One-year results from cryopreserved mitral allograft transplantation into the tricuspid position in a sheep experimental model.

Mitral allografts are still used only exceptionally in the mitral or tricuspid position. The main indication remains infectious endocarditis of atrioventricular valves for its flexibility and low risk of infection. The aim of our study was to evaluate 1-year results of mitral allografts transplantation into the tricuspid position in a sheep model. Mitral allografts were processed, cryopreserved, and transplanted into the tricuspid position anatomically (Group I - 11 animals) or antianatomically (Group II - 8 animals). All survivors (4 from Group I, and 3 from Group II) were checked at 3, 6, and 12 months by echocardiography with the exception of one survivor from Group II (which was examinated only visually). Examination throughout follow-up included for mitral allograft regurgitation and annuli dilatation. At postmortem, the papillary muscles were healed and firmly anchored to the right ventricular wall in all subjects. Transventricular fixation of the papillary muscles with buttressed sutures was proven to be a stable, reproducible, and safe method for anchoring mitral allograft leaflets. There were no significant differences between the two implantation methods. Annulus support of mitral allografts might be very useful in this type of operation and could prevent annular dilatation.

Intracoronary injection of autologous bone marrow-derived mononuclear cells in patients with large anterior acute myocardial infarction and left ventricular dysfunction: a 24- month follow up study.

OBJECTIVE AND BACKGROUND: Despite the use of reperfusion therapies, outcomes in patients with large myocardial infarction (MI), late reperfusion and left ventricular (LV) dysfunction are poor. We investigated long-term safety and efficacy of intracoronary injections of autologous bone marrow-derived mononuclear cells (BMNCs). METHODS: 27 patients with anterior MI (age 59±12 years, mean baseline LV ejection fraction (LVEF) 39±5 %), who underwent percutaneous coronary intervention 4-24 hours after the onset of symptoms, were randomly assigned either to intracoronary BMNCs injection (n=17, BMNCs group, out of which 14 underwent long-term follow-up), or to standard therapy (n=10, Control group). The LVEF, the LV end-diastolic and end-systolic volumes (LVEDV, LVESV) were assessed by echocardiography at discharge, Month 4 and 24. Myocardial perfusion was assessed using SPECT at baseline and Month 4. RESULTS: At 24-month, there was no difference in rates of serious clinical events (36 % vs 50 %, p=0.54). At Month 4 LVEF improved to similar extent in both groups (absolute change +5.8 % vs +7.6 %, p=0.75), with similar infarct size reductions (-10.9 % vs -12.2 %, p=0.47). However, at Month 24, LVEF further improved in BMNCs patients (+12 % vs +8.5 %, p=0.03). This effect resulted from a more pronounced reduction in LVESV (-2.6 ml vs -1.8 ml, p=0.26) and a smaller increase in LVEDV (+16.7 ml vs +17.9 ml, p=0.27) suggesting beneficial long-term effects on LV remodeling. CONCLUSIONS: BMNCs injections in patients with MI and LV dysfunction were associated with a significant improvement of global LVEF during long term follow-up compared to standard therapy (Tab. 3, Fig. 1, Ref. 50). Full Text in PDF www.elis.sk.

CCAAT/enhancer-binding protein alpha (CEBPA) polymorphisms and mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia.

The CCAAT/enhancer-binding protein alpha, encoded by the intronless CEBPA gene, is a transcription factor that induces expression of genes involved in differentiation of granulocytes, monocytes, adipocytes and hepatocytes. Both mono- and bi-allelic CEBPA mutations were detected in acute myeloid leukaemia and myelodysplastic syndrome. In this study we also identified CEBPA mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia. We found 16 various deletions with the presence of two direct repeats in CEBPA by analysis of 431 individuals. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493- 498_865-870), GG (486-487_885-886), and GCCAAGCAGC (508-517_907-916), all according to GenBank Accession No. NM_004364.2. In one case we identified that a father with ischaemic heart disease and his healthy son had two identical deletions (493_864del and 508_906del, both according to GenBank Accession No. NM_004364.2) in CEBPA. The occurrence of deletions between two repetitive sequences may be caused by recombination events in the repair process. A double-stranded cut in DNA may initiate these recombination events in adjacent DNA sequences. Four types of polymorphisms in the CEBPA gene were also detected in the screened individuals. Polymorphism in CEBPA gene 690 G>T according to GenBank Accession No. NM_004364.2 is the most frequent type in our analysis. Statistical analysis did not find significant differences in the frequency of polymorphisms in CEBPA in patients and in healthy individuals with the exception of P4 polymorphism (580_585dup according to GenBank Accesion No. NM_004364.2). P4 polymorphism was significantly increased in ischaemic heart disease patients.

