BACKGROUND: Myelodysplastic syndrome with isolated chromosome 5q deletion (5q- syndrome) is a clonal stem cell disorder characterized by ineffective hematopoiesis. MicroRNAs (miRNAs) are important regulators of hematopoiesis and their aberrant expression was detected in some clonal hematopoietic disorders. We thus analyzed miRNA expressions in bone marrow CD34+ cells of 5q- syndrome patients. Further, we studied gene expressions of miR-143, miR-145, miR-378 and miR-146a mapped within the 5q deletion. RESULTS: Using microarrays we identified 21 differently expressed miRNAs in 5q- patients compared to controls. Especially, miR-34a was markedly overexpressed in 5q- patients, suggesting its role in an increased apoptosis of bone marrow progenitors. Out of four miRNAs at del(5q), only miR-378 and miR-146a showed reduced gene expression in the patients. An integrative analysis of mRNA profiles and predicted putative targets defined potential downstream targets of the deregulated miRNAs. The list of targets included several genes that play an important role in the regulation of hematopoiesis (e.g. KLF4, LEF1, SPI1). CONCLUSIONS: The study demonstrates global overexpression of miRNAs is associated with 5q- phenotype. Identification of hematopoiesis-relevant target genes indicates that the deregulated miRNAs may be involved in the pathogenesis of 5q- syndrome by a modulation of these targets. The expression data on miRNAs at del(5q) suggest the presence of mechanisms for compensation of a gene dosage.

• MeSH
• antigeny CD34 biosyntéza   genetika   MeSH
• chromozomální delece MeSH
• Krüppel-like faktor 4 MeSH
• lidé MeSH
• lidské chromozomy, pár 5 genetika   metabolismus   MeSH
• makrocytární anemie genetika   metabolismus   MeSH
• mikro RNA biosyntéza   genetika   MeSH
• myelodysplastické syndromy genetika   metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD34 MeSH
• KLF4 protein, human MeSH Prohlížeč
• Krüppel-like faktor 4 MeSH
• mikro RNA MeSH

We evaluated the efficiency, safety and risks of three techniques which were used for autologous PBPC collections: (a) large-volume leukapheresis (LVL), (b) standard collections, and (c) a new modified technique which was named as "Mixed" collections. In spite of the fact that the standard and LVL collection techniques are used routinely, there may occur special conditions in which the procedures cannot be recommended. Some patients may suffer from serious clinical complications and they cannot tolerate either standard procedures with administration of higher doses of ACD-A, or the high extent of procedure in the course of LVL. We tried to find the safe and efficient collection technique which could help this group of patients to overcome their problems. The "Mixed" collection technique could be such a choice. The numbers of 136 autologous PBPC collections were performed in 98 patients who suffered from hemato-oncological diseases. We evaluated the results of (a) 93 LVL (more than 3 TBV, total blood volumes of the patients were processed; anticoagulation: ACD-A and Heparin), (b) 16 Standard procedures (less than 3 TBV were processed; anticoagulation: ACD-A), and (c) 27 "Mixed" collections (less than 3 TBV of patients were processed; anticoagulation: ACD-A+ Heparin). Collections were performed by the use of separator Cobe Spectra, Caridian. In patients (a) with a good effect of mobilization (precollection CD 34+ cells in blood higher than 20×10(3)/mL) we prepared almost the same median dose of CD 34+ cells from the standard and "Mixed" collections, 3.8 and 4×10(6)/kg, respectively. In LVL the median yield of CD 34+ cells was 8.2×10(6)/kg. In patients (b) who were mobilized weakly (precollection CD 34+ cells in blood lower than 20×10(3)/mL), LVL enabled to prepare 1.5×10(6) of CD 34+/kg from one collection, while the median yield of CD 34+ cells from the standard and "Mixed" collections was 0.9 and 1.2×10(6)/kg. All the standard, LVL and "Mixed" procedures were tolerated well without any serious adverse reactions. We detected 22 adverse reactions, but only three reactions were associated directly with the procedure. Mild hypocalcemia (2) and hypotensive reaction (1) were transient and treated efficiently. Procedures could continue and were finished according to the planned programme. Other reactions were related either to the insufficient function of central venous catheter or to the poor clinical condition of the patients. LVL enabled to get a higher yield of CD 34+ cells than the Standard and "Mixed" collections in well mobilized patients as well as in weakly mobilized patients. We observed the similar efficiency in standard and "Mixed" collections in well mobilized and weakly mobilized patients. We can recommend LVL in all patients who can tolerate it due to a greater chance of collecting higher yields of progenitor cells. In the weakly mobilized patients LVL offers a greater chance of collecting at least a minimum amount of CD 34+ cells needed for transplantation. "Mixed" collections may be used as an alternative technique under the circumstances in which standard or LVL cannot be recommended - like in patients who do not tolerate a high amount of citrate or a high extent of the procedure, e.g. patients with cardiac arrhytmia, impaired liver or renal function or unstable vital signs.

