Design, synthesis and biological activities of new brassinosteroid analogues with a phenyl group in the side chain.

We have prepared and studied a series of new brassinosteroid derivatives with a p-substituted phenyl group in the side chain. To obtain the best comparison between molecular docking and biological activities both types of brassinosteroids were synthesized; 6-ketones, 10 examples, and B-lactones, 8 examples. The phenyl group was introduced into the steroid skeleton by Horner-Wadsworth-Emmons. The docking studies were carried out using AutoDock Vina 1.05. Plant biological activities were established using different brassinosteroid bioassays in comparison with natural brassinosteroids. Differences in the production of the plant hormone ethylene were also observed in etiolated pea seedlings after treatment with new brassinosteroids. The most active compounds were lactone 8f and 6-oxo derivatives 8c and 9c, their biological activities were comparable or even better than naturally occurring brassinolide. Finally the cytotoxicity of the new derivatives was studied using human normal and cancer cell lines.

An overview of the Czech Bone Marrow Donor Registry.

The Czech Bone Marrow Donor registry (CBMD) was founded in 1991 in the National HLA centre at Prague's Institute for Clinical and Experimental Medicine. In the same year, the CBMD submitted its data to the Bone Marrow Donors Worldwide (BMDW). Another line of CBMD's international cooperation is accomplished through computer linkup with the European Donor Secretariat (E.D.S) network. Donors are being recruited constantly through blood transfusion units and other volunteers are enrolled through the mass media. All the methodology used is developed in compliance with the standards of the European Federation for Immunogenetics (EFI). CBMD closely cooperates with clinical centres for transplantation of bone marrow, stem cells (PBSC) and cord blood from unrelated donors. More than 7,000 potential bone marrow typed in HLA-A, B locus have been registered. Besides potential bone marrow donors, frozen cells of cord blood are kept by CBMD. Search requests from registries all over word come via E.D.S. daily except for weekends. Since its foundation in 1991, nearly 20,000 international requests have been handled. During the last two years, 5 CBMD donors provided their bone marrow to Czech patients, one donor provided stem cells (PBSC) and one donor provided bone marrow + stem cells (PBSC). To date, more than 20 transplantations from unrelated donors have been performed in Prague's transplant centres.

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.

Frequency analysis of cytotoxic T lymphocyte precursors in search for donors in bone marrow transplantation.

The usefulness of cytotoxic T lymphocytes precursors (CTLp) frequency analysis in the search for donors in bone marrow transplantation was studied. The frequency of anti-recipient CTLp was approached by limiting dilution assay in HLA matched unrelated, HLA partially matched related and HLA genotypically identical donors. The majority of patients examined were affected with different hematological malignancies. Alloreactive CTLp recognizing non-HLA gene products were not detected in pretransplant examination of two pairs of HLA identical siblings. However, an increased incidence of allospecific CTLp was identified in HLA matched MLC negative unrelated pairs. Thus, CTLp assay allowed to uncover the residual Class I incompatibilities that remained hidden in standard serotyping. In two matched unrelated pairs with high pretransplant CTLp frequency the severe acute graft-versus-host disease (GVHD) developed after bone marrow transplantation. Examination of other relatives in patients lacking an HLA identical sibling showed the importance of Class I incompatibility for CTLp generation as well. The lack of correlation between CTLp frequency and HLA-D disparity could suggest that Class II antigens do not participate in CTLp induction. With one exception we had good correlation between MLC and DNA analysis of Class II antigens demonstrating that MLC gives interpretable results even in unrelated pairs. Our results demonstrate the significance of CTLp frequency assay in detection of residual Class I incompatibilities in matched unrelated pairs and in assessment of Class I compatibility in related pairs. For that it should be used in the final selection of BMT donors.

Agranulocytosis in a patient with primary Sjögren's syndrome.

The authors describe a case of primary Sjögren's syndrome, which was complicated with severe autoimmune agranulocytosis quite sensitive to immunosuppressive therapy. Agranulocytosis is a very rare complication of this autoimmune rheumatic disease as opposed to leucopenia. A remarkable feature of the presented case is the fact that correct diagnosis of primary Sjögren's syndrome has not been settled for almost 25 years. The disease has manifested only in the form of arthropathy imitating rheumatoid arthritis.

