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PubMed2004

Establishment and characterization of a cytogenetically complex Chinese multiple myeloma-derived cell line with homozygous p53 deletion and cyclin E overexpression.

We describe the establishment and characterization of a new myeloma-derived cell line (MM17), originating from the sacral plasmacytoma of a 54-year-old Chinese woman diagnosed with multiple myeloma (MM). MM17 was confirmed morphologically and immunophenotypically to be clonal plasma cells positive for CD38 and CD138 and negative for EBV marker. Authenticity was confirmed using comparative genomic hybridization and DNA fingerprinting studies on bone marrow aspirate, sacral tumor tissue and MM17. Combined G-banding and multicolor fluorescence in situ hybridization analyses demonstrated a primarily hypodiploid karyotype with two sidelines sharing common stemline aberrations: +6, -7, -10, -13, -14, -17, -X, der(1;17)(q10;q10), t(2;7)(q23;q11.2), t(8;14)(q24;q32) and ins(16;1)(q13;?q22q41); and a number of hypertriploid cells. The involvement of p53 alteration and cyclin E overexpression, both with relevance to the induction of chromosomal instability, was investigated in MM17 and together with two other MM derived cell lines (U266 and IM-9) for cyclin E expression. Homozygous deletion of p53 gene hitherto not reported in MM, was detected. Both MM17 and U266 with complex cytogenetic aberrations demonstrated overexpression of cyclin E1 and E2, whereas IM-9 with a normal karyotype showed cyclin E2 but not E1 overexpression. These data suggested that E1 but not E2 overexpression was associated with chromosomal abnormalities observed in MM17 and U266, which provides the first supporting evidence for the link of cyclin E and chromosomal instability in MM. This is the first characterized Chinese MM-derived cell line with homozygous p53 deletion which may serve as a valuable in vitro system for studying MM pathogenesis particularly for Chinese.

Cell Line, Tumor↗
PubMedDate not supplied

Tracking lymphocytes in vivo.

The ability to track antigen (Ag)-specific lymphocyte populations in vivo has greatly increased our understanding of the location and functional status of these cells throughout the course of an immune response. Recent technical advances have enhanced researchers' capability to follow migration, activation and cellular interactions of Ag-specific lymphocytes in situ. It is now possible to monitor changes in T cell subsets, co-stimulatory molecules, and chemokine expression within the physiological context of secondary lymphoid organs. Furthermore, the Ag-presenting cell-T cell interaction can be studied,thus dissecting the role and timing of Ag presentation of particular dendritic cell subsets in the initiation of the immune response. The capacity to adoptively transfer small populations of Ag-specific T lymphocytes has also increased our knowledge of the physiologically important role of regulatory T cells in autoimmunity and immunosuppression. New fluorescence imaging techniques such as multicolor video microscopy, laser scanning cytometry, and multiphoton tissue imaging have provided new ways in which researchers can track cellular changes within Ag-specific lymphocytes in vivo. This review summarizes some of the ways in which these techniques have led to discoveries in the role of signaling cascades, cell cycle progression, and apoptosis in maintaining an Ag-specific immune response.

Animals↗
PubMed2004

Remote sensing as a tool for mapping mosquito breeding habitats and associated health risk to assist control efforts and development plans: a case study in Wadi El Natroun, Egypt.

