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Biomedical subjects

C Holding

Publications and source records attributed to C Holding.

13 recordsLinked to original sources
PubMed2003

Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls.

OBJECTIVES: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. RESULTS: Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. CONCLUSIONS: OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.

Acute Disease↗
PubMed2001

Human embryonic genes re-expressed in cancer cells.

Human preimplantation embryonic cells are similar in phenotype to cancer cells. Both types of cell undergo deprogramming to a proliferative stem cell state and become potentially immortal and invasive. To investigate the hypothesis that embryonic genes are re-expressed in cancer cells, we prepare amplified cDNA from human individual preimplantation embryos and isolate embryo-specific sequences. We show that three novel embryonic genes, and also the known gene, OCT4, are expressed in human tumours but not expressed in normal somatic tissues. Genes specific to this unique phase of the human life cycle and not expressed in somatic cells may have greater potential for targeting in cancer treatment.

Base Sequence↗
PubMed2001

Isolation of novel developmental genes from human germ cell, oocyte and embryo cDNA by differential display.

Due to the difficulties inherent in research on human embryos, almost nothing is known about genes active in human early development. Although the human genome project will provide resources that theoretically provide access to every human gene, those genes specific to human early development may be difficult to define. Also, by definition, genes specific to early development will not be represented in cDNA databases derived from human somatic cells. Yet these unknown human developmental genes are likely to be of key importance for several areas of human health, including assisted reproduction and contraception, embryo stem cell research and tissue transplantation, ageing and cancer. In order to identify and isolate these human developmental genes, we have prepared amplified cDNA from human primordial germ cells, oocytes and embryos, and used differential display to compare patterns of gene expression in these embryonic cells and in the cells of somatic tissues of a 10-week human fetus. This paper reviews the highly sensitive procedures used to create amplified cDNA representing expressed genes in a single cell and the use of differential display to identify developmental genes. Several such genes have been isolated, but their full-length sequences and function are yet to be elucidated. Genes active in human early development are expected to play key roles in the maintenance of the archetypal stem cell state, potential immortality and the invasiveness of trophectoderm and primordial germ cells. They represent candidate genes regulating these functions for targeting in clinical research in human reproduction, stem cell differentiation and cancer.

Animals↗
PubMed2001

Gene expression studies on human primordial germ cells and preimplantation embryos.

Research on the regulation of gene expression in human germ cells and preimplantation embryos is restricted due to the scarcity of samples and the requirement for highly sensitive molecular techniques to investigate the few cells available. To overcome these difficulties, we have developed a reliable procedure capable of generating amplified cDNA preparations from single cells. Using this procedure, we prepared cDNA from primordial germ cells (PGCs) isolated from the gonads of fetuses at 10 weeks gestation and from preimplantation embryos at the 1-cell, 4-cell, 8-cell and blastocyst stages. Our cDNA preparations allow us to investigate the expression profile of an almost unlimited number of different genes in the same sample preparation. This is of great advantage for studies of a panel of genes in a particular family or functional group, or with related mechanisms of regulation, e.g., developmental genes, oncogenes, cell cycle-control genes and imprinted genes. We have used these cDNA preparations in conjunction with differential display to identify genes specifically expressed in PGCs and preimplantation embryos in a sex- and developmental stage-specific manner. Genes specifically expressed in PGCs, oocytes and embryos were further analysed for their expression in embryonal carcinoma cells and in their differentiated derivatives following treatment by retinoic acid. Our strategy will disclose genes essential for gametogenesis and embryonic development which may only be expressed at certain stages of their development. The germ cell- and embryo-specific cDNA molecules, cDNA libraries and microarrays are a valuable resource for other researchers in this field.

Blastocyst↗
PubMed2000

The distribution of P2X receptor clusters on individual neurons in sympathetic ganglia and their redistribution on agonist activation.

The distribution of P2X receptors on neurons in rat superior cervical ganglia and lability of P2X receptors on exposure to agonists were determined. Antibody labeling of each P2X subtype P2X(1)-P2X(7) showed neurons isolated into culture possessed primarily P2X(2) subunits with others occurring in order P2X(7) > P2X(6) > P2X(3) > P2X(1) > P2X(5) > P2X(4). Application of ATP and alpha,beta-meATP to neurons showed they possessed a predominantly nondesensitizing P2X receptor type insensitive to alpha,beta-meATP, consistent with immunohistochemical observations. P2X(1)-green fluorescent protein (GFP) was used to study the time course of P2X(1) receptor clustering in plasma membranes of neurons and internalization of receptors following prolonged exposure to ATP. At 12-24 h after adenoviral infection, P2X(1)-GFP formed clusters about 1 microm diameter in the neuron membrane. Application of ATP and alpha,beta-meATP showed these neurons possessed a predominantly desensitizing P2X receptor type sensitive to alpha,beta-meATP. Infection converted the major functional P2X receptor type in the membrane to P2X(1). Exposure of infected neurons to alpha,beta-meATP for less than 60 s led to the disappearance of P2X(1)-GFP fluorescence from the cell surface that was blocked by monensin, indicating the chimera is normally endocytosed into these organelles on exposure to agonist.

