Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “clonal analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources
PubMed2002

Clonality analysis of follicular lymphoma using laser capture microdissection method.

Whether a common and a single clone present, or not, among follicles of follicular lymphoma (FL) was examined in 12 cases with FL. Histologic grade was I in 6 cases, II in 3, and III in 3. DNA was selectively extracted from the neoplastic follicles of paraffin-embedded samples with use of laser capture microdissection method, and used for PCR-based analysis of rearrangement of immunoglobulin heavy chain variable region gene. Three different follicles in each case of FL were microdissected. Semi-nested PCR was performed using two sets of primers (Fr2A and Fr3A). In PCR with Fr2A primers, nine of 12 cases showed a common band among neoplastic follicles. The remaining three cases showed no PCR products. With Fr3A primers, eight of 12 cases showed a common band among follicles of the same case. The other four cases showed oligoclonal bands, among them presence of a common band was difficult to assess. Oligoclonal bands were more frequently observed in PCR with Fr3A than that with Fr2A and in grade I or II than in grade III cases. In total, 11 of 12 cases showed a common band in PCR with either Fr2A or Fr3A primers. In two cases, DNA extracted from whole section was amplified with both Fr2A and Fr3A or only Fr3A primers, showing smear or oligoclonal bands. These results showed the presence of a single clone of cells in neoplastic follicles of FL and the usefulness of PCR-based rearrangement analysis of immunoglobulin heavy chain gene combined with microdissection methods for differential diagnosis of FL from follicular hyperplasia.

Adult↗
PubMed1992

A clonal analysis of lung T cells derived by bronchoalveolar lavage of healthy individuals.

The characteristics of the T-cell population in the healthy human lung have been investigated by analysing the properties of T-cell clones derived from bronchoalveolar lavage (BAL) samples and comparing them with T cells cloned from the blood of the same individuals. The proportions of CD4+ and CD8+ T cells in the starting populations from BAL and blood were similar although only 14% of BAL T cells were CD45RA+ compared to 70% of blood T cells. The precursor frequency of T-cell clones derived from BAL was less than from blood. The cytokine profiles [after phytohaemagglutinin (PHA) stimulation] of the clones derived from both sources were markedly different and these differences lay in the CD4+ population. BAL-derived CD4+ clones produced interferon-gamma (IFN-gamma) more frequently than did those from blood while blood-derived clones were more likely to produce interleukin-2 (IL-2) than those from BAL. IL-4 was produced by the majority of BAL- or blood-derived clones (93% and 88% respectively) either along with IFN-gamma (BAL) or IL-2 (blood). The cytokine profiles of BAL-derived T-cell clones are consistent with those derived from lung interstitium and suggest that the BAL T-cell populations reflect those in the lung wall. Whether the unique properties of lung T cells are acquired after leaving the blood or whether there is selective entry of T-cell subpopulations into the lung remains to be determined.

Adult↗
PubMed1992

Clonal analysis of bcr-abl rearrangement in T lymphocytes from patients with chronic myelogenous leukemia.

The cytogenetic hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome (Ph1), which reflects a chromosomal translocation t(9;22) and a rearrangement of the ABL and bcr genes. This marker is found in all cells arising from the same malignant precursor cell and can be detected in CML cells of the myeloid, monocytic, erythroid, and B-lymphocyte lineage. It is, however, controversial as to whether T lymphocytes of CML patients carry this gene rearrangement. An answer to this question would clarify whether the translocation in CML occurs in a pluripotent hematopoietic stem cell or in a precursor cell already committed to certain lineages, but not the T-cell lineage. To address this question, we established T-cell clones from peripheral venous blood cells of four patients with CML and screened these clones for bcr-abl fusion transcripts by means of polymerase chain reaction and Southern blot analysis. In four T-cell clones of three of these patients, the bcr-abl transcript could be detected. None of 12 T-cell clones of the fourth patient disclosed detectable bcr-abl amplification product. Both CD4+ as well as CD8+ clones displayed fused bcr-abl sequences. These data imply that in CML some but not all T lymphocytes may originate from the Ph1-positive stem cell.

