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[Neural stem cells in the brain].

Stem cells in the central nervous system were usually considered as relevant for evaluation only in embryonic time. Recent advances in molecular cloning and immunological identification of the different cell types prove the presence of neurogenesis of the new neurons in adult mammals brains. New neurons are born in two areas of the mammal and human brain--sybventricular zone and subgranular zone of dentate gyrus. New born granular neurons of dentate gyrus have a great importance for memory and learning. New neurons originate from precursors which in culture and in situ could also transform into astrocytes and oligodendrocytes, thus fulfill criteria of neural stem cells. In culture, mitotic activity of these stem sells depends on fibroblast growth factor 2 and epidermal growth factor. Depletion of cultural medium of these factors and addition of serum, other growth factors (Platelet-derived growth factor and ciliary neurotrophic factor) leads to generation of neurons and astrocytes. Isolation and clonal analysis of stem cells is based on immunological markers such as nestin, beta-tubulin III, some types of membrane glicoproteids. Identification and visualization of stem cells in brain revealed two populations of cells which have properties of stem cells. In embryonic time, radial glia cells could give origin to neurons, in mature brain cells expressing glial fibrillar acidic protein typical marker of astrocytes fulfill criteria for stem cells. Neural stem cells could transform not only into mature neurons and glial cells but also into blood cells, thus revealing broad spectrum of progenitors from different embryonic tissues. Further progress in this field of neurobiology could give prosperity in the cell therapy of many brain diseases.

Animals↗
PubMed2002

The ash2 gene is involved in Drosophila wing development.

The absent small and homeotic (ash2) gene is a member of the trithorax group of positive transcriptional regulators of the homeotic genes. In this work we present evidence that loss-of-function of ash2 results in patterning alterations in the developing wing. Homozygous adults of the weak allele ash2(112411) develop extra cross-veins. However, clonal analysis of a stronger allele, ash2(11), shows that this allele results in reduction of intervein tissue and increase of longitudinal veins and cross-vein tissue in the wing except the region between vein L3 and L4. These results suggest that ash2 function is required for both activation of intervein tissue and repression of vein tissue. Moreover, we have found that cross-vein development can be rescued in the absence of crossveinless-2 when the levels of ash2 were reduced.

Alleles↗
PubMed2002

The T cell receptor repertoire in psoriatic synovitis is restricted and T lymphocytes expressing the same TCR are present in joint and skin lesions.

OBJECTIVE: To investigate the T cell receptor (TCR) repertoire in psoriatic synovitis and to determine whether T lymphocytes in joint and skin lesions show the same Vbeta CDR3 region. METHODS: The expression of Valpha and Vbeta families was evaluated by reverse transcriptase-polymerase chain reaction. The CDR3 region of some Vbeta families was analyzed by cloning and sequencing. RESULTS: We found a diverse variable beta chain usage within psoriatic synovial fluid of 11 patients although some Valpha and Vbeta families were more frequently expressed without evidence of clonality. Analysis of TCR in skin and synovial lesions of 3 patients showed identical CDR3 sequences, indicating that T cells bearing the same TCR are present at the 2 sites of chronic inflammation. CONCLUSION: These data suggest that common or similar crossreactive antigens present in the 2 locations are responsible for the expansion of the same TCR-bearing T cells possibly already activated by a superantigen. This supports the hypothesis that both polyclonal and oligoclonal lymphocyte activation contribute to the initiation and persistence of psoriatic arthritis.

Adolescent↗
PubMed2002

Deregulation of caspase 8 and 10 expression in pediatric tumors and cell lines.