[Myocardial infarction, left ventricle remodeling and cellular therapy]. / Infarkt myokardu, remodelace levé komory a bunecná terapie.

The paper brings an overview of results of the most important and significant clinical studies dealing with the issues of bone marrow stem cell implantation in patients with acute myocardial infarction. On the world scale, research has been focused on this area for several years. Much hope is put primarily on the possibility to prevent the process of progressive remodelling of the left ventricle, the substitution of necrotic or fibrotic tissue and the resulting prevention of development and progression of heart failure. In the centre of attention are especially patients whose long-term prognosis is often very poor in spite of progress in contemporary medicine.

Cryopreserved semilunar heart valve allografts: leaflet surface damage in scanning electron microscopy.

OBJECTIVE: Allograft heart valves (AHV), biological valves of human origin, offer potential advantages over conventional xenografts in terms of superior hemodynamics and, perhaps, better durability. The most important factors for long-term AHV clinical performance are the processing and cryopreservation methods. The aim of this study was to evaluate the impact of current processing protocol on valve tissue morphology, mainly to address the effect of successive processing steps on the leaflet surface structure. For the detection of fine changes in endothelial covering and underlying layers, our own modification of the scanning electron microscopy (SEM) technique was utilized. MATERIAL AND METHODS: The study was based on an investigation of 20 AHV (40 specimens). Fourteen valves came from heart-beating donors (multiorgan harvesting) when the heart could not be transplanted for any reason (donor criteria, availability of recipient and/or logistics). Six were obtained at the time of routine postmortems--non heart-beating donors (NHBD). All specimens were initially fixed in Baker's solution. Tissue samples were dissected, dried with hexamethyldisilazane (HMDS), gold-coated, studied and photographed by SEM (Tesla BS 301). In order to define the integrity of the endothelium, subendothelial layers and the quality of the surface under SEM, a special six-level score system was introduced: 1-intact endothelium, 2-confluent endothelium with structural inhomogeneity, 3-disruption of intercellular contacts, 4-separation of endothelial cells, 5-complete loss of endothelium, 6-damage of subendothelial layers). AHV samples were divided into 4 groups for comparison. One aortic AHV "fresh" control sample obtained from a heart-beating donor was evaluated without any processing and was compared with (i) tissue from AHV obtained from NHBD with warm ischemia of 12 and 48 hours, (ii) samples stored at +4 degrees C in saline for 24 h, (iii) antibiotic-treated tissue for 24 h at 37 degrees C and finally with (iv) cryopreserved valves stored in liquid nitrogen (-196 degrees C) for 6-38 months. RESULTS: Our alternative for drying samples by the HMDS method proved to be suitable for thin membranes of human semilunar valves. We were able to detect early changes in the endothelium after harvesting and denudation of the endothelial covering during preservation with and without freezing. The surface of the AHV samples revealed the typical features and score system determined endothelial cell damage. Control "fresh" sample: score 2, (i) NHBD samples with warm ischemia of 12 h: score 3-4, with warm ischemia of 48 h: score 4-5, (ii) samples stored at +4 degrees C in saline for 48 h: score 5-6, (iii) antibiotic-treated tissue for 24 h at 37 degrees C: score 5, (iv) cryopreserved valves stored in liquid nitrogen for 6-38 months: score 5-6. CONCLUSION: SEM (using HMDS drying) together with other methods may be helpful for the morphological control of processing, cryopreservation and liquid nitrogen storage of AHV. Severe AHV leaflet endothelial destruction was proven on AHV grafts. These changes arose already in the initial steps of tissue processing, just after the donor heart harvesting and then at the time of antibiotic valve graft treatment. These results are considered as the starting point for the development of a better preservation protocol.

