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P C Vincent

Publications and source records attributed to P C Vincent.

At least 19 recordsLinked to original sources
PubMed1992

Binding of proteins from embryonic and differentiated cells to a bidirectional promoter contained within a CpG island.

We have analysed binding sites of nuclear protein factors to a CpG island (HTF9), which contains the promoter for a pair of overlapping, divergently-transcribed "housekeeping" genes. Using DNaseI protection assays with extracts from a range of differentiated and undifferentiated cell lines, including mouse embryonic stem (ES) and embryonal carcinoma (EC) cells, we located multiple protein binding sites on HTF9. Most of the sites were outside the defined core promoter and could bind to previously identified transcription factors. These included constitutive, inducible and apparently tissue-specific factors in an extremely asymmetric array relative to the transcription start sites of the two genes. A number of sites showed different binding specificities or affinities in different cell types, including ES cells. However, we found no factors that were specific for both ES and EC cells, and no protein-binding site protected exclusively in undifferentiated embryonic cells.

3T3 Cells↗
PubMed1991

Characterization of endocrine-rich monolayers of human fetal pancreas that display reduced immunogenicity.

Fibroblast-free insulin-secreting monolayers of human fetal pancreas (14-20 wk of gestation) were formed by plating isletlike cell clusters (ICCs) obtained from partially digested pancreases on plates coated with bovine corneal matrix. Human fetal pancreatic cells, freshly digested with collagen, displayed a 17-fold response to human peripheral blood lymphocytes (HPBLs) in mixed-lymphocyte culture. After 14 days in culture, monolayers derived from ICCs exhibited a smaller, twofold response to HPBLs. By comparison, in monolayers produced from single-cell suspensions, fibroblast overgrowth remained a problem. The endocrine component of the monolayers was 65 +/- 13 and 43 +/- 8%, respectively, with the number of beta-cells being 51 and 9%. Cells from both monolayers displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+, and 0.6-1.3 microM 12-O-tetradecanoylphorbol-13-acetate but not to 20 mM glucose. Monolayers derived from ICCs synthesized DNA, proinsulin, and protein. This study showed that it is possible to establish an endocrine-rich monolayer of human fetal pancreas that has greatly reduced immunogenicity. The existence of residual activity to HPBLs suggests some additional form of immunosuppression is required to prevent rejection of this tissue when grafted into diabetic patients. Subculturing and cryopreservation may also be needed to achieve adequate numbers of beta-cells for clinical transplantation.

Calcium↗
PubMed1990

Pig fetal pancreatic monolayers. A model of potential use in transplantation.

Endocrine-rich monolayers of pig fetal pancreas that are free of fibroblasts have been established with the ultimate aim of providing guidelines for the culture of the human equivalent. The immunogenic potential of the monolayers--hence their capacity to be grafted--has also been analyzed. Fetuses ranging from 50 to 90 days were used, and, following digestion with collagenase (4 mg/ml, 15-20 min), the pancreatic suspension was plated onto tissue culture vessels containing RPMI 1640. The fetal calf serum concentration was kept low (5%) initially to inhibit fibroblast proliferation, but subsequently increased to 7%. Monolayers from a typical litter of 8-10 fetal pigs produced 6-8 x 10(8) viable epithelial cells by day 10 of culture, of which 75% were endocrine cells. This represents an 8-fold increase in a two-week period. The ratio of beta:alpha:delta:pancreatic polypeptide cells was 19:33:18:5. These monolayers synthesized both DNA, (pro)insulin and protein, and displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+ and 1.3 microM 12-0-tetradecanoyl-phorbol-13-acetate. Static stimulation with 20 mM glucose however, did not elicit a response in insulin secretion. These cells displayed no reaction to allogeneic lymphocytes in a mixed lymphocyte culture, whereas freshly obtained porcine epithelial cells did. Methods may need to be found to increase the proportion of B cells in this enriched endocrine cell population. In general however, guidelines have been established that may be useful in developing a monolayer of human fetal pancreatic cells with the eventual aim of transplantation. The reduction in immunogenicity of the pig fetal pancreatic cells suggests that they too might be a potential source for transplantation.

Animals↗
PubMed1989

Cytogenetic abnormalities in benign lymphoid hyperplasia: a dual-parameter study using chromosome analysis and flow cytometry.