Methylation of the promoter regions of CpG-rich sites in genes is the major mechanism for the silencing of many genes in tumors. Methylation of the key apoptosis-related gene caspase 8 (CASP8) has been reported in some childhood tumors and in neuroendocrine lung tumors. We examined the methylation status of 181 pediatric tumors and found frequent methylation in rhabdomyosarcomas (83%), medulloblastomas (81%), retinoblastomas (59%), and neuroblastomas (52%). Methylation frequencies were low in Wilms' tumors (19%) and absent in hepatoblastomas, acute leukemias, osteosarcomas, Ewing's sarcomas, and ganglioneuromas and in normal tissues. Methylation of CASP8 and the tumor suppressor gene RASSF1A were highly significantly correlated in all tumor types by both the chi(2) and the Fisher's exact tests (P < 0.0001 for both tests). Because the region of the gene examined by us and others is not located in the promoter region and lacks features of a CpG island, we explored the relationship between methylation and gene silencing in detail using 23 pediatric tumor cell lines. Studies included relating the methylation of the region to gene expression at mRNA and protein levels, enzymatic assays of gene function, clonal analysis of PCR amplicons of the region, and exposure to a demethylating agent. These studies indicated that methylation correlated with the loss of gene function in most cases; however, other mechanisms of gene inactivation were present in some cases. Posttranscriptional inactivation of the closely related gene caspase 10 was present in many cell lines. Our results suggest that deregulation of the death receptor pathway to apoptosis is frequent in many types of pediatric tumors and their cell lines.

Azacitidine↗
PubMed2003

Locally born olfactory bulb stem cells proliferate in response to insulin-related factors and require endogenous insulin-like growth factor-I for differentiation into neurons and glia.

After late embryogenesis, new neurons are continuously added to the olfactory bulb (OB) from stem cells located in the forebrain subventricular zone. Nonetheless, stem cells have not been described within the embryonic olfactory bulb. Here we report the isolation of local olfactory bulb stem cells from the embryonic day 12.5-14.5 mouse embryo. These cells were 99.2% nestin positive and proliferated extensively in culture to at least 150 cell doublings. Clonal analysis demonstrated that neurons (TuJ1(+)), astrocytes (GFAP(+)), and oligodendrocytes (O4(+)) could be generated from single-plated cells, indicating that they are multipotent. At least 90% of proliferating cells expressed insulin-like growth factor-I (IGF-I), (pro)insulin, and their cognate receptors; these growth factors collaborated with fibroblast growth factor-2 plus epidermal growth factor (EGF) to promote stem cell proliferation and sphere formation. Cells from Igf-I(-)/- mice, however, proliferated as extensively as did Igf-I(+/+) cells. Differentiation and survival of stem cell-generated neurons and glia showed strong dependence on exogenous IGF-I, but oligodendrocyte differentiation also required insulin at low concentration. Furthermore, the percentages of stem cell-generated neurons, astrocytes, and oligodendrocytes were markedly lower in the cultures prepared from the Igf-I(-)/- mice compared with those of Igf-I(+/+). Concordantly, lack of IGF-I resulted in abnormal formation of the olfactory bulb mitral cell layer and altered radial glia morphology. These results support the presence within the embryonic mouse olfactory bulb of stem cells with specific requirements for insulin-related growth factors for proliferation or differentiation. They demonstrate that IGF-I is an endogenous factor regulating the differentiation of stem and other precursor cells within the olfactory bulb.

Animals↗
PubMed2003

A new role for the cell adhesion molecule L1 in neural precursor cell proliferation, differentiation, and transmitter-specific subtype generation.

Adhesion molecules play important roles in the development and regeneration of the CNS and PNS. We found that the immunoglobulin superfamily recognition molecule L1 influences proliferation and differentiation of neural precursor cells. Substrate-coated L1 reduced proliferation of precursor cells in a dose-dependent manner and increased neuronal and decreased astrocytic differentiation when compared with poly-l-lysine or laminin substrates. Enhancement of neuronal differentiation was more effective if L1 was offered via the cell surface of transfected fibroblasts compared with substrate-coated purified L1. Furthermore, L1 decreased cholinergic-subtype differentiation and accelerated GABAergic differentiation of precursor cell-derived neurons in comparison with poly-l-lysine or laminin. Generation of dopaminergic neurons was not influenced by L1. Experiments with precursor cells generated from L1-deficient mice indicate that L1 acts via heterophilic interaction on proliferation and differentiation of L1-negative precursor cells and via a homophilic or L1 coreceptor-mediated interaction on maturation of precursor cell-derived L1-positive neurons. Clonal analysis revealed that L1 equally inhibits proliferation of monopotential, bipotential, and multipotential precursor cells, but selectively enhances neuronal differentiation of multipotential and bipotential neuron-astrocyte precursors. Our observations support a new role for L1 or L1 ligands in neural precursor cell proliferation and differentiation.