Blotting, Southern↗
PubMed1992

Identification and characterization of osteoclast progenitors by clonal analysis of hematopoietic cells.

We have identified a distinct population of colony-forming cells that give rise to mononuclear cells expressing an enzyme marker and other features of the osteoclast in bone marrow cultures stimulated by conditioned medium of a murine tumor cell line. These colony-forming cells were defined as osteoclast colony-forming units (CFU-O). The tumor cell-derived activity was recently isolated and was named osteoclast colony-stimulating factor (O-CSF). To understand the development of osteoclast progenitors and to clarify the relationship of osteoclast progenitors to other hematopoietic progenitors, we examined CFU-O in hematopoietic tissues obtained from normal adult mice, mouse fetuses, and mice with 5-fluorouracil (5FU) treatment. CFU-O were present in the adult mouse bone marrow, adherent cell-depleted marrow, in the spleen, and in the day 14 fetal liver, with an incidence similar to other hematopoietic progenitors. The culture period required for the development of CFU-O-derived colonies in vitro and the manner in which CFU-O responded to 5FU suggested that CFU-O belonged to a relatively primitive progenitor population; they are clearly more immature than macrophage progenitors that respond to macrophage-CSF, but more mature than multilineage progenitors that respond to stem cell factor. Our studies have defined and characterized an osteoclast progenitor and distinguished it from other hematopoietic progenitors for the first time.

Adenocarcinoma↗
PubMedDate not supplied

[Clonal analysis of some multiple antibiotic resistant E. coli strains isolated from river and polluted waters].

Several multiple antibiotic resistant E. coli strains isolated from river and polluted waters were compared for their genetic relatedness. Antibiotic susceptibility testing was performed for gentamycin, kanamycin, ampicillin, tetracycline, chloramphenicol, ceftazidime and cefotaxime, as described by Kirby-Bauer disk diffusion method following NCCLS recommendations. Minimum inhibitory concentrations (MICs) were performed using dilution method in Mueller-Hinton broth with a 0.06-64 micrograms/ml concentration range for all antimicrobials and bacterial inoculum of about 1-2 x 10(8) CFU/ml. For the data analysis NCCLS breakpoints for resistance and sensitivity were used. Genomic DNA was isolated from E. coli strains by CTAB method and digested to completion with HindIII enzyme. Genetic characterization was performed by agarose gel electrophoresis and spectrophotometric analysis. Genetic similarity and clustering were calculated using NISIS program. All E. coli strains isolated from river and polluted waters show a high incidence of multiple antibiotic resistance phenotype, 16% of them being resistant to 7, 6 and 4 antibiotics, 40% to 5 and 8% to 2 antibiotics, respectively. A moderate resistance was observed to kanamycin (higher than 30%) and cefotaxime (68%). The percentage of resistant E. coli strains ranged from 76% (to ampicillin, gentamicin and chloramphenicol) to 85% (to ceftazidime). The best results (resistance about 99%) were obtained with tetracycline. Screening for plasmids relieved the presence into 4 E. coli strains of several plasmids ranging from 3.8 kpb to more than 50 kpb. The number of fragments produced by HindIII digestion of genomic DNA ranged from 11 to 25, with sizes of approximately 22 to more than 750 kb. The phenotypic data shows the dynamic flow of multiple antibioresistant E. coli strains in aquatic media (river and polluted waters). Electrophoretic patterns analysis reflects the incidence and diversity of analyzed plasmids. DNA fingerprinting with genomic DNA RE suggested that, depending of the isolation source, E. coli strains could be grouped in two distinct populations with a different plasmid diversity.

Aminoglycosides↗
PubMed1992

Clonal analysis of myelodysplastic syndrome: monosomy 7 is expressed in the myeloid lineage, but not in the lymphoid lineage as detected by fluorescent in situ hybridization.