The metabolism of glucose of nerve cells cultivated under different conditions.

1. The consumption of glucose and formation of lactate was studied in medium of long-term cultivated nerve tissue. Fragments of chicken brain embryo dissociated and reaggregated brain cells were cultivated in Rose chambers, Falcon plastic dishes and Erlenmayer flasks. 2. Dissociated cells were cultivated in Petri dishes in media containing 100 mg/100 ml glucose. Consumption of glucose and formation of lactate increases until the 9th day. Glucose is completely exhausted in the medium up to the 2nd week of cultivation. 3. The time curve of both glucose consumption and lactate production is similar in cultures cultivated in Rose chambers, Petri dishes and Falcon plastic dishes. Cultures in Rose chambers utilize glucose at later stages anaerobically, whereas in Petri dishes and Falcon plastic dishes approximately 25% is utilized by aerobic glycolysis. 4. Cells dissociated by trypsinization and sieving are metabolically more active than cells separated mechanically (sieving only). During later stages of cultivation of enzymatically dissociated cells in 100 mg/100 ml glucose, lactate is utilized like a substrate, because of concentration of glucose in the medium is not being sufficient. 5. The concentration of glucose is essential for utilization by the aerobic pathway. In dissociated cells, cultivated in media enriched by 400 mg/100 ml of glucose in Falcon plastic dishes 75% of aerobic glycolysis is found during first 10 days and 50% in later stages. In the same system, cultivated in 100 mg/100 ml of glucose, glucose is exhausted up to the 12th day and lactate is utilized as a substrate. 6. In the close system of cultivation, i.e. in Rose chambers, 50% of glucose is utilized by the aerobic pathway if the medium contains 400 mg/100 ml of glucose. Early cultivation period of dissociated cells in Falcon plastic dishes is slowed, because cells adhere slowly to the plastic ground. 7. Structural development of cultures and differentiation of cells was studied during the cultivation period. Cells cultivated in elevated glucose concentration exhibit signs of better differentiation.

Structure and ultrastructure of cultivated glial cells from Corpus callosum.

1. Explants and dissociated cells from Corpus callosum (c. c.) of rats and rabbits were cultivated in Petri dishes and Rose chambers. 2. Different types of glial cells were found in the cultivated Corpus callosum (c. c.) explanted from 12 days old rats: a) adendritic glial cells, typical for migrating oligodendroglial cells, b)-migrating large, nondifferentiated astrocytes with pronounced phagocytosing activity, c) macro- and microglial cells which differentiated during cultivation. 3. The population of differentiated glial cells is mostly composed of oligodendroglia, less of astrocytes and microglial cells are rare. 4. Differentiation of dissociated cells from c. c. in homogenous and mixed population was studied. The appearance of first processes of macroglial cells is postponed to 6 to 10 days of cultivation. No substantial difference was observed between homogenous and mixed population. A higher incidence of macrophages was observed in the later. 5. Glial cells differentiate surrounded by degenerated nerve fibers and myelin, exhibiting phagocytoses and cleaning reaction.

Differentiation of corpus callosum glial cells and factors influencing their maturation in vitro.

1. Explants of Corpus callosum (c. c.) from 12-day-old rats were cultivated under different experimental conditions. 2. Migration and differentiation is activated by the presence of neighbouring explants, toward which glial cells predominantly migrate. Glial cells migrate if closely adhering to the supporting collagen and the process of differentiation is enhanced by presence of underlying cell layers. 3. Migratory activity of glial cells decreases and is delayed with age of donors. Migrating cells have a similar appearance as in cultures from 12 days old donors. The presence of immature types of glial cells in c. c. of adult animals was proved. 4. Glucose was found to be an adequate metabolical substrate, utilisation of glucose being lower than in cultivated neurons. In the absence of glucose or serum in the medium, neither migration nor differentiation of glial cells was observed. 5. The addition of embryonal extract and embryonic brain extract enhanced only initial stages of cell migration and differentiation.

The search for HLA-compatible bone marrow donors. Procedure currently used in the Institute of Hematology and Blood Transfusion, Prague.