Limited mosquito ground surveys were combined with remote sensing and GIS technologies to identify mosquito breeding habitats in Natroun lakes area and to delineate associated health risks. Mosquito larval surveys were carried out in a small area to characterize positive breeding habitats and determine their geographic coordinates. Mosquitoes (Anopheles multicolor, Culex antennatus and Cx. theileri) were found breeding in water-flooded habitats with dense vegetation cover spatially associated to existing lakes. Chemical analysis indicated that mosquito breeding water was found to be polluted by several sources including agriculture, industrial and domestic sources. This information served as a training set to characterize the spectral signature of mosquitogenic (mosquito-producing) habitats using reflectance data of the Thematic Mapper (TM) sensor aboard Landsat 5 satellite. Following characterization of the spectral signature, satellite data were used to predict, potential mosquito breeding patches over the whole study area. Field surveys were then carried out to assess the accuracy of predicted habitats and those surveys have indicated that all checked sites were positive for mosquito larvae demonstrating an accuracy of 100%. Based on an average adult mosquito flight range of 2 km, GIS was used to create buffer zones around breeding habitats describing areas at risk from mosquito nuisance and disease transmission. The obtained results could thus provide a new basis for directing the control of mosquito vectors as they provide health authorities with precise maps of mosquito breeding habitats in a timely manner. Moreover, the generated map delineating risk areas could be used by project developers to either re-site the project or invest in mosquito control activities in order to avoid health risks and ensure sustainability of their development. The approach adopted in this investigation demonstrated the practical and successful application of remote sensing and GIS in assisting health and development decision making.

Animals↗
PubMed1992

Thymic shared antigen-1. A novel thymocyte marker discriminating immature from mature thymocyte subsets.

In a previous study, we raised a mAb (MTS 35) reacting with a plasma membrane Ag expressed on both cortical thymocytes and a subset of thymic medullary epithelial cells. In view of the shared expression of this molecule, we have defined it as thymic shared Ag-1 (TSA-1). Considering its selective reactivity with cortical, but not medullary thymocytes, the relevance of TSA-1 as a marker of immature T cells was investigated in detail in this study, using multicolor flow cytometric analysis. TSA-1 was found on all immature thymocyte subsets (CD3-4-8-, CD3-4+8-, CD3-4-8+, CD3-4+8+, CD3low4+8+). Conversely, CD3high4+8- and CD3high4-8+ thymocytes, early thymic migrants and peripheral T cells were TSA-1-. More refined gating and analysis of the transitional CD3intermediate/high4+8+ thymocytes, proposed candidates for negative selection, demonstrated that approximately one half were TSA-1-. In fact, there was a directly inverse relationship between TSA-1 and CD3 expression on thymocytes. In the periphery, TSA-1 was detected on B lymphocytes. TSA-1 is PI-linked and has a molecular mass of 17 kDa nonreduced, or 12 to 13 kDa reduced. Through cross-correlation analysis, this molecule was distinct from H-2K, PNA-R, CD5, CD11a/18, Thy-1, HSA, Ly6A/E, Ly6C, ThB, CD25, CD44. Hence TSA-1 appears to be a unique marker which exquisitely separates mature from immature thymocytes.

Animals↗
PubMed2004

Cytogenetic characterization of seven human cancer cell lines by combining G- and R-banding, M-FISH, CGH and chromosome- and locus-specific FISH.

Leukemia cell lines K562, KG1a, U937, HL60, Jurkat and solid tumor cell lines A549 and M4Beu are widely used in studies of cell cycle, apoptosis and adhesion mechanisms in cancer cells. Although the K562 and U937 cell lines were previously subjected to a detailed cytogenetic characterization, only a few molecular cytogenetic investigations have been performed on the other five cell lines. We combined several molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH), multicolor FISH (M-FISH), and comparative genomic hybridization (CGH) to demonstrate the precise genetic aberrations in tumor genomes of these seven cell lines. This information may be useful for multiple studies on these cell lines, providing a genetic basis for the interpretations of experimental findings.

Cell Line, Tumor↗
PubMedDate not supplied

[Molecular cytogenetic techniques and their application in clinical diagnosis].