Conflicting results have been published on whether or not myelodysplastic syndromes (MDS) affect all cell lineages. Involvement of myeloid and erythroid cell lineages has been regularly observed, but it remains controversial whether the different lymphoid cell lineages are involved. In this study of eight patients with MDS associated with monosomy 7, fluorescent in situ hybridization (FISH) was used to enumerate the chromosomes 7 in interphase cells. With the probe D7Z1, the rate of false-positive detection of monosomy 7 was 3% +/- 2% in normal cells. T- and B-cell lines were established from eight patients with MDS and monosomy 7. As determined by FISH in interphase cells, 1.9% (0% to 3%) of the cells in the B-cell lines showed one fluorescent spot and 1.1% (0% to 2.9%) of the cells in the T-cell lines. These values do not differ from normal values. However, the possibility that normal cells were selected when the T- and B-cell lines were established could not be excluded. Therefore, peripheral blood cells were obtained, separated according to surface markers specific for lymphoid and myeloid cell lineage with a cell sorter, and analyzed for the expression of monosomy 7 by FISH. Antibodies recognizing T cells (CD3), B cells (CD20), natural killer (NK) cells (CD57), monocytes and granulocytes (low and high expression of CD11b antigen), and myeloid progenitors (CD33) were used to separate cells. The expression of monosomy 7 in the T cells, NK cells, and B cells did not differ from control values. These results in the lymphoid subpopulations are in stark contrast with the observations in the myeloid populations; the percentage of cells with monosomy 7 ranged from 9% to 78% (controls: 6% +/- 2%) in cells with low CD11b expression, 20% to 89% in cells with a high expression of the CD11b antigen (controls: 7% +/- 3%), and 23% to 91% in the CD33 positive cells (controls: 5% +/- 3%). The results of this study suggest that monosomy 7 does not usually affect lymphoid subpopulations but is restricted to committed progenitor cells with the capacity to differentiate into mature myeloid cells.

Adolescent↗
PubMed1991

Clonal analysis of the peripheral T cell compartment of the SCID-hu mouse.

Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.

Animals↗
PubMed1990

Clonal analysis of gene expression by PCR.

A method for determining the heterogeneity of gene expression from isolated macrophage colonies is described. Clones derived using specific growth factors were isolated by soft agar cloning and used as sources of mRNA for analysis. Gene expression was monitored by the reverse transcription polymerase chain reaction method to permit a number of specific genes to be simultaneously amplified. The technique described here involves the use of internal primers in addition to the usual two external primers typically used in gene amplification. Such internal primers greatly increased the accuracy and precision of amplification by adding an extra constraint such that the gene of interest was reliably amplified while decreasing background noise. This method permits gene expression to be monitored in cell colonies, thereby allowing the extent of phenotypic heterogeneity in developing macrophages (or other cell types) to be determined. This technique may also be useful in gene expression studies involving other cell types under the influence of specific growth factors.

Animals↗
PubMed1991

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.

Antigens, CD↗
PubMed1991

Clonal analysis of T-cell deficiencies in autotransplant recipients.

We have used limiting dilution culture methods to determine the frequency of mitogen-responsive T cells in peripheral blood of patients after bone marrow autotransplantation, and have compared their responsiveness to that of allotransplant recipients and normal controls. Autotransplant patients were found to have low responder cell frequencies in tests for lymphokine-secreting helper function, and for IL-2 dependent proliferator and cytotoxic function. Multiple regression analysis showed that function was lower in autotransplant patients than in allorecipients, and lower in male patients for all three functional assays. Patients with clinically significant infection tended to have lower proliferative function in both transplant groups and lower cytotoxic function in the allotransplant population. Graft-versus-host disease was associated with lower T-cell function, but was present only in the allotransplant group; therefore, it cannot account for the even lower levels of function observed in the autotransplant population. Because we observe deficits in T-cell regeneration in autotransplant recipients that are even more severe than in allorecipients, we postulate that cellular immunodeficiency after bone marrow transplantation may reflect limitations in thymic-dependent repopulation rather than an effect of genetic disparity between host and donor (eg, clinical or subclinical graft-versus-host).