Autologous intra-arterial infusion of bone marrow mononuclear cells in patients with critical leg ischemia.

AIM: The injection of bone marrow mononuclear cells (BMMC) into the gastrocnemius muscle has given promising results in patients with critical limb ischemia (CLI). In this article, we have assessed whether a less invasive procedure, i.e. intravascular BMMC infusion, could be effective in this population of patients. METHODS: A total of 28 limbs in 24 patients with CLI were treated. An amount of 276-700 mL of marrow blood was harvested from posterior iliac crests and BMMC were obtained by standard procedure used for bone marrow transplantation. After performance of digital subtraction angiography, BMMC were injected laterally through a 4 Fr sheet. Primary outcome was efficacy of the procedure measured as healing of defects, frequency of high amputations and change of ischemia grade; among secondary outcomes were safety of the procedure, angiographic changes and changes in quality of life. RESULTS: One year after treatment, all patients were alive and only 2 patients have undergone high amputation. Eleven of 14 defects have healed (78%) and Fontaine grade of ischemia has changed from median grade 3.5 to median grade 2 (P<0.0001). Collateral vessel development has improved by mean 1.13 and 1.3 points on a four-point semiquantitative scale in calf and foot, respectively (P<0.0001). There were no grade III-IV adverse events. According to the SF-36 quality of life questionnaire, 1 year after the procedure patients have reported significant improvement in all measured items. CONCLUSION: Intra-arterial infusion of BMMC can lead to significant and long-lasting subjective and objective improvements in patients with CLI. The results merit validation by randomized controlled studies in patients with less critical limb ischemia.

Prognosis of pediatric patients transplanted for Ph+ chronic myeloid leukemia in the period from 1989 to 2006 in the Czech Republic.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by clonal proliferation of primitive hematopoietic stem cell. The median age at diagnosis is 55 to 60 years with less than 10% of patients younger 20 years. Incidence of CML in children in the Czech Republic is 0.106 cases/100 thousands per year. Here we report outcome of 38 pediatric patients (median age 12.5 years; range 1.8 - 17.3) with Ph-positive CML diagnosed between years 1989 to 2006. Primarily chronic phase of the disease was diagnosed in 32 (84%) patients. 32 (84.2%) patients underwent hematopoietic stem cell transplantation (HSCT) with the median age at transplantation of 14.9 years (range 6.9 - 20.5 years). Out of transplanted patients 16 (50%) obtained graft from unrelated donor, 13 (41%) from matched sibling donor, 2 from haploidentical family donor and autologous transplantation has been performed in one case. 6 patients were not transplanted, 4 of them died (median 1.2 years from diagnosis), 2 are alive 0.6 and 17.8 years from the diagnosis. Overall survival (OS) in 25 patients after HSCT at our department during the whole period is 66.7% with 15/16 being in stable continuous molecular-genetic remission (94%). During the period of time results of transplantations have been significantly improved (p=0.0071). OS after HSCT until year 1997 is 25% while from year 1998 until now is 87.5%. All centers OS of patients after HSCT is 71%. Results of HSCT in children with CML obtained from the year 1998 at our center are fully comparable with results achieved in large and experienced centers. HSCT remains the only proven and effective method for the treatment of CML. Clinical studies assessing the role of tyrosine kinase inhibitors in children instead of early HSCT should be planned carefully in order to avoid sub-optimal outcomes.

One-day kinetics of myocardial engraftment after intracoronary injection of bone marrow mononuclear cells in patients with acute and chronic myocardial infarction.