Animals↗
PubMed2003

Stromal cell heterogeneity in fibroblast growth factor-mediated stromal-epithelial cell cross-talk in premalignant prostate tumors.

Homeostasis of normal prostate and two-compartment nonmalignant prostate tumors is dependent on two-way communication between epithelial and stromal compartments. Independence of epithelial cells on controlling instructions from stroma is a hallmark of extremely malignant epithelial cell tumors. To better understand the evolution of stromal independence during malignant progression, we performed a clonal analysis of stromal cells derived from a well-defined model of two-way stromal-epithelial cell communication that loses response to stroma during prostate tumor progression. Directionally specific signaling from stroma to epithelium contributes to homeostasis between the two compartments. Stromal cells were characterized in respect to expression and activity of isotypes of the fibroblast growth factor (FGF) family of ligands and receptors in addition to morphology and cytoskeletal markers. One stromal subtype (DTS1) exhibited a fibroblast-like morphology and did not display smooth muscle cell (SMC) alpha-actin. The other (DTS2) exhibited SMC alpha-actin and an SMC-like morphology in vitro. Both subtypes expressed FGF7 and equally low levels of FGFR2IIIc mRNA, whereas fibroblast growth factor receptor (FGFR) 1 predominated in DTS1 cells. DTS1 cells also expressed FGF10 and no detectable FGFR3, whereas the absence of FGF10 and presence of FGFR3 distinguished DTS2 cells. Epithelial cell-derived FGF9 bound to FGFR and stimulated growth of specifically FGFR3-positive DTS2 cells, not the FGFR3-negative DTS1 cells. These results demonstrate stromal cell heterogeneity in signal reception of FGF from epithelium. This correlated with potential heterogeneity in the response back to epithelial cells. Epithelium-dependent control of a stromal cell phenotype within a tumor may be a determinant of whether tumors remain in nonmalignant homeostasis or progress to malignancy.

Adenocarcinoma↗
PubMed1992

Remission induction of acute promyelocytic leukemia by all-trans-retinoic acid: molecular evidence of restoration of normal hematopoiesis after differentiation and subsequent extinction of leukemic clone.

Sequential molecular and cellular changes during remission induction were investigated in a case of acute promyelocytic leukemia (APL) treated with all-trans-retinoic acid (ATRA). By means of clonality analysis, the assessment of the retinoic acid receptor alpha gene alterations, as well as conventional cytologic studies, it was demonstrated that remission induction of APL by ATRA proceeds in two steps: first, the differentiation of the leukemic clone to mature granulocytes and its subsequent extinction; second, proliferation/differentiation of the residual normal clones to restore polyclonal hematopoiesis.

Adult↗
PubMed1992

T cell repertoire and tolerance after fetal stem cell transplantation.

We studied the T cell repertoire and the mechanism of tolerance in two patients with severe combined immunodeficiency transplanted with HLA mismatched fetal liver stem cells. They are 17 and 5 years old now, healthy, and show normal immunoresponses to recall antigens. Their T cells are of donor origin, whereas monocytes and B cells remained of the host. The NK cells have different sources since in one patient they derive from the donor and in the other one from the host. Despite the HLA mismatch between donor and host cells, no acute or chronic graft-versus-host disease was observed. In vitro experiments with PBMC showed specific nonresponsiveness for the HLA antigens expressed by the host cells. However, an extensive clonal analysis showed that CD4+ and CD8+ host-reactive T cell clones recognizing class II and class I HLA molecules of the host, respectively, were present in the peripheral blood of both patients. Limiting dilution experiments indicated that the frequency of CD8+ host-reactive cells was in the same range as that observed for alloreactive T cells. In contrast, no donor reactive CD8+ T cells could be isolated. Host-reactive CD4+ and CD8+ T cell clones were normal in their capacity to produce IL-2, IFN-gamma, GM-CSF and IL-5, but they failed completely to synthesize IL-4. In addition, CD4+ T cell clones from patient RV secreted very high levels of IL-10. Interestingly, exogenous IL-10 was able to inhibit the proliferative responses of the CD4+ host-reactive T cell clones. Our data demonstrate that host-reactive cells are not deleted from the donor T cell repertoire following allogenic fetal liver stem cell transplantation. Therefore, in vivo tolerance between the host and the donor is maintained by a peripheral autoregulatory mechanism in which cytokines may play a role.