Adult↗
PubMed1990

[Clonal analysis of karyotypic instability and metastatic potential in tumor cell populations].

Rat organospecific transplantable RA-2 rhabdomyosarcoma strains RA-2H and RA-2L were investigated using single cell cloning technique for "Micronuclear containing cell frequency" (MCF) and "Metastatic potential" (MP). The RA-2H was obtained from RA-2 during selection for increasing metastatic potential, whereas the RA-2L was the result of selection in opposite direction. MP was assessed by lung colony formation technique and in the case of RA-2H, it was one hundred times greater than in the case of RA-2L (2490 +/- 280 and 22 +/- 11 lung colonies i.v. after injection of 10 cells, respectively). Average MCE for RA-2H was 3 and for RA-2L 5.3%. Also, significant differences were observed between these substrains in percentage of anaphase and telophase cells with bridges and fragments (24 +/- 4 for RA-2H and 56 +/- 5% for RA-2L, P less than 0.05). The selection of RA-2H clones with high level of MCF was successful. In the population obtained MCF was increased up to 7.8% and MP decreased (79 +/- 44). So, the selection for decreasing of MP led to significant increase in genome instability characteristics and the selection for increasing of MCF--to the abrupt decline of MP, which proved the existence of negative correlation between these two characters in populations studied. It is concluded that correlation between malignancy and genome instability in tumor populations with different levels of malignancy is not, as a rule, positive.

Animals↗
PubMed1990

Clonal analysis of human meningiomas and schwannomas.

Meningiomas and schwannomas are two of the most common tumors of the human nervous system. To determine whether these tumors arise from a single cell or from multiple cells, we used molecular genetic techniques to study X chromosome inactivation in meningiomas and schwannomas isolated from females including one who had neurofibromatosis type 2. The tumors were also screened for loss of heterozygosity at several loci on chromosome 22 using polymorphic DNA markers. Among nine meningiomas, at least three of which showed loss of alleles on chromosome 22 and five of which retained heterozygosity for the chromosome 22 alleles examined, all nine tumors were monoclonal. Among eight schwannomas, at least seven of which retained heterozygosity for chromosome 22 loci, seven were monoclonal. We conclude that human meningiomas and schwannomas arise from a single cell.

Chromosomes, Human, Pair 22↗
PubMed1990

Clonal analysis of infiltrating T lymphocytes in liver tissue in viral hepatitis A.

The pathogenic mechanism leading to liver tissue injury in hepatitis caused by hepatitis A virus is unclear. We have randomly established T-cell clones from liver biopsies from four patients with hepatitis A. A total of 578 clones was phenotypically analysed. During the acute phase of the disease CD8+ clones dominated over CD4+ clones, whereas in a biopsy taken late after onset of clinical syndromes more CD4+ than CD8+ clones were obtained. Interestingly, in a patient with a second exacerbation of the disease, more than 20% of all clones had the CD3+ WT31- CD4- CD8- 'NK-like' phenotype. All CD8+ clones had cytotoxic activity and approximately 50% of all CD8+ clones showed specific cytotoxicity against autologous fibroblasts infected with hepatitis A virus. The CD8+ cells also produced IFN-gamma in response to these target cells. Variable IFN-gamma production was observed with all types of T-cell clones. These results suggest that the liver injury in hepatitis A is not caused by a viral cytopathogenic effect but is due to an immunopathological reaction of sensitized cytotoxic T lymphocytes against infected hepatocytes. In addition, these studies show an enrichment of CD4-8-T-cell receptor alpha beta-chain-negative T lymphocytes at the site of an inflammation and suggest a role of these cells in an anti-viral reaction.

Acute Disease↗