OBJECTIVE: To investigate the kinetics of myocardial engraftment of bone marrow-derived mononuclear cells (BMNCs) after intracoronary injection using 99mTc-d,l-hexamethylpropylene amine oxime (99mTc-HMPAO) nuclear imaging in patients with acute and chronic anterior myocardial infarction. DESIGN: Nuclear imaging-derived tracking of BMNCs at 2 and 20 h after injection in the left anterior descending (LAD) coronary artery. SETTING: Academical cardiocentre. PATIENTS: Five patients with acute (mean (SD) age 58 (11) years; ejection fraction range 33-45%) and five patients with chronic (mean (SD) age 50 (6) years; ejection fraction range 28-34%) anterior myocardial infarction. INTERVENTIONS: A total of 24.2 x 10(8)-57.0 x 10(8) BMNCs (20% labelled with 700-1000 MBq 99mTc-HMPAO) were injected in the LAD coronary artery. RESULTS: At 2 h after BMNC injection, myocardial activity was observed in all patients with acute (range 1.31-5.10%) and in all but one patient with chronic infarction (range 1.10-3.0%). At 20 h, myocardial engraftment was noted only in three patients with acute myocardial infarction, whereas no myocardial activity was noted in any patient with chronic infarction. CONCLUSIONS: Engraftment of BMNCs shows dynamic changes within the first 20 h after intracoronary injection. Persistent myocardial engraftment was noted only in a subset of patients with acute myocardial infarction.

Allograft semilunar cardiac valves processing and cryopreservation - morphology in scanning electron microscope.

OBJECTIVE: The most important factors of long term clinical performance of biological heart valve prostheses are methods of processing and cryopreservation. That is why we decided to evaluate the impact of current Allograft Heart Valves (AHV) Bank protocol on valve tissue morphology. Scanning electron microscope (SEM) is a valuable tool for investigation of biological surfaces. In case of cardiac valves it is especially suitable for detection of fine changes in endothelial covering and underlying layers. MATERIAL AND METHODS: "Fresh" aortic and pulmonary AHV samples, harvested from "heart-beating" cadaveric donors, were compared with (1) tissue from AHV obtained from non heart-beating donors, (2) samples stored in 4 degrees C saline for 24 h, (3) antibiotic treated tissue for 24 h at 37 degrees C and finally (4) cryopreserved valves, stored in liquid nitrogen (-196 degrees C) for 6-38 months. All samples were dissected, dried with hexamethyldisilazane (HMDS), gold coated, studied and photographed by SEM (Tesla BS 301). RESULTS: Our alternative method of drying samples by the HMDS method proved to be suitable for thin membranes of human semilunar valves. We were able to detect early changes in the endothelium after harvesting, and denudation of the endothelial covering during preservation with and without freezing. CONCLUSION: SEM (using HMDS drying) along with other methods may be helpful for the morphological control of processing, cryopreservation and liquid nitrogen storage of AHV. According to the current findings we have to avoid washing of AHV in saline after harvesting.

[High dose therapy with autologous stem cell transplantation in patients with Hodgkin's lymphoma: long-term follow-up in patients treated in one center]. / Vysokodávkovaná terapie s autologní transplantací krvetvorných bunek u nemocných s Hodgkinovým lymfomem: dlouhodobé sledování nemocných lécených v jednom centru.

