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Biomedical subjects

I Titley

Publications and source records attributed to I Titley.

11 recordsLinked to original sources
PubMed2005

An improved definition of mouse mammary epithelial side population cells.

BACKGROUND: Mammary epithelial side population cells have been suggested as candidate mammary stem cells. To date, for technical reasons, these cells have been poorly defined and cross-comparison of data between different laboratories has been difficult. Here, we set out to define mammary side population cells in a way that improves the ability to carry out such comparisons. METHODS: Mouse mammary epithelial cells were stained with Hoechst 33342. Light scatter, PI staining and clonogenicity of different regions of the Hoechst profile were examined. Time-course analyzes of Hoechst 33342 loading were carried out. RESULTS: Detailed examination of the light scatter and PI staining of Hoechst 33342-stained mammary cells enabled single live side population and non-side population cells to be defined with greater accuracy. Comparison of ABC pump inhibitors identified potential discrepancies in results obtained using these inhibitors. Time-course analyzes enabled side populations cells to be identified as a dynamic cell population that could be defined accurately by using the relationship between Hoechst 33342-staining profiles of consecutive time points. DISCUSSION: Defining the side population of solid tissues as a 'stabilized side population percentage' will enable a more rigorous study of the side population phenomenon and improve evaluation of results from different laboratories.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Cyclin D1 by flow cytometry as a useful tool in the diagnosis of B-cell malignancies.

The translocation (11;14)(q13;q32) and its molecular counterpart the BCL-1 rearrangement are features observed in mantle cell lymphoma (MCL) and less commonly in other B-cell disorders. This rearrangement leads to cyclin D1 overexpression, which may be the main pathogenic event in these tumours and is therefore recognised as a diagnostic marker. We developed a flow cytometry method to detect cyclin D1 overexpression using the monoclonal antibody (MoAb) 5D4, and characterised its frequency in 93 B-cell malignancies. The competitive reverse transcriptase polymerase chain reaction (RT-PCR) for cyclin D1, D2 and D3 was then performed on 40 of these cases to assess the validity of the flow cytometry method. Fluorescence in situ hybridisation (FISH) to detect t(11;14)(q13;q32) was carried out on 31 cases and results were compared with cyclin D1 expression by flow cytometry. Twenty five cases showed cyclin D1 expression using 5D4, including MCL (12/13, 92%), chronic lymphocytic leukaemia (CLL) (4/30), B-prolymphocytic leukaemia (B-PLL) (1/4), splenic lymphoma with villous lymphocytes (SLVL) (4/13), hairy cell leukaemia (HCL) (1/7) and other B-non Hodgkins Lymphoma (B-NHL) (3/15). There was a good correlation between flow cytometry results and RT-PCR in 36/40 cases (90%), and with FISH for t(11;14) in 25/31 cases (80%). We concluded that the detection of cyclin D1 expression by flow cytometry in cell suspensions could be applied routinely to the study of B-lymphoproliferative disorders and may be of value for their diagnosis and management.

Biomarkers, Tumor↗
PubMed1999

Functional and molecular analysis of hematopoietic progenitors derived from the aorta-gonad-mesonephros region of the mouse embryo.

Herein, we show that CD34, c-kit double-positive (CD34(+)c-kit(+)) cells from the aorta-gonad-mesonephros (AGM) region of the developing mouse are multipotent in vitro and can undergo both B-lymphoid and multimyeloid differentiation. Molecular analysis of individual CD34(+)c-kit(+) cells by single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) shows coactivation of erythroid (beta-globin) and myeloid (myeloperoxidase [MPO]) but not lymphoid-affiliated (CD3, Thy-1, and lambda5) genes. Additionally, most cells coexpress the stem cell-associated transcriptional regulators AML-1, PU.1, GATA-2 and Lmo2, as well as the granulocyte colony-stimulating factor receptor (G-CSF-R). These results show that the CD34(+)c-kit(+) population from the AGM represents a highly enriched source of multipotent hematopoietic cells, and suggest that limited coactivation of distinct lineage-affiliated genes is an early event in the generation of hematopoietic stem and progenitor cells during ontogeny.

Animals↗
PubMed1998

Regulation of the apoptotic response to radiation damage in B cell development.

B lymphocyte precursor cells are ultrasensitive to DNA damage induced by irradiation and drugs and die by apoptosis at very low levels of exposure. Previous studies have shown that this high level sensitivity is p53-dependent, associated with very low level expression of Bcl-2 protein and can be reversed by expression of a bcl-2 transgene. We show here that transition from the pro-B to pre-B and then mature B cell stages of murine lymphopoiesis is accompanied by changes in proliferating cells in sensitivity to X-irradiation induced apoptosis and that this is paralleled by variation in the ratio of anti-(Bcl-2/Bcl-chiL) to pro-(Bax) apoptotic proteins. These are however not fixed or invariant features of developmental stage as they can be modulated by interactions via adhesive interactions with stromal cells, stromal proteins and growth factors. We interpret these data in the context of the stringent developmental regulation of clonal lymphopoiesis and the contingency programming of cells that have extensive proliferative potential with a very low threshold for apoptosis following DNA damage.

Animals↗
PubMed1998

Successful treatment of aplastic anemia with G-CSF and high dose erythropoietin.

We report the successful treatment of pancytopenia with G-CSF and high dose erythropoietin (Epo) in an elderly patient diagnosed with aplastic anemia (AA). Furthermore this effect is dose dependent for Epo in vivo. Detection of apoptosis by gel electrophoresis shows that high dose Epo protects bone marrow mononuclear cells from spontaneous apoptosis in vitro. These findings may explain some of the mechanisms of aplastic anemia.