• MeSH
• antigeny CD34 biosyntéza   MeSH
• dospělí MeSH
• kmenové buňky cytologie   MeSH
• konzervace krve MeSH
• lékařská onkologie metody   MeSH
• leukaferéza metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mobilizace hematopoetických kmenových buněk metody   MeSH
• nádory terapie   MeSH
• riziko MeSH
• senioři MeSH
• transplantace periferních kmenových buněk přístrojové vybavení   metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD34 MeSH

The aim of our study was to evaluate the occurrence of chondrocytes containing alpha-smooth muscle actin in human normal and diseased cartilage. Immunohistochemistry using monoclonal antibodies for alpha-smooth actin, muscle-specific actin, S-100 protein, CD 34, and desmin was performed on samples of human articular cartilage obtained at autopsy following sudden death, during total hip and knee replacement for osteoarthritis, or after femoral neck fracture in patients without symptoms of osteoarthritis. Moreover, the layers of residual cartilage from chondral posttraumatic defects obtained during preoperative arthroscopy and of newly formed cartilage after autologous-chondrocyte transplantation (Hyalograft C) obtained during second-look arthroscopy were also examined by immunohistochemistry and RT PCR. Our study showed that a significant percentage of articular chondrocytes express alpha-smooth muscle actin in healthy, diseased, and regenerated articular cartilage. Alpha-actin positive chondrocytes (18%) were observed predominantly in the upper zone of normal articular cartilage. By contrast, only approximately 10% of cartilage cells in the deep region stained for this contractile actin isoform. Actin-positive chondrocytes (myochondrocytes) are formed predominantly in response to injury to the osteoarthrotic cartilage, at sites of defective healing, and in newly formed cartilage after autologous chondrocyte transplantation. Fibrocartilage is present in some of these conditions, and it is known that this tissue contains chondrocytes with actin. The presence of myochondrocytes in the surface layer of normal articular cartilage indicates that this region probably plays an important role in maintaining cartilage integrity. Myochondrocytes may utilize the contractile actin isoform in manipulating the extracellular matrix of articular cartilage. It is also possible that actin-containing chondrocytes have a higher potential for regeneration in contrast to chondrocytes that do not contain this contractile material in their cytoplasm.

• MeSH
• aktiny biosyntéza   MeSH
• antigeny CD34 biosyntéza   MeSH
• chondrocyty metabolismus   transplantace   MeSH
• desmin biosyntéza   MeSH
• dospělí MeSH
• imunohistochemie MeSH
• kloubní chrupavka cytologie   metabolismus   transplantace   MeSH
• lidé středního věku MeSH
• lidé MeSH
• osteoartróza metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny S100 biosyntéza   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• antigeny CD34 MeSH
• desmin MeSH
• proteiny S100 MeSH

BACKGROUND: The success of treatment using monoclonal antibodies in oncology is influenced by, among other factors, the level of target antigen expression on tumor cells. The authors analyzed the intensity of the CD52 antigen expression in patients with chronic lymphoproliferative diseases and compared them with B-lymphocytes of a healthy population and CD34(+) cells in peripheral blood stem cells (PBSC) grafts. METHODS: Recently diagnosed and previously untreated patients with B-cell chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), or small lymphocytic lymphoma (SLL) were evaluated and compared with control group and CD34(+) cells. The intensity of CD52 was expressed in molecules of equivalent soluble fluorochrome units (MESF) and antibody-binding capacity (ABC). RESULTS: In the group of patients with B-CLL, the CD52 level on tumor cells (245 x 10(3) MESF; 107 x 10(3) ABC) was significantly lower than on B-lymphocytes of the control group (446 x 10(3) MESF; 194 x 10(3) ABC; P < 0.001) and SLL tumor cells (526 x 10(3) MESF; 229 x 10(3) ABC; P < 0.001). The CD52 antigen was expressed on a majority of CD34(+) cells, but its expression intensity was low (101 x 10(3) MESF; 44 x 10(3) ABC). CONCLUSIONS: Our data demonstrate differences in the intensity of the CD52 antigen expression between B-lymphocytes and tumor lymphocytes of B-CLL patients, and between B-CLL and SLL tumor cells. CD52 antigen is expressed at low level on CD34(+) cells.