BACKGROUND: Majority of patients with Hodgkin's Lymphoma (HL) can be cured by first line therapy. The high dose therapy (HDT) with autologous stem cell transplantation (ASCT) is the option which can be used in the situation when the conventional therapy failed. METHODS AND RESULTS. Beginning 1994 till 2005 84 pts with HL who did not respond the conventional chemotherapy underwent 105 HDT procedures with ASCT. The median age at the time of HDT was 30.5 years. The reason for salvage therapy followed by HDT with ASCT was the failure to achieve 1st complete remission-- CR (n 16) or the subsequent relapse or progression (n 68). The disease status at the time of HDT after conventional salvage chemotherapy was assessed as chemosensitive in 65 pts (77.4%) and chemoresistant in 19 pts (22.6%). The most frequent HDT regimen used was BEAM (82 HDT), 22 pts entered into the tandem HDT program. Bone marrow only was used as the source of progenitor cells in 4 ASCT, peripheral blood progenitor cells (PBPC) only were used in 85 ASCT and the combination of both in 16 ASCT. The disease status after the HDT with ASCT was assessed (77 pts were qualifiable) as CR in 39 pts (50.6%), PR in 31 (40.3%) and as stable disease or progression in 7 pts (9.1%). Treatment related mortality in HDT with PBPC was 3.9%. The median follow up is 5.3 years. The five year probability of event free survival (EFS) is 43.1% and overall survival 53.2%. The EFS and OS probability respectively for the chemosensitive patients was 48.6% and 62.9% respectively. The status at HDT and the results after it have prognostic significance. There were observed 39 deaths and 26 of them were caused by disease progression. Secondary tumor was observed in 5 pts and in all of them it caused the death. CONCLUSIONS: The HDT with ASCT allows the long-term survival without disease progression in about a half of the patients with reasonable toxicity.

[Gene therapy of the graft versus host reaction]. / Genová terapie reakce stepu proti hostiteli.

Graft-versus-host-disease (GVHD) is a frequent and dangerous complication of allogenic transplantations of bone marrow. Gene therapy offers a way to deal with the problem. It is based on the introduction of suicide genes (SG) into the donor's T lymphocytes, which are responsible for the development of GVHD. If it develops, the presence of SG in the effector cells gives an opportunity to get rid of them, because their products are capable of changing otherwise innocuous substances into highly cytotoxic metabolites. For the transduction of SG retrovirus-based vectors are used. The authors tried to employ for this purpose recombinant adeno-associated viruses (rAAV). The attempt was unsuccessful. When using rAAV as vectors, the efficacy of transduction was very low. Further experiments indicated that this failure was due to the absence of receptor for AAV in T lymphocytes. It seems clear that until the surface of rAAV is modified to facilitate their penetration into T cells, they cannot replace retroviruses for transfer of SG into this cell type.

[Viability of parathyroid gland tissue measured by flow cytometry]. / Viabilita tkáne prístítných telísek merená prutokovou cytometrií.

BACKGROUND: Postoperative hypoparathyroidism after the total parathyroidectomy (PTX) remains a problem, no matter our experiences with 243 operations on parathyroid glands (PG). Implantation of "fresh" tissue, cryopreservation and reimplantation of cryopreserved tissue are performed with uncertain results. The aim of this project was to compare viability of cryopreserved tissue of parathyroid glands with "fresh" tissue obtained during parathyreoidectomy and with tissue from cadaverous donors. METHODS AND RESULTS: Group 1 included 55 cryopreserved samples obtained from 41 patients after PTX (22M, 19F, a mean age of 46 +/- 11 years). Average duration of storage in liquid nitrogen was 84 +/- 49 months. Group 2 included "fresh" tissue of PG, harvested during PTX. Viability was measured in different time in samples from 42 patients with hyperparathyroidism (11M, 31F, a mean age of 55 +/- 13 years). Group 3 included tissue of 14 cadaverous donors obtained during multiorgan harvesting (7M, 7F, a mean age of 31 +/- 5 years, WIT 32 min). Viability was measured by flow cytometry with propidium iodide after dissociation of tissue. Evaluation of PG tissue was proven by histology. Average viability in group 1 was 36.9 +/- 24.7%, no correlation with the duration of storage in liquid nitrogen was found. Average viability in group 2 was 51.4 +/- 24%. Viability in group 3 was 66.8 +/- 32%. Group 1 vs. group 2 were different with p < 0.05, group 2 vs. 3 did not reach significance (with marginal p = 0.06) and group 1 vs 3 were different with p < 0.001. CONCLUSIONS: The highest viability was found in tissue of cadaverous donors, the lowest in cryopreserved tissue (with no correlation to the duration of storage in liquid nitrogen).

[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.