Cytogenetics analysis is at present the basic element of the diagnostic process of genetic disorders which are caused by chromosomal abnormalities. Since the chromosome banding technique has been introduced in the 1970s, it has been available as a diagnostic tool of a number of clinical syndromes. It enabled to prove the causal association between specific chromosomal abnormalities and clinical features observed in patients. However, since banding resolution is not always sufficient for the identification of chromosomal abnormalities, additional techniques for solving diagnostic dilemma of classical cytogenetics are needed. A new field of cytogenetics -- molecular cytogenetics, the product of a combination of cytogenetics and molecular biology, has increased the resolution and diagnostic utility of cytogenetic analysis. The basic method of molecular cytogenetics is fluorescence in situ hybridization (FISH). It enables a specific detection of unique sequences, chromosomal regions or entire chromosomes in metaphase, interphase cells or in tissue sections. In this article FISH technique and its modifications such as multicolor FISH (M-FISH, SKY, CCK), Primed In Situ Labelling (PRINS) and Comparative Genomic Hybridization (CGH) are presented. The range of applications and the use of these techniques for the identification of chromosomal abnormalities in relation to diagnostic possibilities of the classical methods of karyotyping is also discussed.

Chromosome Aberrations↗
PubMed2004

Clinical significance of soluble p53 protein in B-cell chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: p53 status and CD38 antigen are biological factors influencing response to therapy and clinical course in B-cell chronic lymphocytic leukemia (B-CLL). This study tests the hypothesis that soluble p53 alone and in association with CD38 can enucleate B-CLL subsets at worse prognosis. DESIGN AND METHODS: Wild and mutant forms of p53 protein were evaluated in 197 B-CLL patients at diagnosis or before progression by an immunoenzymatic method in plasma using an anti-p53 monoclonal antibody. CD38 expression was analyzed by a multicolor flow cytometric assay. RESULTS: Higher levels of both soluble p53 (sp53) and CD38 were significantly correlated with intermediate and high Rai stages, with higher beta2-microglobulin and soluble CD23 values, determined at diagnosis. Shorter overall survival (OS) and progression-free survival (PFS) were both observed in sp53+ and CD38+ patients (p<0.0001). Simultaneous positivity or negativity for sp53 and CD38 identified two subsets of patients, the former with a worse prognosis and the latter with a better prognosis with regard to PFS (p<0.0001) and OS (p<0.0001). The predictive value of sp53 and CD38 was retained among the patients within the intermediate Rai risk group. INTERPRETATION AND CONCLUSIONS: sp53 and CD38 together with ZAP-70 were confirmed to be independent prognostic factors in multivariate analysis. With regard to PFS, ZAP-70, sp53 and CD38 were confirmed to be independent prognostic factors. Concerning OS, ZAP-70, CD38 and age (< or > 60 years) were independent prognostic factors whereas sp53 showed only a tendency towards statistical significance.

ADP-ribosyl Cyclase 1↗
PubMed2005

Advanced molecular and cytogenetic technologies in birth defect diagnosis and prevention.

Fluorescence in situ hybridization (FISH) has become an important diagnostic tool as an adjunct to classical cytogenetics. FISH utilizes DNA probes comprised of specific nucleic acid sequences tagged with fluorescent molecules to identify the number and location of specific DNA sequences in human cells. These probes can be used to determine various numerical and structural chromosomal aberrations, in many cases, gene dosage and/or structure alterations. Chromosomal abnormalities are responsible for a considerable number of birth defects, and more than 50% of spontaneous abortions. These numbers have been significantly higher since the advent of FISH technology that allows the detection of submicroscopic chromosome alterations. The clinic application of FISH technology in postnatal, prenatal, and preimplantation diagnoses has been playing an important role in the diagnosis and prevention of birth defects. As new technologies evolve, more and more new FISH techniques-such as subtelomeric FISH, multicolor FISH (M-FISH), comparative genomic hybridization (CGH), and microarray-are used in clinical diagnoses, the role of FISH technology in both research and clinical aspects of birth defects will surely continue to expand.

Chromosome Aberrations↗
PubMed2005

[Peripheral blood T cell immuno-tolerance in PBSCT donors induced by rhG-CSF in vivo].