This is a prospective study of lymphoid tissue showing benign reactive hyperplasia (18 lymph nodes and 2 tonsils), using cytogenetic analysis of cells stimulated with T- or B-cell mitogens. The reason for this study was the detection of an abnormal chromosomal population in cells from an enlarged lymph node excised from a 7-year-old female who on further investigation was found to be clinically well and after one year's close follow-up had not developed any further signs or symptoms of malignancy. In addition, DNA content was measured by flow cytometry (FCM) on fresh cell suspensions in 17 cases and fixed cell suspensions in 3 cases. Structural and numerical clonal chromosome abnormalities were found in 9 of the 20 samples, but no common specific defect was identified. FCM showed an abnormal DNA content in 10 of the 20 samples studied; 3 of these showed clonal chromosome abnormalities. Surface membrane immunoglobulin studies were carried out on 15 of the 20 samples using cell suspensions and frozen tissue sections. In 5 of the 15 cases, monoclonal surface immunoglobulin was detected. There was no direct correlation between the surface membrane immunoglobulin studies and the chromosome and FCM analyses. We conclude that aneuploidy is a common feature in reactive lymphoid tissue, but both cytogenetics and FCM are needed to identify it.

Aneuploidy↗
PubMed1989

Characterization of cell lines derived from a multiply aneuploid human bladder transitional-cell carcinoma, UCRU-BL-13.

A series of cultured cell lines (designated UCRU-BL-13) has been established from different serial passages of a multiply aneuploid human bladder transitional-cell carcinoma xenografted in nude mice. Serial passage of the xenografts in vivo and of the cell lines in vitro was accompanied by shifts in the tumor ploidy, with dominance of different major peaks. Despite this, the expression of tumor markers remained constant, and consistent chromosomal markers were observed both in the 8th xenograft passage and in a subline in tissue culture established over a year apart. Chromosomal numbers reflected the predominant aneuploid peaks observed; consistent numerical and structural changes included a marker derived from chromosome 1, 8p-, -10, 11q+, and 17q+. The cell line derived from the initial xenograft comprised a mixture of transitional, adenocarcinoma and squamous carcinoma cells in early passage, but adenocarcinoma cells were absent from later passages. The lines expressed the B-blood-group antigen, histocompatibility antigens, receptors for transferrin and EGF, and reacted with a series of monoclonal antibodies (MAbs) directed to malignant human epithelial cell lines. These lines provide a model for studying the evolution of tumor heterogeneity and drug resistance in bladder carcinoma exhibiting multiple aneuploidy.

Aneuploidy↗
PubMed1989

Hodgkin's disease.

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Hodgkin Disease↗
PubMed1989

CFU-GM inhibitors in neutropenia.

Peripheral blood lymphocytes from 20 patients with neutropenia not consistent with aplastic anaemia were tested for their ability to inhibit the proliferation of normal granulopoietic precursor cells (CFU-GM) in agar culture. Two patients, both with features of an autoimmune disorder, had lymphocytes which were more inhibitory than normal lymphocytes to both normal and their own CFU-GM. Two other patients had lymphocytes which were more inhibitory than normal lymphocytes to either their own CFU-GM or normal CFU-GM but not both. Eight patients had lymphocytes which were significantly less inhibitory than normal lymphocytes to either normal or their own CFU-GM, but only one showed this feature against both normal and their own CFU-GM. One patient had a highly potent plasma inhibitor of CFU-GM--this patient had received multiple transfusions and had a leucocyte antibody of a broad specificity. No clinical or haematological features were common to any of these groups of patients which reflects the heterogeneity of patients studied and stresses the importance of controls.

Adolescent↗
PubMed1989

A randomised trial of empirical antibiotic therapy in febrile neutropenic patients with hematological disorders: ceftazidime versus azlocillin plus amikacin.

One hundred and two patients with neutropenia (less than 1 x 10(9)/L) secondary to primary hematological disorders or chemotherapy for hematological malignancies were prospectively randomised, upon the development of fever or other signs of infection, to receive empirical antibiotic treatment with either ceftazidime (+/- flucloxacillin) (n = 52) or azlocillin plus amikacin (+/- flucloxacillin) (A&A, n = 50). The two groups were equivalent with respect to clinical and laboratory parameters prior to antibiotic therapy and flucloxacillin was added to approximately 25% of the patients in each group on the clinical suspicion of Gram positive infection. When assessed at 96 hours, the complete response rates were 59.6% for the ceftazidime treated patients and 44% for A&A treated patients. Partial response rates were 17% and 20% respectively. This difference was not statistically significant. Eight patients died whilst on the trial, three of those initially randomised to ceftazidime and five initially randomised to A&A. Moderate to severe hypokalemia was encountered significantly less often in the ceftazidime treated group (p less than 0.01), whilst other parameters of toxicity were equivalent. No primary or acquired resistance to ceftazidime was encountered. Separate analysis of those patients who did not receive flucloxacillin yielded identical results. We conclude that ceftazidime (+/- flucloxacillin) is as efficacious as azlocillin plus amikacin (+/- flucloxacillin) in the empirical antibiotic management of such patients and is associated with a lower incidence of moderate to severe hypokalemia.