Comparison of two different methods for CD34+ selection and T cell depletion in peripheral blood stem cell grafts--our experiences with CellPro, E rosetting and CliniMACS technique.

The aim of this study was to establish a suitable method for in vitro T cell depletion in peripheral blood stem cell grafts for mismatched/haploidentical transplantation in children and adults with severe hematological disorders and for autologous transplantation in patients with autoimmune diseases refractory to conventional immunosuppressive treatment. Two different selection techniques have been used: CD34+ selection using immunoaffinity columns (CellPro Ceprate) followed by T cell depletion by E-rosetting or CD34+ selection using submicroscopic paramagnetic beads (CliniMACS device) with T cell depletion in a one step procedure. The mean purity and recovery of CD34+ cells and efficiency of T cell removal in the final product were compared. From March 1995 to December 1998 we prepared twelve allografts using Cell Pro system for eight children with high-risk hematological malignancies and six autografts for six patients with severe autoimmune diseases. From January 1999 to October 2000 we prepared fifteen allografts using CliniMACS system for ten children with high-risk hematological diseases and inborn metabolic disorders or primary immunodeficiences, five allografts for three adult patients with high-risk hematological malignancies and two autografts for two patients with autoimmune diseases. In allogeneic transplantation the median purity of CD34+ cells in the final products after CellPro and E-rosetting was 85.6% (55.3%-95.7%); median recovery was 24.8% (17%-35%), median transplanted doses of T cells per kilogram of body weight were 0.66x10(4) (0-2.8); in autologous transplantation the median purity of CD34+ was 92.6% (55.6%-96%), median recovery was 28% (22%-46.2%), median transplanted doses of T cells per kilogram of body weight were 0.39x10(4) (0.0-3.6). After CliniMACS technique the median purity of CD34+ cells was 94.87% (69.15%-99%),medianrecoverywas 58% (30%-79.6%), median transplanted doses of T cells per kg of body weight were 0.254x10(4) (0-14.15); in autologous transplantation the median purity of CD34+ was 94% (94%-94%, median recovery was 97.4% (95%-99.8%), median transplanted doses of T cells per kilogram of body weight were 0.87x10(4) (0.49-1.24). We consider both methods of CD34+ selection and T cell depletion suitable for peripheral blood stem cell processing before mismatched hemopoietic stem cell transplantation in patients without identical donor or before autologous transplantation for severe autoimmune diseases. However, magnetic separation using CliniMACS system results in higher levels of purity and recovery with efficient T cell depletion.

[Hematopoietic stem cell transplantation in autoimmune diseases in rheumatology practice]. / Transplantace krvetvorných bunek autoimunitních chorob v revmatologii.

Autoimmune diseases (AID) result from the impairment of the effector and/or recognition phase of the immune response. The autoimmune process plays a crucial role in the pathogenesis of the systemic lupus erythematosus (SLE), systemic sclerosis (SSc), rheumatoid arthritis (RA), and their treatment is therefore largely based on immunosuppression. However, some patients do not respond to its standard doses. The disease becomes intractable with the survival rate comparable to that of some haematological malignancies, or patients become soon handicapped with very poor quality of life, depending on continual administration of high doses of steroids. The new hope for those patients becomes therapy with high dose myelo- and immuno-ablative chemotherapy with autologous hematopoietic progenitor cell support (PBPC). Tens of patients with intractable forms of AID were transplanted in the pilot clinical studies with promising results. The most frequent indications included: SLE, SSc, and RA. Final conclusion of the therapeutic effects will be drawn from the analysis of larger trails.

[Immunoablation and hematopoietic stem cell transplantation in the treatment of multiple sclerosis]. / Imunoablace s transplantací krvetvorných bunek v lécbe roztrousené sklerózy.