Adolescent↗
PubMed1992

Polyclonal hematopoiesis in interferon-induced cytogenetic remissions of chronic myelogenous leukemia.

Interferon (IFN) therapy of early chronic myelogenous leukemia (CML) frequently produces partial or complete cytogenetic remission of the disease. Patients with complete cytogenetic remission often continue on therapy for several years with bone marrow showing only diploid (normal) metaphases. We studied hematopoiesis in five female patients with major cytogenetic remissions from CML during IFN therapy. Clonality analysis using the BstXI PGK gene polymorphism showed that granulocytes were nonclonal in all patients during cytogenetic remission. BCR region studies showed rearrangement only in the one patient whose remission was incomplete at the time of sampling. Granulopoiesis is nonclonal in IFN-induced remissions of CML and may be derived from normal hematopoietic stem cells.

Clone Cells↗
PubMed1992

Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2.

One model to explain the high frequency of alloreactive T cells proposes that allogeneic MHC molecules are recognized together with host cell-derived peptides. A model system was developed to investigate the relevance of this mechanism by expression of H-2Dd or H-2Ld in 174xCEM.T2 (T2) cells. This human cell line contains a mutation in its Ag-processing pathway that should restrict the association of endogenous peptides with cell surface class I molecules. CTL generated by stimulating C57BL/6 (H-2b) responder cells with H-2Dd or H-2Ld transfectants of the human B cell line C1R or the murine T cell lymphoma EL4 were assayed for their ability to recognize alloantigenic determinants on these transfectants. The major fraction of the H-2Dd-specific allogeneic CTL response, generated in a MLC or under clonal limiting dilution conditions, was composed of T cells that recognized H-2Dd expressed on C1R or EL4 cells, but failed to recognize this molecule on T2 cells. Clonal analysis indicated that approximately one-third of these CTL recognized determinants that were unique to H-2Dd expressed on C1R stimulator cells whereas the remainder recognized determinants that were also found on EL4 transfectants. Less than 10% of H-2Dd-reactive CTL recognized the T2 transfectant, and these clones also killed C1R-Dd and EL4-Dd. This result suggests that the great majority of H-2Dd-specific alloreactive CTL recognize determinants that are formed by a complex of H-2Dd with endogenous peptides that are absent or significantly reduced in T2 cells. Based on recognition of human or murine transfectants, these CTL exhibit some level of specificity for the structure or composition of the bound peptides. Examination of allogeneic CTL specific for H-2Ld revealed populations similar to those described for H-2Dd. In addition, a major new population was present that recognized determinants shared between C1R-Ld and T2-Ld but not present on EL4-Ld. These results are consistent with the idea that the alloreactive response to H-2Ld is also largely dependent on the presence of bound peptide. However, they also may indicate that the H-2Ld molecule expressed on T2 cells is occupied by one or more peptides that are shared with other human, but not murine, cells. The significance of these results to current models of alloreactivity is discussed.

Animals↗
PubMed1992

Novel surface molecules involved in human NK cell activation and triggering of the lytic machinery.

Three new monoclonal antibodies (MAbs) termed 7A6, PP35 and A6/143 were isolated after mouse immunization with CD3- CD16+ NK clones. The screening procedure was based on the ability of MAbs to trigger cytolytic activity of the immunizing clones in a re-directed killing assay against the P815 murine mastocytoma cell line. The 7A6 MAb reacts with 58 kDa surface molecules that appear to belong to the same molecular family defined by the previously described NK-sub-set-specific GL183 and EB6 MAbs. However, unlike from these MAbs, the 7A6 MAb reacted with (and activated) all CD3- NK lymphocytes, independent of their sub-set assignment (based on the expression or lack of expression of EB6, GL183 and CD16). The PP35 MAb reacted with a 70 kDa surface molecule expressed on all CD3- NK cells, as well as on TCR gamma/delta + cells and on a small sub-set of TCR alpha/beta + CD8+ lymphocytes. The PP35 MAb induced activation of essentially all NK cells, although clonal analysis revealed quantitative differences in the magnitude of the cytolytic responses elicited in different clones. Finally, the A6/143 MAb reacted with a molecule of 115 kDa expressed by all human PBL. Similarly to 7A6 and PP35 MAbs, the A6/143 MAb activated all sub-sets of cloned NK cells.

