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.

[Heparin-induced thrombocytopenia in patients and prevention with low molecular weight heparin]. / Heparinem indukovaná trombocytopenie u pacientu s prevencí nízkomolekulárním heparinem.

Prospective study implicates problems of heparin-induced thrombocytopenia (HIT). This is one of the most important complications in heparin treatment and prophylaxis. The set was formed of patients with severe diagnosis and larger intra-abdominal operation that was associated with relative high risk of thrombotic event. That's why there was need of heparin prophylaxis. We used Enoxaparin in dose 0.2-0.4 ml subcutaneously. The controlled parameters were platelet counts, detection of antibodies using ELISA Assay method (GTI-PFA), indirect platelet immunofluorescent test for monitoring platelet disorders. We had no known case of clinical symptoms of HIT. The change in platelet counts was due to postoperative course, or binding on blood transfer or basic haematological disease. The test detecting antibodies was positive in eight percent without clinical correlation, without important thrombocytopenia. Low-molecular-weight heparin has certain advantages over unfractionated heparin, as well know from clinical and laboratory monitoring in other studies, including larger safety (in relationship with HIT) with very effective prophylaxis.

[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.

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.

[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.

[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 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.

[Treatment of chronic myeloid leukemia with bone marrow transplantation at the Institute of Hematology and Blood Transfusion in Prague]. / Lécba chronické myeloidní leukémie transplantací kostní drene v UHKT Praha.

The outcomes of bone marrow transplantation (BMT) performed at the Institute of Haematology and Blood Transfusion from April 1988 to December 1994 in 31 patients with chronic myelogenous leukemia are presented. Age of the patients range from 18 to 49 years, median 34 years. Male:female ratio was 1.58:1. The conditioning regimen consisted of Cyclophosphamide and total body irradiation (TBI) or Busulfan and Cyclophosphamide. The results are evaluated as of January 1, 1995. Nineteen patients (61.3%) are alive, 12 patients (38.7%) died. The causes of death are discussed. The median time of follow up all patients is 10.4 months, range 0.3-81.5. The median time of follow up of surviving patients is 21.8 months, range 2.5-81.5. Probability of 2 years survival by Kaplan-Meier analysis is 58 +/- 10%. Of the 24 transplanted in the first chronic phase, 18 patients are alive. Of the 7 transplanted in advanced phase of the disease, 1 patient is alive. Of the 27 patients, who received bone marrow from an HLA identical sibling, 19 are alive. Of the 4 patients who received bone marrow from other donor than an HLA identical sibling, none is alive. Acute GvHD III.-IV. grade developed in 5 patients (16.1%), moderate and severe chronic GvHD developed in 11 patients (31.5%). Cytogenetic relapse was diagnosed in 1 patient, hematological relapse in 2 patients. Karnofsky scores of patients surviving after BMT range from 30% to 100%, median 90%.

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.

[Identification of HLA-D allele regions using DNA analysis]. / Identifikace alel HLA-D oblasti analýzou DNA.

The successful bone marrow transplantation presents the highest demands on the compatibility between donor and recipient in the antigen products of HLA system, namely in transplantations performed in unrelated individuals. Traditionally used serotyping gives rather exact results in class I HLA antigen determination, but when typing of class II antigens, at least 25% errors occur. Our papers presents a comparison of different molecular genetics methods used in class II HLA antigen typing. First results of class II HLA typing using RFLP technique, as performed in the Institute of Hematology and Blood Transfusion, are summarized.

[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.

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 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.

[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.

[Immunologic monitoring of bone marrow transplantation. Experience with 16 cases]. / Imunologické zabezpecení transplantace kostní drene. Zkusenosti s 16 prípady.

HLA-A, B antigens and the ABO group were examined in 184 patients with aplasia of bone marrow and leukaemia and in 373 of their relatives, mostly siblings. A HLA-A, B donor, identical or compatible, was found for 35.87% patients, a HLA-DR identical for 84.21% of 38 patients who had a HLA-A, B identical relative. Bone marrow was transplanted to 16 patients (10 with bone marrow aplasia, 6 with acute or chronic leukaemia), with one exception bone marrow from a sibling. The bone marrow was accepted in all patients but two where the transplantation was made despite MLC positivity. From the results ensues that it is essential for successful transplantation of bone marrow to ensure maximal identity between donor and recipient as regards the ABO group, HLA antigens and negativity of the MLC reaction. The negativity of the MLC reaction is more important than HLA-DR identity; when assessing only one HLA-DR antigen in a donor identical with the patient, it cannot be ruled out that on the lymphocytes of the donor there exists another one which was not detected. The authors discuss the causes of different results of the MLC reaction and HLA-A, B, DR typing.

[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.