliferation was dose-dependent. Hy 0.02 PM inhibited ConA-stimulated lymphocyte transformation only if the mitogen was added after 24 hours from the Hy priming. An inhibition effect registered also in IL-2 stimulated cells in both situations of cttokine addition from the onset or after 24 hours of culture. In the EL4 cell cultures Hy demonstrated a dose-dependent antiproliferative action, effective at doses between 0.4-0.004 &I. Conofuslons: Kinase-inhibitor Hy showed an antipmliferative effect both in normal stimulated T-lymphocytes and tumoral thymocytes. The inhibition of normal T-cell transformation seems to invotve the signal transduction reactions in case of ConA a&Nation and respectively the membrane receptors in case of IL-2 activation. The antipmiiierative effect in tumour EL4 cells could be due to a dose-dependent intracellular Hy-induced enzyme inhibiiion.
P.5.23.08
Directed immunosuppression therapy in tracheal transplantation
A. Karpitzky ‘, S. Panko, V. Anytchkin, A. Oladko, G. Sapko, A. Lyzikov, V. Taller. Medical Institute of Vitebsk, Belams, ’ Research Institute for Radiation Medicine of Vitebsk, Selarus
Intmductlon:Existing methods of suppressing transplant rejection reactions based on general immunosuppression, are sufficiently effective in the transplantation of organs and tissues into the internal medium of the organism. Materialsand Methods:We have developed a method of allotransplantation of the trachea with omentopexia. followed by a special method of immunosuppression therapy with specially developed prolonged action forms of Cyclospodn A. The experiments were carried out on 56 mongrel dogs in 3 series (1) replacement of circular tracheal defects by unconsewed allografts 28 dogs (2) replacement of circular tracheal defects by unconserved allografts, along with omentopexia and the use of prolonged action forms of Cyclospodn A - 23 dogs; (3) replacement of circular tracheal defects by unconserved transplants with a specially developed protective covering along with omentopexia and directed transport of Cyclosporin A -15 dogs. Results: The given method consists of allotransplantation of the trachea, followed by the insertion of a specially developed prolonged action microcapsulated form of Cyclospodn A into the transferred omentum (series 2). This significantly reduces (upto 8.7%) the number of transplantation complications (lysis of carttlage rings, stenosis), which caused the death of all operated animals from series 1 within 25 days of surgery. Likewise, the doses and frequency of insertion of the immunosuppressant are also reduced. The pmtectiie covering of the tracheal allotransplant @ties 3) increases the selectivity of drugs of this group because of its impermeability to immunopasittve cells and prevents the rejection of the tracheal tube allotissue. Endcecopic, radiological and macroscopic control of the tmcheobronchial tree in animals from series 2 and 3 shows that the transplants are viable, their carcass rlgidt and lumen are maintained. Conclusion:Allotmnsplantatlon of the trachea in combination wtth omentopexia and immunosuppression therapy with prolonged action forms of Cyclosponn A re-establishes the blood supply to the transplanted airway segment, prevents insufficiency of the tmcheo-transplant joints, causes long term inhibition of rejection and maintains the viability of the allogmft. P.5.23.07
Selenium influence on immunological reactions
RR. Tuhvatshin, V.A. Nasymv, R.A. Zulkameyev, G.B. Usenkulova. Central Research Laboratory Bishkek. Kjqyzstan The aim of research work is to study selenatum nattium glutaminant (SNGA) and selenitum natrium glutaminant (SNGI) influence on indices of humoml and cells, immunity. Subjects were 3 groups of white mouses. The first group accept selenatum natrtum glutaminant, the second - selenitum natrium glutaminant, and the 3rd control. 1st and 2nd group of white mouse use SNGA and SNGI 7 and 15 days in dose of 0.4 mg/kg. Immunologic reaction was examined on the fifth day after peritoneal immunisation of sheep erythrocytes(SE) 2 x 10s cells by counting; (1) antitelgenemtion cells in spleen (AGC), (haemagglutininum serum (HS), (3) msettforming cells in spleen (RHS). Resuh Group with selenit natrium glutaminant during 15 days the number of AGC increase to 467 f 58.3 (as control 180 f 44.9 (p = 0.01) and during 7 days - no changes, titre HS was excluded control in 2.3 times; (2) The selenat natrium glutaminant use dudng 15 days was increased AHC in 3 times (p = 0.01). and tttre of HS in 3.4 times. We conclude that SNGA and SNGI in dose of 0.4 mg/kg activated anttbodygeneration, by increasing the AHC number in spleen, and had poor influence on T activity of cell immunity.
1P.5.23.08 1 Binary sample of platinum drugs and interferon conjugates U. Plotnikov. Virus Leuk. Dept., Lab. of Biomodefs, Tomsk, Russia At present progress on the field of drug antitumor and antivirus therapy is essentially related to the development and use of advanced preparations of new generation. We have worked out a new technology (know-how) of obtaining binary conjugates of the know cytostatic preparation of cisplatinum (cis-DDP) and promising platinum and 6-mercaptopurin complex of the common group of Pt-8-mp. As a carder, in the given case, we used human interferon-gamma. As test systems, we used leukemic K-562, HUT-102 cells, the reaction of lymphocyte blastic transformation. Platinum content in conjugates was established by means of the neutron-activation analysis and chemical methods. The binary immunoconjugates we developed have shown a dose-dependent cytotoxic activity (60-95% of damaged). It should be noted that the above technology allows launching production of new cytostatic preparations on a large scale for scientific and clinic use. We think it expendent to epmloy the above immunological vector