The study was aimed to investigate the mechanism of T cell tolerance in human peripheral blood induced by rhG-CSF in vivo. Dendritic cell (DC) subsets, CD8(+)CD28(-) T suppressor cells and the expression of CD28 on T cells of peripheral blood before and after mobilization were analyzed by multicolor flow cytometry. The results showed that after mobilization by rhG-CSF in vivo, the relative counts of CD3(+)CD28(+) cells increased significantly (P < 0.01), and so did the CD8(+)CD28(+) cells (P < 0.01). The mean fluorescence intensity of CD28 expression on CD3(+) cells decreased greatly (P < 0.05), but there were no significant changes of the relative fluorescence intensity of CD28 overall expression on T cells (P > 0.05). The percentages of DC2 before mobilization were significantly lower as compared with normal bone marrow (P < 0.01). After using rhG-CSF, the DC2 count was significantly higher in the apheresis graft than in peripheral blood and bone marrow before mobilization (P < 0.01), while the DC1:DC2 ratios were lower (P < 0.01) and there was no significant difference of DC1 before and after mobilization (P > 0.05). The percentages of CD8(+)CD28(-) T suppressor cells increased significantly also after mobilization (P < 0.05). It is concluded that the higher numbers of DC2 and CD8(+)CD28(-) T suppressor cells in peripheral blood grafts may contribute to the ability of tolerance in peripheral blood T cells induced by rhG-CSF in vivo.

Adolescent↗
PubMed2005

Atypical lipomatous tumor with rare structural rearrangements involving chromosomes 8 and 12.

Atypical lipomatous tumor (ALT), an intermediate malignant neoplasm of soft tissues, is characterized by the presence of supernumerary ring and giant marker chromosomes. These supernumerary chromosomes consistently contain amplified 12q-material in association with amplified segments from a variety of other chromosomes. However, a few cases of ALT with other types of chromosomal rearrangements have been reported earlier. We report on new types of structural aberrations in a case of ALT. In a pseudodiploid karyotype, there were two aberrant chromosomes, both consisting of alternating chromosome 8 and 12 sequences as shown by multicolor fluorescence in situ hybridization (FISH). The complex rearrangement was not only the result of multiple breaks and reunions of these chromosomes, but was also associated with a gain of chromosome 12 sequences. FISH analyses revealed that the number of MDM2 signals was slightly elevated (median, 5). There were three intact copies of HMGA2 and one additional copy of the 5' part of the gene. These findings are consistent with previous reports that the ALT phenotype may be associated with a low or moderate level of gene amplification, whereas truncation of HMGA2 has been observed in both ALTs and benign lipomas. The aberrations in the present case were stable, although rare cells with higher MDM2 copy numbers were detected. Whether ALTs with these types of aberrations have a lower risk of tumor progression than ALTs with the notoriously mitotically unstable ring and giant marker chromosomes remains to be investigated.

Biopsy↗
PubMed2004

Larval habitats of main malaria vectors in Hormozgan province and their susceptibility to different larvicides.

Hormozgan Province is located in the south of Iran bordering the Persian Gulf. In this area, malaria is still considered a major health problem. In the province, eleven species of Anopheles were identified: An. pulcherrimus, An. sergenti, An. apoci, An. multicolor, An. subpictus, An. turkhudi, An. fluviatilis, An. stephensi, An. d'thali, An. superpictus, and An. culicifacies, among which only the last five species play an important role in malaria transmission. Larval habitats of malaria vectors varied from clean to brackish water, as well as sewage water, under palm trees, marshland, pools, pounds, drainage, irrigation canals, wells, and water containers. Malathion, fenitrothion, chlorpyrifos, and temephos were tested against An. stephensi, An. fluviatilis and An. d' thali larvae at diagnostic doses recommended by WHO. The results showed susceptibility to malathion, chlorpyrifos and temephos, but fenitrothion was not able to yield 100% mortality.

Animals↗
PubMed2005

[Molecular cytogenetic analysis for a familial complex chromosomal rearrangement].