Adolescent↗
PubMed1988

Localisation of satellite DNA sequences on human metaphase chromosomes using bromodeoxyuridine-labelled probes.

Human highly repeated satellite sequences, cloned into M13, were used as templates to prepare single-stranded DNA probes containing bromodeoxyuridine (BrdUrd) in place of thymine. The probes were hybridised to human metaphase chromosomes and visualised using an indirect immunological detection procedure. The sensitivity and accuracy of the technique were tested using a BrdUrd-labelled probe of known copy number and location: a segment from the 2.5 kb Y chromosome repeat. The procedure proved to be reliable and fast, with a sensitivity similar to that of other in situ hybridisation techniques. The technique was then used to determine the chromosomal locations of a 100 bp repeat from human satellite 3. The satellite 3 probe hybridised to a large number of chromosomes and, surprisingly, the intensity of label at all locations remained unchanged when the slides were washed at a higher stringency. The resolution of the technique was very high and allowed accurate localisation of the satellite sequence. Hybridisation was observed in two regions of the subcentromeric heterochromatin of chromosome 9, in two locations at the centromere and short arm of all the acrocentric autosomes, and at the centromere and long arm of the Y chromosome. In addition the probe hybridised to centromeric heterochromatin in chromosomes 1, 16, 17 and 20. We believe that single-stranded BrdUrd-labelled probes should be very useful for detecting RNA transcripts in cells, and discuss ways by which the procedure could be modified to locate single copy DNA on chromosomes.

Base Sequence↗
PubMed1987

A simple and rapid immunological technique for visualising chromosome-mediated gene transfer (CMGT).

A method is described for visualising chromosome-mediated gene transfer (CMGT) by detecting chromosomes labelled with bromodeoxyuridine (BrdU) using a monoclonal antibody to BrdU. In this experiment, the CCRF-CEM T cell line was grown in the presence of BrdU and the labelled chromosomes were isolated and transfected into human embryonic fibroblasts. Uptake and retention of chromosomes were compared for transfection with either PEG or DMSO treatments. Following transfection the labelled chromosomes could be visualised in recipient cells using a monoclonal antibody to BrdU, followed by immunoperoxidase staining. Chromosome uptake into cells was similar for both DMSO and PEG treatments and was a relatively frequent event; about 1 in 5 recipient cells had labelled material present. This technique can be used to assess the technical aspects of the earliest stages of chromosome-mediated gene transfer.

Antibodies, Monoclonal↗
PubMed1987

Evidence following splenic radiotherapy for a highly dynamic traffic of CFU-GM between the spleen and other organs in chronic granulocytic leukaemia.

Five patients with Ph1 +ve chronic granulocytic leukaemia and massive splenomegaly were given induction therapy with splenic irradiation, and their peripheral blood leucocyte count and granulocyte macrophage progenitor (CFU-GM) concentration monitored during the following six hours. In each patient there was a greater fall in CFU-GM than would have been expected from the fall in leucocyte count, but no evidence of a plasma inhibitor was found to explain the disproportionate reduction in CFU-GM. The difference between the estimated and observed decrease in CFU-GM/1 following splenic irradiation indicates a highly dynamic traffic of CFU-GM from the spleen to other organs in chronic granulocytic leukaemia.

Adult↗
PubMed1987

The CFU-C assay in patients with neutropenia and, in particular, drug associated neutropenia.

One-hundred and four patients with a diagnosis of aplastic anaemia (28) or neutropenia (76) referred to our laboratory for assessment of granulopoiesis were studied. Bone marrow myeloid progenitor cell (CFU-C) frequency was measured and in 85 patients, inhibitor studies with either in-vitro drug addition or plasma co-culture were performed. Of 28 patients with aplastic anaemia, 22 (79%) had low numbers of CFU-C, while four (14%) had numbers within the normal range and two (7%) had elevated progenitor cell frequency. In contrast, of the 76 patients with neutropenia who were studied only 29 (38%) had low CFU-C numbers, 33 (43%) had a CFU-C frequency within the normal range and 14 (18%) had elevated CFU-C numbers. Thirty-nine patients had ingested potentially myelotoxic drugs and in eight of these it was possible to demonstrate drug associated inhibition of CFU-C proliferation in vitro. The drugs most commonly associated on a historical basis with myelosuppression were chloramphenicol, antimalarials, sulphonamides, anticonvulsants and nonsteroidal anti-inflammatory agents.