The search for compatible donors is based on the HLA types of donors and recipients. HLA-A, -B and -DRB1 antigens or alleles must be unequivocally typed in donors and recipients. The typing of HLA-DQB, -DPB and -C gene products is also useful to characterize the HLA phenotype, but it is not absolutely necessary in the donor selection. The standard serological methods using alloantisera and one-dimensional isoelectric focusing are used for the typing of HLA class I antigens. Recently, DNA analysis of class I alleles has been introduced. In HLA class II typing the serological analysis was generally replaced by DNA analysis. In addition to typing techniques that determine the individual HLA alleles or HLA gene products, the cellular matching techniques are used in the selection procedure (mixed lymphocyte culture, cytotoxic T lymphocyte precursor frequency assay, and IL-2-producing helper T lymphocyte precursor frequency assay). The cellular matching techniques determine the compatibility in the regions of HLA comprising several HLA loci; some of them may detect minor histocompatibility (non-HLA) gene disparities as well.

[Detection of HLA-A and HLA-B antigens on thrombocytes. Comparison of 3 methods]. / Prukaz HLA-A, B antigenu na trombocytech. Srovnání trí metod.

HLA antigens A1, 2, 9, 10, B5, 7, 8, 12, 35 were determined on 28 samples of healthy persons' thrombocytes by means of three tests; the microabsorption and immunofluorescence tests and the ELISA technique. Identification with the ELISA test was exceptional. Unlike the microabsorption test, the immunofluorescence test was employed to identify HLA antigens with only some sera as a rule. Identical positive results of both the microabsorption and the immunofluorescence tests were rather infrequent. The authors could not corroborate literary reports on difficulties in the identification of the antigens HLA-B8 and HLA-B12, certain problems were encountered in the case of the HLA-B7 antigen only. A discussion is presented of the causes of the different results of each of the tests, and of the relevance of the results for transfusions of platelet concentrates chosen on the basis of lymphocytic cross-matching.

[Antibodies against granulocyte precursors in patients with neutropenia: use of the ML-1 myeloid line]. / Protilátky proti prekursorum granulocytu u nemocných s neutropenií: vyuzití myeloidní linie ML-1.

With the aid of indirect immunofluorescent test, the authors investigated antibodies in the serum of patients, which reacted against immature myeloid cells and mature granulocytes. Myeloid line ML-1, longitudinally maintained in vitro, was used as source of immature myeloid cells. 248 sera were examined and antibodies against the immature myeloid cells were detected in 8% of them. In 6 selected sera of the patients with neutropenia, the authors determined the Ig class of the identified antibody against the immature cells of the ML-1 myeloid line, against the myeloid cells of the ML-1 line differentiated with retinoic acid, respectively even the Ig class of the antibody against neutrophils. The IgM class antibody was found to have the highest frequency. Using absorption studies, the authors provided evidence that the antibody against the myeloid cells can be absorbed only by means of the cells of the myeloid line ML-1, which is demonstrated by the absence of the surface character in the mature neutrophils against which the antibody was directed. The authors discuss the outcome of their investigation aimed at making a more accurate diagnosis of immune neutropenia.

[Rabbit anti-thymocyte globulin, the cause of false-positive results in the microlymphocytotoxicity test?]. / Králicí antithymocytární globulin, prícina falesné pozitivity výsledku mikrolymfocytotoxického testu?

The authors describe a positive crossover reaction between serum of a female patient treated with rabbit antithymocytic globulin and donor lymphocytes. In the serum of the patient with inhibition of the bone marrow HLA cytotoxins, thrombolysins, granulocytotoxins and granuloagglutinins were detected. Complete and incomplete thromboagglutinins were not detected. Two days after the positive tests no type of antibodies was detected in the patient's serum. The authors discuss the reasons for the development of the crossover reaction.

[Donor selection for allogenic bone marrow transplantation from 1991 to 1995. (The Prague Center for Bone Marrow Transplantation)]. / Výber dárcu pro alogenní transplantaci kostní drene v letech 1991-1995 (Prazské centrum transplantace kostní drene).