OBJECTIVE: To determine a complex chromosomal rearrangement by advanced molecular cytogenetic techniques and analyze its clinical effect. METHODS: A complex chromosomal rearrangement (CCR) involved in chromosomes 5, 16 and 20 in a 29-year-old male carrier was determined by chromosomal microdissection and multicolor fluorescence in situ hybridization (M-FISH), and family degree investigation was further performed. RESULTS: The karyotype of the case was a complex chromosomal translocation among chromosomes 5, 20 and 16, and accompanied with a band of chromosome 20 inserted into chromosome 5. His mother and sister both had the same abnormal karyotype by familial investigation. CONCLUSION: The combined use of M-FISH and chromosome microdissection is a powerful tool to determine CCR. The complex chromosomal rearrangement could be transmitted stably in the family, but still the carriers could give birth to a healthy baby by chance.

Adult↗
PubMed2005

Multitarget FISH and LOH analyses at chromosome 3p in non-small cell lung cancer and adjacent bronchial epithelium.

Multitarget fluorescence in situ hybridization (FISH; LAVysion, Vysis, Downers Grove, IL) targeting chromosomes 6p11-q11, 7p12, 8q24, and 5p15.2 was compared with results of microsatellite studies at chromosome 3p to identify molecular changes associated with tobacco use and tumor development in non-small cell lung cancer (NSCLC). Analyses were performed on 26 NSCLCs and matched benign bronchial epithelium; samples from 10 patients without NSCLC served as control samples. Significant molecular differences between tumor tissue and corresponding benign bronchi were found using FISH (P = .001) and loss of heterozygosity (LOH) analysis (P = .031). Bronchial epithelium from patients with NSCLC was genetically different from epithelium from patients without NSCLC in FISH analysis (P = .025). Receiver operating characteristic curve analysis revealed an optimal cutoff value of 5% atypical cells for bronchial epithelium. There was no statistical correlation with the patient's smoking history, and LOH analysis of bronchi did not differentiate between patients with and without NSCLC. Multicolor FISH analysis is able to detect a tumor-associated molecular field effect in bronchi adjacent to NSCLC.

Adult↗
PubMed2005

New rearrangement t(3;17)(q26.3;q12) in an AML patient with a poor outcome.

In more than 90% of acute promyelocytic leukemia (APL) cases a reciprocal translocation t(15;17)(q22;q12) can be observed. The RARalpha gene on 17q12 is known to have other translocation partners than PML (in 15q22) in a minority of APL cases. Here, we describe a previously unrecorded chromosomal translocation involving the RARalpha gene and an unknown partner on chromosome 3. The chromosomal rearrangement was studied in detail by 24-color-FISH using whole chromosome painting probes plus multicolor banding. Thus, the breakpoint could be characterized as t(3;17) (q26;q12). In this case 10% of blasts showed AML-M3 characteristics although typical rearrangements with RARalpha were not detected by molecular methods. The characterization of the present and other comparable APL-cases with exceptional translocation partners of PML or RARalpha will help to enlighten the understanding of the pathogenesis of APL.

Adult↗
PubMedDate not supplied

Impact of cytogenetic and molecular cytogenetic studies on hematologic malignancies.

Conventional cytogenetic analysis of chromosome abnormalities in hematologic malignancies is hampered by the low mitotic index and poor quality of metaphases. A range of techniques based on fluorescence in situ hybridization (FISH) has greatly enhanced the identification of non-random translocations and deletions, pinpointing regions which contain genes involved in leukemogenesis. One of the main advantages of FISH is its ability to use non-dividing interphase cells as DNA targets, enabling the screening of large numbers of cells and providing access to a variety of cells with different hematopoetic activity. Furthermore, multicolor FISH (SKY, M-FISH and CGH microarrays) combines the screening potential of cytogenetics with the accuracy of molecular genetics, allowing the visualization of the entire human genome in 24 different colors.

Chromosome Aberrations↗
PubMed2005

M-FISH applications in clinical genetics.