• MeSH
• aktivace lymfocytů imunologie   MeSH
• antigen CD52 MeSH
• antigeny CD34 biosyntéza   MeSH
• antigeny nádorové analýza   imunologie   metabolismus   MeSH
• B-lymfocyty imunologie   MeSH
• biologické markery analýza   MeSH
• CD antigeny analýza   imunologie   metabolismus   MeSH
• chronická lymfatická leukemie krev   diagnóza   imunologie   MeSH
• chronická nemoc MeSH
• dospělí MeSH
• glykoproteiny analýza   imunologie   metabolismus   MeSH
• hematopoetické kmenové buňky imunologie   MeSH
• leukemie B-buněčná krev   diagnóza   imunologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfom z plášťových buněk krev   diagnóza   imunologie   MeSH
• lymfoproliferativní nemoci krev   diagnóza   imunologie   MeSH
• monoklonální protilátky imunologie   MeSH
• nádorové biomarkery analýza   imunologie   MeSH
• prediktivní hodnota testů MeSH
• průtoková cytometrie metody   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antigen CD52 MeSH
• antigeny CD34 MeSH
• antigeny nádorové MeSH
• biologické markery MeSH
• CD antigeny MeSH
• CD52 protein, human MeSH Prohlížeč
• glykoproteiny MeSH
• monoklonální protilátky MeSH
• nádorové biomarkery MeSH

Expression of major cytochrome P450 forms (P450) was followed in preparation of purified hematopoietic CD34+ stem and progenitor cells. Levels of transcripts as well as mature proteins were traced by quantitative real-time polymerase chain reaction and by Northern and Western blotting. P450 1B1 and P450 2E1 proteins and respective mRNAs were found in all cases. On the other hand, no expression of P450 3A4, P450 3A7, and P450 2C9 was found. The results showed that expression of various P450 enzymes starts at different stages of cell differentiation. Both P450 forms found are known to be connected with cancer cells and with activation of procarcinogens (P450 1B1, polycyclic aromatic hydrocarbons; P450 2E1, nitrosamines, and solvents). Hence, cells at the early stage of differentiation already may be influenced by interaction with xenobiotics. This fact should also be taken into consideration when hematopoietic cell transplant therapy is applied.

• MeSH
• antigeny CD34 biosyntéza   MeSH
• cyklofilin A biosyntéza   genetika   MeSH
• exprese genu genetika   MeSH
• hematopoetické kmenové buňky enzymologie   imunologie   metabolismus   MeSH
• imunoblotting MeSH
• izoenzymy biosyntéza   genetika   MeSH
• komplementární DNA izolace a purifikace   metabolismus   MeSH
• lidé MeSH
• messenger RNA biosyntéza   genetika   MeSH
• průtoková cytometrie MeSH
• RNA izolace a purifikace   metabolismus   MeSH
• systém (enzymů) cytochromů P-450 biosyntéza   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD34 MeSH
• cyklofilin A MeSH
• izoenzymy MeSH
• komplementární DNA MeSH
• messenger RNA MeSH
• RNA MeSH
• systém (enzymů) cytochromů P-450 MeSH

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.

• MeSH
• antigeny CD34 biosyntéza   MeSH
• dítě MeSH
• hematopoetické kmenové buňky patologie   MeSH
• lidé MeSH
• mobilizace hematopoetických kmenových buněk přístrojové vybavení   metody   MeSH
• nádory terapie   MeSH
• T-lymfocyty metabolismus   MeSH
• transplantace hematopoetických kmenových buněk metody   MeSH
• viabilita buněk MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antigeny CD34 MeSH

The AC133 antigen is selectively expressed on subset of CD 34+ cells isolated from leukapheresis products from high risk breast cancer patients receiving chemotherapy plus G-CSF. MiniMACS AC133+ isolated cells contained a mean of 85% (80-90) AC133+ cells. Enriched AC133+ cells coexpressed 80% CD34+, 6.6% CD33+ and 2% CD15+. Separated AC133+ cells contained 600 GFU-GM/10(4) cells and 70 BFU-E/10(4) cells. Flow-cytometric analysis indicated that AC133+ cells were isolated from cells population with low granularity (SS), while CD33+ a CD15+ cells had a high granularity. After a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL11, the expansion of cells increased 19.4 times. The mean percentage of blasts decreased from 100% at the start of culture to 81% on day 3 and 30% on day 7. Promyelocytes were slow to appear with 10% present on day 3, but thereafter increased to 33% on day 7. The appearance of myelocytes and metamyelocytes lagged 3 days behind promyelocytes and continued to increase during culture to become the predominant (30%) cell type on day 7. Very few neutrophils (2%) were observed in any of the cultures on day 7. Monocytes or macrophages were not detected on day 7. By day 7 megakaryocytes were present at low levels (10%). The mean value of CFU-GM in the culture after day 7 of ex vivo expansion in the presence of SCF+IL-3+IL-11 had increased 45-fold, BFU-E 5-fold. After 7 days of expansion with IL-3+SCF+IL-11 cells expressed a mean of 12% CD34+, 8% AC133+, 59% CD33+ and 30% CD15+. The aim of this experiment was to determine whether ex vivo culture of peripheral blood AC133+ cells could generate sufficient numbers of progenitors to potentially abrogate cytopenia after transplantation and passive purging of tumor cells.