Animals↗
PubMed2004

Aberration of X chromosome in liver neoplasm detected by fluorescence in situ hybridization.

BACKGROUND: A diverse range of cytogenetic alterations of autosomal chromosomes has been reported to date. However, few studies have addressed the abnormalities of X chromosome in hepatocellular carcinoma (HCC) except sporadic reports on the deletion of band F1 in X chromosome, and the clonal analysis of methylation pattern of the X chromosome-linked human androgen receptor gene. Identification of specific X chromosome alterations during the course of neoplastic development would be essential to defining the genetic basis of HCC. Therefore, we studied the regularity of aberration of X chromosome in liver cancer. METHODS: Hepatocarcinoma cellular lines and tumor tissues were detected respectively through DNA probes of X chromosome after fluorescence in situ hybridization (FISH). RESULTS: Increased copies of X chromosome were observed in all samples, and four signals of hybridization were of the major type. CONCLUSIONS: Increased copy number of X chromosome frequently occur in liver cancer. The relationship between copy number of X chromosome and liver cancer genesis needs further investigation. This study is the first of its kind determining the copy number of X chromosome in liver cancer by using FISH.

Adult↗
PubMed2004

[Diagnostics of invasive meningococcal, haemophilus and pneumococcal disease by PCR assay].

OBJECTIVES: Development of extended polymerase chain reaction (PCR) for non-culture detection of Nesseria meningitidis, Haemophilus influenzae and Streptococcus pneumonie from invasive infections. MATERIALS AND METHODS: A method of PCR was optimalised on strains of Nesseria meningitidis, Haemophilus influenzae b and Streptococcus pneumonie. Detection of pathogens was evaluated on 230 samples from patiens with invasive infection. RESULTS: Positive results of PCR were found in 103 samples of 230 (44.7 %). The percentage of positivity was higher in CSF samples (57.0 %) than in serum (33.8 %) or blood (33.3 %) samples. CONCLUSION: PCR method enables etiological diagnostics in cases, where antibiotic treatment was started. PCR results are available earlier than the results of cultivation. Multilocus sequence typing (MLST) of PCR products enables clonal analysis of etiological agents even in cases with negative results of cultivation.

DNA, Bacterial↗
PubMed2005

[Cytogenetic aberrations of esthesioneuroblastoma studied by comparative genomic hybridization].

OBJECTIVE: To characterize the cytogenetic alterations of esthesioneuroblastoma (ENB). METHODS: Comparative genomic hybridization (CGH) was performed on genomic DNA extracted from 12 patients with primary ENB, 4 patients with tumor recurrence and 7 with metastasis. Equal amounts of biotin-labeled tumor DNA and digoxigenin-labeled normal reference DNA were hybridized to normal meta phase chromosomes. Tumor DNA was visualized by fluorescein (FITC) and normal DNA by rhodamin (TRITC ) and detected by fluorescence microscopy. The signal intensities of the different fluorochromes were quantitated as gray levels along the single chromosomes. The over-and under-represented DNA segments were determined by computation of FITC/TRITC ratio images and average ratio profiles. RESULTS: Consensus deletion regions were most frequently observed on chromosomes 1p, 2q, 3p/q, 4p/q, 5p/q, 6q, 8p/q, 9p, 10p/q, 11p, 12q, 13q, 18q, and 21q. DNA over-representations were identified on chromosomes 1p, 7q, 9q, 11q, 14q, 16p/q, 17p/q, 19p/q, 20p/q and 22p/q. The genetic pattern of ENB was distinct from that of other small round-cell tumor types and neuroblastomas. The deletion on chromosome band 1p21-p31 was associated with bad prognosis. In particular, all patients died whose tumors had combined 1p21-p31 deletion, with tumors in clinical stage C or D, and of low differentiation (grade III or IV). Clonality analysis revealed a high concordance between pairs of primaries and metastases. CONCLUSION: CGH analysis identifies characteristic cytogenetic aberrations of esthesioneuroblastoma associated with its malignant phenotype.