BACKGROUND: The prerequisite of successful transplantation of haematopoietic stem cells is to find a suitable, i.e. HLA matched bone marrow donor. In the submitted paper the authors give an account of the strategy of selection of bone marrow donors, as practised by the Prague transplantation group. METHODS AND RESULTS: In 1991 to 1995 (end of first quarter) the authors sought suitable bone marrow donors for 421 patients where an allogenic bone marrow transplantation was indicated. During that period 95 transplantations were performed (22.5% of all indicated cases), incl. 82 from siblings (86.3%) of the implemented allogenic bone marrow transplantations). A HLA matched donor from the wider family circle was selected for 15 donors, transplantation were performed in 6 patients (6.4% of the implemented allogenic bone marrow transplantations). An unrelated bone marrow donor was sought for 41 patients as no donor was found in the wider family. Transplantation was implemented in 7 of these patients (7.3% of the implemented transplantations. CONCLUSIONS: With the increasing experience of our transplantation centre, no doubt, the ratio of other than sibling transplantations of bone marrow will increase. At present it accounts for 13.7%. The task of our HLA group must be selection of donors matched as well as possible with the recipient according to all available tests of tissue compatibility - from the wider family circle as well as from the register of unrelated donors.

[The significance of the thrombocyte crossmatching test]. / Význam trombocytární krízové zkousky.

The authors investigated the dependence of the results of the crossover test with thrombocytes and lymphocytes of the donor and serum of the recipient on the rise of thrombocytes 1 and 24 hours following administration of a thrombocyte concentrate. The clinical condition of the patient at the time of thrombocyte administration was taken into account. The donor of thrombocytes isolated in a blood element separator was selected from a panel of cca 800 subjects with previously assessed HLA antigens. A total of 36 administered concentrates was evaluated. From the assembled findings it may be concluded that the result of thromboconcentrate transfusion is significantly influenced by adverse clinical factors in the patient such as splenomegaly, haemorrhage, febrile conditions. A positive crossover lymphocytotoxic test can predict reliably the ineffectiveness of thrombocyte administration. A positive crossover test with the donor's thrombocytes does not affect significantly the success of the transfusion. It seems thus that for the fate of administered thrombocytes the presence of HLA antibodies is decisive and not the presence of specific thrombocytic antibodies.

[Analysis of HLA class I molecules using isoelectric focusing. Its application in selection of bone marrow donors]. / Analýza molekul HLA-I. trídy metodou izoelektricke fokuzace. Vyuzití pri výberu dárce kostní drene.

BACKGROUND: Differences between HLA proteins class I are assessed by serological typing using HLA allo-antisera. This method suffices for the assessment of HLA signs in allogenic transplantations of bone marrow in genotypically identical siblings. However, it does not suffice in the selection of donors from the wider family of a related or not related donor from the register of voluntary bone marrow donors. In order to assess the polymorphism of the HLA-class I not detected by serological typing other more sensitive techniques are introduced. In the authors department one-dimensional isoelectric focusing was introduced as an auxiliary method for evaluation of identity of molecules in HLA-class I between bone marrow recipient and donor. METHOD AND RESULTS: The authors introduced the method of one-dimensional isoelectric focusing which uses for detection of molecules of HLA-class I a polyclonal antibody against heavy chains of HLA molecules class I and a secondary antibody labelled by alkaline phosphatase. Forty-one pairs of bone marrow donors and recipient were examined. In six instances, i.e. in almost 15%, the authors detected by isoelectric focusing disagreement in HLA molecules class I. The authors present three interesting pairs of donors and recipients where isoelectric focusing helped with the selection of a suitable bone marrow donor. CONCLUSIONS: The isoelectric focusing is at present another method which helps to reveal differences in HLA class I molecules. When methods of DNA analysis are introduced to recognize alleles of the HLA-I class, correlation with serologically and biochemically assessed variants wil be an essential guide in the evaluation of the final typing of HLA molecules class I.

[A registry of potential bone marrow donors]. / Registr potencionálních dárcu kostní drene.

Transplantation of bone marrow is relatively successful treatment in some haematological diseases. The most suitable donor of bone marrow is a HLA genotypic identical sibling; however, roughly only 30% of patients have a suitable related donor. In order to find a HLA identical non-related donor for a maximum number of patients it is necessary to create as extensive registers as possible of potential bone marrow donors. The authors mention the possibility to obtain these donors from the ranks of regular blood donors and they mention the advantages of obtaining a register from this group of subjects.

[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 hematopoietic stem cells in the treatment of children with high-risk acute leukemia]. / Alogenní transplantace kmenových bunek krvetvorby v lécbe detí s vysoce rizikovou akutní leukémií.