• MeSH
• antigen AC133 MeSH
• antigeny CD34 biosyntéza   krev   imunologie   MeSH
• CD antigeny MeSH
• cyklofosfamid škodlivé účinky   MeSH
• dospělí MeSH
• etoposid aplikace a dávkování   MeSH
• faktor růstu kmenových buněk farmakologie   MeSH
• faktor stimulující kolonie granulocytů aplikace a dávkování   MeSH
• faktory růstu hematopoetických buněk farmakologie   MeSH
• glykoproteiny biosyntéza   krev   imunologie   MeSH
• hematopoetické kmenové buňky cytologie   účinky léků   imunologie   MeSH
• interleukin-11 farmakologie   MeSH
• interleukin-3 farmakologie   MeSH
• leukaferéza MeSH
• lidé středního věku MeSH
• lidé MeSH
• mobilizace hematopoetických kmenových buněk MeSH
• nádory prsu krev   farmakoterapie   imunologie   MeSH
• peptidy krev   imunologie   MeSH
• protokoly antitumorózní kombinované chemoterapie terapeutické užití   MeSH
• rekombinantní proteiny farmakologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen AC133 MeSH
• antigeny CD34 MeSH
• CD antigeny MeSH
• cyklofosfamid MeSH
• etoposid MeSH
• faktor růstu kmenových buněk MeSH
• faktor stimulující kolonie granulocytů MeSH
• faktory růstu hematopoetických buněk MeSH
• glykoproteiny MeSH
• interleukin-11 MeSH
• interleukin-3 MeSH
• peptidy MeSH
• PROM1 protein, human MeSH Prohlížeč
• rekombinantní proteiny MeSH

In this study flow cytometric and morphologic methods of apoptosis detection in human promyelocytic leukemia cell line HL-60 were compared. HL-60 cells were harvested at 4, 7, 16, 24 a 48 hours after induction of apoptosis by 3 % ethanol. Little changes were observed both by flow cytometry (decrease of forward scatter, increase of unprocessed cells staining with APO2.7 antibody) and viability determination by Trypan-blue staining until after 7 hours. However, after 4 hours morphologic changes were observed in the nuclear and cytoplasmic structures using Diff-Quik stained cytospin preparations and standard light microscopic techniques (50% apoptotic cells). The same results were obtained by flow cytometric measurement of sub-diploid DNA content (sub-G1 cells), and an increase of staining with APO2.7 antibody in cells permeabilised by digitonin prior to staining. After 7 hours almost all cells exhibited apoptotic morphology. After 16 hours the cell size (forward scatter) decreased significantly, and 54% of unprocessed cells were APO2.7 positive. After 24 hours only 6% of cells were alive (high forward scatter) and these cells were APO2.7 negative. The HL-60 cells did not proliferate during the cultivation in 3% ethanol, and after 48 hours all stained by Trypan blue. HL-60 leukemic cells were CD34-/AC133-, CD33+/CD15+, and only 2% of the cells were CD95+. Induction of apoptosis by ethanol did not enhance CD95 antigen expression.

• MeSH
• antigen AC133 MeSH
• antigen CD33 MeSH
• antigen Lewis X biosyntéza   MeSH
• antigeny CD34 biosyntéza   MeSH
• antigeny CD95 biosyntéza   MeSH
• antigeny diferenciační myelomonocytární biosyntéza   MeSH
• apoptóza účinky léků   MeSH
• buněčné jádro účinky léků   MeSH
• buněčný cyklus účinky léků   MeSH
• časové faktory MeSH
• CD antigeny biosyntéza   MeSH
• cytoplazma účinky léků   MeSH
• DNA metabolismus   MeSH
• ethanol farmakologie   MeSH
• glykoproteiny biosyntéza   MeSH
• HL-60 buňky MeSH
• kinetika MeSH
• lidé MeSH
• monoklonální protilátky metabolismus   MeSH
• peptidy MeSH
• průtoková cytometrie MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen AC133 MeSH
• antigen CD33 MeSH
• antigen Lewis X MeSH
• antigeny CD34 MeSH
• antigeny CD95 MeSH
• antigeny diferenciační myelomonocytární MeSH
• CD antigeny MeSH
• CD33 protein, human MeSH Prohlížeč
• DNA MeSH
• ethanol MeSH
• glykoproteiny MeSH
• monoklonální protilátky MeSH
• peptidy MeSH
• PROM1 protein, human MeSH Prohlížeč