Adolescent↗
PubMed2004

[Experimental study on biological character changes of human epidermal cells during proliferation culture in vitro].

UNLABELLED: [Abstract] OBJECTIVE: The study was (1) to investigate the biological character changes of human epidermal cells during proliferation culture in vitro and (2) to provide data for construction of engineered skin. METHODS: The foreskin was collected from 20 healthy children. The epidermal cells were isolated with digestion of the foreskin and cultured in vitro. Growth curve was obtained from the data of cell counting. Cell growth kinetics was observed. Meanwhile, clonal analysis and cell size measurement was performed. The rate of keratin 19 (K19) and involucrin expression-positive cells was counted by flow cytometer. Expression of K19 and involucrin mRNA was detected by RT-PCR. RESULTS: When passaged to P6, the keratinocytes from child foreskin could be expanded by (700 +/- 37) times. Flow cytometer results showed that the percentage of K19 expression-positive cells decreased from (66.97 +/- 3.14)% to (4.65 +/- 1.38)% while the percentage of involucrin expression-positive cells increased from (11.65 +/- 1.62)% to (97.03 +/- 2.66)% at P0 and P6 respectively. RT-PCR results showed that expression of K19 mRNA decreased from P0 to P6 while involucrin mRNA kept stable with passage in vitro. CONCLUSIONS: Human epidermal cells of passage 5 maintain proliferation phenotype, which are suitable for skin tissue engineering. Decrease of proliferation phenotype content is partially responsible for the proliferation capacity loss of in vitro cultured epidermal cells.

Cell Proliferation↗
PubMed1992

Astrocyte precursors in neonatal rat spinal cord cultures.

Cultures of newborn rat spinal cord contain multiple types of astrocytes. By using a combination of cultures enriched for glial precursors and clonal analysis, we have identified a particular astrocyte precursor that gives rise to morphologically distinct classes of astrocytes. This astrocyte precursor labels with the monoclonal antibody A2B5, is highly migratory, proliferates in response to serum and platelet-derived growth factor, and differentiates into process-bearing astrocytes, many of which subsequently assume a "pancake"-shaped morphology. A2B5+ astrocyte precursors share antigenic and migratory characteristics with previously described O2A progenitor cells but differ in their response to regulatory factors, including serum and coculture with type 1 astrocytes. More importantly, these astrocyte precursors do not give rise to oligodendrocytes. In their proliferative response to serum and their capacity to differentiate into astrocytes, these glial precursors resemble type 1 astrocyte precursors from optic nerve. However, unlike type 1 astrocyte precursors, these cells are A2B5+, highly migratory, and do not give rise to fibroblast-like astrocytes. Neonatal rat spinal cord cultures contain approximately twice the number of the A2B5+ astrocyte precursors than O2A progenitor cells. By contrast, the majority of A2B5+ cells in postnatal day 7 optic nerve cultures are O2A progenitors. The presence of large numbers of A2B5+ astrocyte precursors in rat spinal cord cultures may reflect the more complex cytoarchitecture of the spinal cord compared to the optic nerve.

Animals↗
PubMed2005

Post-transplantation lymphoproliferative disorder of recipient origin in a boy with acute T-cell leukemia with detection of B-cell clonality 3 months before stem cell transplantation.

Post-transplantation lymphoproliferative disorder is an infrequent complication after hematopoietic stem cell transplantation. It is hypothesized that lack of T-cell surveillance following transplantation permits reactivation of latent EBV leading to polyclonal B-cell expansion and finally outgrowth of a predominant clone. Most cases are of donor origin. Here, we describe an 8-year old boy with early onset post-transplantation lymphoproliferative disorder following matched-unrelated stem cell transplantation for high-risk T-cell leukemia whose disease was unusual for two reasons. First, his B-cell clone was of host origin and, in contrast to the few PTLD of host origin described so far, not associated with autologous reconstitution. Secondly, using clonal analysis, we could retrospectively show that the B-cell clone emerged during consolidation chemotherapy for T-cell leukemia, 3 months before stem cell transplantation.

Antibodies, Monoclonal↗