Until recently, presence of de novo marker or derivative chromosomes was quite problematic for genetic counseling especially in prenatal diagnosis, because characterization of marker and derivative chromosomes by conventional cytogenetic techniques was nearly impossible. However, recently developed molecular cytogenetic technique named Multicolor Fluorescence in Situ Hybridization (M-FISH) which paints all human chromosomes in 24 different colors allows us to characterize marker and derivative chromosomes in a single hybridization. In this study, we applied M-FISH to determine the origin of 3 marker and 3 derivative chromosomes. Marker chromosomes were found to originate from chromosome 15 in two postnatal and one prenatal case. Of these, one of the postnatal cases displayed clinical findings of inv dup (115) syndrome and the other of infertility, and the prenatal case went through amniocentesis due to the triple test results. Karyotypes of the patients with derivative chromosomes were designated as 46,XY,der (21)t(1;21)(q32;p11), 46,XX,der(8)t(8;9)(p23;p22) and 46,XX,der(18)t(18;20)(q32;p11.2) according to cytogenetic and M-FISH studies. All of the M-FISH results were confirmed with locus specific or whole chromosome painting probes. The case with der (8)t(8;9) had trisomy 9(p22-pter) and monosomy 8(p23-pter) due to this derivative chromosome. The case with der(18)t(18;20) had trisomy 20(p11.2-pter) and monosomy 18(q32-qter). Parental origins of the derivative chromosomes were analyzed using microsatellite markers located in the trisomic chromosomal segments. Patients' clinical findings were compared with the literature.

Adult↗
PubMed2006

A novel subpopulation lacking Oct4 expression in the testicular side population.

We characterized murine spermatogonial stem cells (SSCs) using a multi-parameter selection strategy, combining Oct4 expression determined by monitoring green fluorescent protein (GFP) expression, and the testicular side population (SP) showing weak fluorescence on Hoechst 33342 dye staining, as markers of stem cell purification. Testicular cells were collected from Oct4/GFP transgenic mice and analyzed using a fluorescence-activated cell sorter (FACS). SP was detected in testicular cell suspensions at an average rate of 0.10%. Multicolor analysis indicated that 96% of SP cells were negative for Oct4. The cells did not express SSC marker genes, but expressed Bcrp1. While the main population was 93% positive for pyronin Y staining, this was limited to 51% in SP. We found a novel subpopulation with reduced RNA content lacking Oct4 expression in testicular SP. These results suggest that the cells isolated by FACS represent a novel population of SSCs in the G0 quiescent state.

Animals↗
PubMed2005

[Comparative study on the treatment effects with rhIL-11 and rhG-CSF in combination or alone on immune function].

OBJECTIVE: To investigate the effect of in vivo administration of rhG-CSF and/or rhIL-11 on mice immune system function. METHODS: T cell subgroups, suppressor T cells (CD8+ CD28-, CD4+ CD25+, CD3+ CD4- CD8- T cells), expression of CD28 on T cells, and spleen T cells intracellular IL4/IFN-gamma secretion were determined by multicolor flow cytometry. MTT was used to determine the T cell proliferation capacity and mixed lymphocyte reactions. RESULTS: In vivo administration of cytokines decreased the percentage of lymphocytes (P < 0.05), rhIL-11 and rhG-CSF in combination significantly decreased the CD4+/CD8+ ratio and increased the percentage of CD8+ CD28- suppressor T cells compared to either cytokine alone (P < 0.01). There was no difference in the percentage of CD3+ CD4- CD8- and CD4+ CD25+ suppressor T cells between either of the cytokines. Furthermore, cytokines treatments significantly decreased the capacities of splenic T cells proliferation and the response to alloantigens compared with the PBS treatment (P < 0.05), the combination group being more significantly decreased (P < 0.01). And cytokines treatment significantly decreased the production of IFN-gamma and increased the production of IL-4 compared with the PBS treatment(P < 0.05). The ratio of IFN-gamma/IL-4 were significantly decreased after the combination compared with either of them alone. CONCLUSION: The combination of rhIL-11 and rhG-CSF is potentially synergistic in the induction of immune tolerance by their effects on the proliferation capacity and function of T lymphocytes.

Animals↗