Aged↗
PubMed1997

BCR/ABL-negative progenitors are enriched in the adherent fraction of CD34+ cells circulating in the blood of chronic phase chronic myeloid leukemia patients.

Philadelphia chromosome-positive (Ph+) hemopoietic cells predominate in patients with chronic myeloid leukemia (CML) in chronic phase, but some Ph presumably normal stem cells persist in most patients. Ph cells are relatively frequent, compared to mature cell populations, in primitive hemopoietic cell populations from CML patients. We have purified CD34+ cells from chronic phase CML blood and separated them into two fractions on the basis of adherence or non-adherence to tissue culture plastic. We also separated CD34+ CML cell populations into HLA-DR(hi) and HLA-DR(lo) fractions and CD38(hi) and CD38(lo) fractions by flow cytometry. The CD34+ cells that adhered to plastic were predominantly CD33-, CD38- and HLA(-)-DR; cells with these phenotypic properties were significantly rarer in the CD34+ non-adherent cell population (P = 0.008-0.02). Expression of p210 BCR/ABL mRNA by adherent, non-adherent, HLA-DR(hi) and HLA-DR(lo)CD34+ cell subpopulations was demonstrated by RT-PCR. Using fluorescence in situ hybridization (FISH) in conjunction with BCR and ABL probes we detected Ph+ and Ph- cells in both adherent and non-adherent CD34+ cell fractions of 15/15 patients studied and in the HLA-DR(lo) or CD38(lo) sorted CD34+ cell fractions. The concentration of Ph- cells in the adherent CD34+ cell fraction was three-fold higher than in the non-adherent fraction (P = 0.001). Ph- adherent cells were detected in untreated CML patients and as late as 6 years after diagnosis of CML in patients treated with hydroxyurea (HU) or interferon-alpha (IFN-alpha). We conclude that whilst appreciable numbers of Ph- primitive hemopoietic progenitors are present in the circulation in untreated patients and also in treated patients in late chronic phase, the majority of cells expressing CD34 but not CD33, CD38 or HLA-DR antigens, are part of the CML clone.

Adult↗
PubMed1996

ts BCR-ABL kinase activation confers increased resistance to genotoxic damage via cell cycle block.

Using a temperature-sensitive mutant of the p210 BCR-ABL gene, transfected into a growth factor-dependent cell line (BaF3), we show that transient BCR-ABL kinase expression increases single cell and clonogenic resistance to apoptosis arising from genotoxic damage induced by ionizing radiation and VP-16/etoposide. This effect is achieved in the absence of any detectable changes in the levels of BCL-2, BAX or BCL-x proteins and is independent of proliferative, MAP kinase-dependent effects of BCR-ABL kinase. In contrast to parental cells that transiently arrest in G2 and then apoptose, p210 BaF3 cells show a pronounced and sustained G2 arrest following radiation coupled with enhanced phosphorylation of cdc2. A cell cycle block in early M phase induced by the mitotic spindle poison, nocodazole, does not provide protection from apoptosis. Reversal of G2 arrest by caffeine abolishes the protective effect of BCR-ABL kinase. These data provide further insight into the transforming properties of BCR-ABL and are relevant to the clinical intransigence of Ph-positive leukaemias.

Antineoplastic Agents, Phytogenic↗
PubMed1995

Extent of variability inherent in measurements of CD34-positive cells in different human haemopoietic tissues.

Estimation of CD34 expression is widely used to detect and quantify progenitor cells in haemopoietic tissues used as stem cell sources for transplantation. Mouse monoclonal antibodies to CD34 recognise different epitopes of the mucin-like sialoglycoprotein. These epitopes can be grouped into three classes by their differing sensitivities to the enzymes: neuraminidase, chymopapain and glycoprotease. We have compared the expression, by flow cytometry, of the three CD34 epitopes on normal adult and fetal haemopoietic tissue and in chronic myeloid leukaemia, and have used four antibodies from each class to assess variability of staining within and between epitope classes. The results reveal variable expression of CD34 both within and between tissue types and antibody classes. As a result of the different levels of detection by different antibodies, the apparent number of CD34-positive cells vary by approximately 6-fold. Enrichment for CD34 cells using magnetic bead technology shows a significant difference in the percentage of CD34 cells detected for two of the epitope types.

Adult↗
PubMed1991

Can flow cytometry reduce the workload for cervical screening? The results of a series of 622 specimens.

A simple method permitting the flow cytometric examination of cervical specimens has been developed and an assessment made of the feasibility of relying on this method to screen women for cervical neoplasia. Examination of four flow cytometric parameters showed differences between morphologically normal and abnormal specimens and allowed identification of a proportion of the normal specimens. The system had a false negative rate of 8%. Our experience with cervical specimens has revealed a number of problems associated with their examination by flow cytometry and these are discussed.

Evaluation Studies as Topic↗
PubMed1989

Fibreoptic bronchoscopy: an assessment of immediate cytological diagnosis using methylene blue stain.

A method of examining cytological material during fibreoptic bronchoscopy using a methylene blue (MB) stain was assessed in 164 consecutive fibreoptic bronchoscopies where cytology specimens were taken. The MB method provided an immediate positive diagnosis in 86% of bronchoscopically visible tumours. Subsequent histology provided a positive diagnosis in 69%, conventional brush cytology in 81% and trap cytology in 77%. The MB method produced no false positive diagnosis of malignancy and the tumour cell type identified by MB stain agreed with the histological cell type in 72% of cases. This technique is considered to be sufficiently specific to provide a method of controlling the quality of specimens taken at bronchoscopy, for further analysis in the laboratory.

Bronchial Neoplasms↗