BACKGROUND: Most children with acute lymphoblastic leukemia (ALL) and increasing number of children with acute myelogenous leukemia (AML) are currently cured with conventional chemotherapy. Despite of this success there is a subset of patients with high-risk features at diagnosis who are predisposed to a very high risk of relapse. Relapse of AML and early bone marrow relapse of ALL can not be cured by conventional chemotherapy. Allogeneic hematopoietic stem cell transplantation (HSCT) is therapeutic option in these children with very high-risk acute leukemia. METHODS AND RESULTS: Between XI/1989-XII/1996 33 children with acute leukemia (ALL: 22, AML: 11) underwent an allogeneic HSCT from HLA identical related donors (HLA-identical sibling: 30, twin: 1, other HLA-identical relative: 2) at the 2nd Dept. of Pediatrics, University Hospital Motol. Median age of our group was 9 years (1.5-19 y.), boys (n = 23) clearly dominated over the girls (n = 10). The resource of stem cells was bone marrow in 31 children, bone marrow and peripheral blood progenitor cells (PBPC) and PBPC in one child respectively. Myeloablative conditioning regimen varied, consisting of total body irradiation and chemotherapy in 21 children and chemotherapy in 12 children. HSCT was performed in first complete remission of acute leukemia in 9 children (AML: 7, ALL: 2), in second remission in 14 children (AML: 2, ALL: 12), in third remission in 4 children (ALL: 4). Six children underwent HSCT in first partial remission (n = 1) and in second (n = 4) or third (n = 1) chemoresistant relapse. Seven (21%) children died due to post-transplant complications. Nine (28%) children suffered from clinically significant acute graft-versus-host reaction (GVH) and 15% (4/27) children who survived 100 days post-transplant suffered from chronic GVH disease. Relapse of leukemia was diagnosed in 39% (12/31) children. Fourteen (42%) children are alive and well in continuous remission with median follow-up 42 months. CONCLUSIONS: Allogeneic HSCT can cure children with very high-risk acute leukemia in the situations where conventional chemotherapy fails. Relapse of leukemia and GVH reaction are most important causes of post-transplant morbidity and mortality.

[The origin of HLA antigens in thrombocytes]. / K puvodu HLA antigenu na trombocytech.

When examining thrombocytic antibodies in serum of patients by the ELISA test concurrently present HLA antibodies were found only in exceptional cases (8), as compared with other tests for the detection of thrombocyte antibodies. One of the possible reasons of this finding could be the mere adsorption of HLA antigens from plasma onto the thrombocyte surface. During the set up of the ELISA test when we work with thrombocytes adhering to the surface of a polystyrene plate and not with a suspension of thrombocytes as in other tests, the spatial arrangement or change of epitopes of HLA antigen could prevent the reaction of HLA antigens with the appropriate antibodies. We tried to test this hypothesis by treating thrombocytes with chloroquine, which is known to remove antigens from the thrombocyte surface, as they are not a firm part of their membrane. Chloroquine actually removed HLA antigens from the thrombocytes as was apparent from: 1. the disappearance of the thrombocyte reactivity with HLA type sera, 2. the inability of thus treated thrombocytes to absorb HLA antibodies from type HLA sera. When resolving the problem of the origin of HLA antigens on thrombocytes we support, based on our results, the view of possible adsorption from plasma, onto the thrombocyte surface. In the discussion the author deals with the possible clinical application of this fact in substitution therapy of refractory patients with thrombocyte concentrates.

[Alloimmune neonatal thrombocytopenia due to anti-P1A1 thrombocyte antibodies]. / Aloimunní novorozenecká trombocytopenie zpusobená anti-P1A1 trombocytární protilátkou.

The authors describe three patients with neonatal thrombocytopenia where they detected in the maternal serum, using the immunofluorescence and immunoenzymatic test, antibodies against thrombocytes and thus confirmed the alloimmune nature of thrombocytopenia. By molecular characterization of the immunoreactivity of the thrombocytic antibody in the mother of neonate no. 1 evidence was provided that the antibody is against glycoprotein IIIa which carried antigen P1A1. In the reference laboratory in Amsterdam the anti-P1A1 specificity of this thrombocytic antibody was confirmed. The authors obtained and tested the first type anti-P1A1 serum in Czechoslovakia which can serve to detect P1A1 antigen negative subjects.