Agranulocytosis↗
PubMed1987

Long-term bone marrow culture induces terminal differentiation of human myeloid leukemic cells.

Bone marrow cells from a patient with acute myeloblastic leukemia were simultaneously cultured in vitro under conditions that favored the survival of either (1) leukemic progenitors (leukemic suspension culture), or (2) normal progenitors (long-term bone marrow culture). Whereas cells that were morphologically primitive and cytochemically leukemic persisted in leukemic suspension culture, they were progressively and completely replaced in long-term bone marrow culture by neutrophilic granulocytes and subsequently by macrophages. However, Auer rods were present in the maturing myeloid cells, including polymorphonuclear neutrophils, between the 7th and 30th days of long-term bone marrow culture, indicating that they were derived directly from the original leukemic population. This observation suggests that, at least in some patients, selection of cells with the potential for terminal differentiation may be the underlying mechanism responsible for the purging properties that have been attributed to long-term bone marrow culture.

Bone Marrow↗
PubMed1987

Fine needle aspiration sampling of bone marrow for DNA flow cytometric analysis.

Fine needle aspiration (FNA) was used to obtain bone marrow samples from 57 patients with a variety of hematologic disorders, and from three normal subjects. We confirm that the technique is well tolerated and show that reliable cytokinetic and cell differential data can be obtained provided marrow fragments are isolated for analysis. This requires aspiration of a larger volume of marrow than originally described but this can still be achieved with minimal discomfort to the patient. The technique thus provides a means by which marrow samples could be obtained at frequent intervals in the monitoring of chemotherapy.

Biopsy, Needle↗
PubMed1986

Significance of secondary cytogenetic changes in patients with Ph-positive chronic granulocytic leukemia in the acute phase.

The karyotypic abnormalities in 29 patients in the acute phase of Ph-positive chronic granulocytic leukemia are described. Of 18 Giemsa banded samples, 11 showed one or more of the typical additional abnormalities found in the acute phase, namely +Ph, +8, or i(17q). Survival data from these patients was combined with three published series providing 135 patients and the effect of one, two, or three of these abnormalities tested. The prognosis was significantly worse in patients with two or more additional abnormalities, compared with those with one or none. Analysis of the subset of patients with only one additional abnormality [+Ph or +8 or i(17q)] suggested a worse prognosis in those with +8 than in those with +Ph or i(17q), although the differences were not significant. There also was a trend for patients in whom all metaphases showed abnormalities in addition to the Ph chromosome to have a worse prognosis than those in whom some or all metaphases contained the Ph only. However, this trend just failed to reach a 5% level of significance.

Adult↗
PubMed1986

Sequence relationships of three human satellite DNAs.

The simple sequence components of three human classical satellite DNAs have been defined, and some segments of each satellite have been sequenced. Each of the classical satellites I, II and III was found to contain, as a major component, a single family of simple repeated sequences. The three simple-sequence families have been called satellites 1, 2 and 3, to indicate the enrichment of each in one of the classical satellites I, II and III, and to differentiate them from these classical satellites, which also contain other repeated components. Satellite 3, the simple sequence component of classical satellite III, when digested with the restriction endonuclease HinfI, forms a ladder based on a repeat of five base-pairs, 5' A-T-T-C-C. The HinfI ladder was shown to be composed of repeated elements with the general sequence 5' (A-T-T-C-C)n-A-TC-T-C-G-G-G-T-T-G. Satellite 2, the simple sequence component of classical satellite II, is digested by HinfI into a large number of very small fragments, of length 10 to 80 base-pairs. These were found to contain the simple repeat 5' A-T-T-C-C, in a highly diverged form. Analysis of satellite 2 sequences suggested that the five base-pair repeat was originally amplified as a higher-order repeat like that of satellite 3. However, the main tandemly repeated segments of satellite 2 in the human genome are much longer, and the simple sequence elements on which they are based are quite degenerate. Satellite 1, the simple sequence component of classical satellite I, is digested by the restriction endonuclease RsaI into a ladder of fragments less than 150 base-pairs in length. These ladder fragments were found to be formed by the loss of RsaI sites from two related A + T-rich sequences, A (17 base-pairs) and B (25 base-pairs), arranged in alternating arrays, -A-B-A-B-A-. Analysis of a large number of cloned fragments from the RsaI ladder of satellite 1 showed that the tandem arrays, -A-B-A-B-A, have a more complex arrangement, with apparent amplification of segments containing particular sequence variants of the repeat units, A and B. No sequence relationship was evident between the repeat elements of satellite 1 and those of satellites 2 and 3.

Base Sequence↗