High dose chemotherapy with autologous hematopoietic cell support is a standard approach in the management of selected hematological malignancies. Autoimmune diseases which do not respond to conventional immunosuppression might benefit from high dose immunoablative chemotherapy. The transplantation of hematopoietic cells is necessary after the high dose chemotherapy to restore bone marrow function. The immune system undergoes a new ontogeny which can result in the development of tolerance. Multiple sclerosis (MS) has so far been the most common indication for this kind of treatment. Experience with preclinical studies on murine experimental allergic encephalomyelitis (EAE), as well as the course of MS following bone marrow transplantation for coincidental malignancy in humans formed the basis of the first clinical studies involving high dose chemotherapy and autologous hematopoietic support. Results of the first studies confirm that the method is feasible in patients with MS, and that the effect is very promising. Nonetheless, more consistent results vis a vis the therapeutic effect should emanate from upcoming studies.

High-dose immunosuppressive therapy with PBPC support in the treatment of poor risk multiple sclerosis.

High-dose immunoablative chemotherapy with autologous haematopoietic cell support might be beneficial in the treatment of intractable forms of MS. We mobilised PBPC in 11 patients with secondary progressive MS and finally eight patients were grafted after high-dose BEAM chemotherapy with either in vitro or in vivo T cell depletion. Median EDSS and SNRS scores at the time of inclusion were 6.5 (6.5-7.5) and 56 (44-65), respectively. PBPC mobilisation was safe with no serious adverse effects, and without significant aggravation of disability. One patient improved significantly (by 1.0 point on EDSS) after the mobilisation. Two mobilisation failures were observed. No life-threatening events occurred during the transplantation. All grafted patients, except one, at least stabilised their disability status. One patient improved significantly (by 1.5 points on EDSS), two patients improved slightly (by 0.5 points on EDSS), one patient worsened by 1.0 point on the EDSS in 10 months. Improvement occurred with a delay of 2-4 months. Median EDSS and SNRS of grafted patients at the last follow up were 6.5 (5.5-8.5) and 64 (39-73), respectively with median follow-up of 8.5 months. Further follow-up is needed to determine the disease course after complete immune reconstitution. Bone Marrow Transplantation (2000) 25, 525-531.

Increased glucagon-stimulated insulin secretion of cryopreserved rat islets transplanted into nude mice.

Cryopreservation is the only available technique for long-term storage of pancreatic islets. The freezing/thawing protocol may cause considerable loss of viable islet tissue and impair its function in vivo. The aim of this study was to investigate glucose and insulin levels after transplantation of fresh and cryo/thawed rat islets. Rat pancreatic islets were isolated following intraductal collagenase injection and Ficoll gradient purification. After isolation, islets were cultured for 24 h and then either transplanted or frozen after stepwise addition of DMSO according to Rajotte et al. and stored in liquid nitrogen. After rapid thawing islets were stepwise transferred into RPMI medium and cultured for another 24 h. The recipients were athymic mice with streptozotocine-induced diabetes. Two hundred fresh (n=13) or cryo/thawed (n=15) islets were transplanted beneath the renal capsule. Glucose levels were measured for 14 days and blood samples for insulin determination were obtained 15 min after i.p. glucagon (10 mg/kg) administration on day 14. Glucose levels were normalized (<9 mmol/l) in all recipients within 3 days since transplantation. On day 14, mean fasting values+/-SE in fresh and cryo/thawed islet groups were 4.0+/-0.6 and 4.4+/-0.4 mmol/l, respectively (P>0.05). Fasting insulin levels were higher in the cryo/thaw than in the fresh islet group (1.67+/-0.33 vs 0.57+/-0.13 ng/ml; P<0.01). Post-glucagon levels did not differ significantly (1.45+/-0.24 vs 0.86+/-0.24 ng/ml; P=0.06). While glucagon significantly increased insulin levels (P<0.01) in the fresh islet group, no change in insulin levels was observed (P>0.05) in the cryo/thaw group. Immunohistochemical staining demonstrated fragmentation of viable islet tissue which was more apparent in the cryo/thaw group. We conclude that in a short-term study cryo/thawed rat islets produce higher insulin levels than fresh islets transplanted into nude mice. This may be due to better islet survival or loss of feed-back regulation.

[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.