Adenosine Triphosphate↗
PubMed2000

Detection of human novel developmental genes in cDNA derived from replicate individual preimplantation embryos.

We have constructed amplified cDNA preparations from replicate samples of human oocytes and individual preimplantation embryos. Differential display of the cDNA preparations shows disparate patterns of gene expression in the individual embryos at all stages of preimplantation development. The variation in patterns of genes expressed is in part due to the low starting cell number undergoing the reverse transcription-polymerase chain reaction (RT-PCR) step in the preparation of the amplified cDNAs. Despite this variability, the use of replicate embryo samples makes it possible to identify and isolate human genes specifically expressed at the different stages of human preimplantation development from the unfertilized oocyte to the blastocyst stage.

Actins↗
PubMed1996

Single-cell analysis of unstable genes.

PURPOSE: We have developed sensitive diagnostic procedures for studies on the normal and mutant alleles of the triplet repeat genes associated with myotonic dystrophy and fragile X in single human somatic cells, gametes and embryos. METHODS: Polymerase chain reaction (PCR) assays for the normal alleles of the myotonic dystrophy and fragile X loci have been refined to the sensitivity of the single cell. In addition, we have developed a simple PCR-based technique, termed ¿Repeat Primer PCR', which can detect the full fragile X expansion in small samples of buccal cells. CONCLUSIONS: The assay for the triplet repeat sequence in the myotonic dystrophy locus could not be used to study stability since we observed additional PCR products derived from in vitro expansion of the triplet repeat sequence during the PCR reaction itself. The implications of in vitro expansion and allele drop-out for studies on the timing of the expansion in development and preimplantation diagnosis of triplet repeat diseases are discussed. The development of a new PCR procedure to identify the expanded alleles of the fragile X locus could prove invaluable for monitoring the timing of repeat expansion in early embryonic development. Triplet repeat polymorphisms provide a means of identifying the maternally and paternally-derived alleles of the myotonic dystrophy gene. Using single cell reverse transcriptase PCR analysis, we have monitored the onset of the myotonic dystrophy gene transcription in early preimplantation embryos. Transcripts from the paternally-inherited allele of the myotonic dystrophy gene are already detectable in the 1-cell stage human embryo.

Alleles↗
PubMed1994

Recycling the single cell to detect specific chromosomes and to investigate specific gene sequences.

We have developed a new procedure, called cell recycling, which combines the two powerful techniques of polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) on the same single cell. A fixed cell is used as the DNA template for PCR, prior to the FISH analysis. Using single blastomeres from mouse embryos as a model system, cell recycling procedures detect the single-copy beta-haemoglobin gene sequence at an efficiency of 70% as well as sex chromosome constitution at an efficiency of 74% in the same single cell. Cell recycling will increase the success rate of pregnancy following preimplantation diagnosis for a specific gene defect by identifying embryos with chromosomal abnormalities and eliminating them from the transfer procedure.

Animals↗
PubMed1993

Development and validation of laboratory procedures for preimplantation diagnosis of Duchenne muscular dystrophy.

In order to develop and validate methods for the preimplantation diagnosis of Duchenne muscular dystrophy (DMD), we have established and evaluated PCR assays for the analysis of four loci within the DMD gene and for two Y chromosome sequences in single cells. A model system using buccal cells picked from mouthwash samples has been used for an extensive evaluation of the sensitivity and specificity of the assays, and each assay has been tested in samples containing single cells, two cells, and three cells per tube. The four DMD and two Y assays have been combined in duplex and triplex reactions to enable simultaneous diagnosis of DMD and of fetal sex. One of the DMD markers is a highly polymorphic simple tandem repeat locus which produces a basic DNA profile, and provides a control for contamination by foreign DNA. Amplification of DMD or Y sequences was observed in 78 to 92% of single male cells, rising to 96% and 97% in tubes containing two or three male cells respectively. Coamplification of both a DMD and a Y sequence together occurred with a mean success of 74% in single male cells, increasing to 93% with two, and 95% with three cells per tube. With appropriate precautions, we believe that it is now possible to proceed to clinical application of these procedures.

Base Sequence↗
PubMed1990

Amplification of a beta-haemoglobin sequence in individual human oocytes and polar bodies.

A 680 base-pair sequence of the human beta-haemoglobin gene was reproducibly amplified in individual unfertilised human oocytes and in first polar bodies isolated from them. Specificity and sensitivity of amplification were achieved by two sequential reactions with two sets of primers, amplifying first a 725 base-pair sequence and secondly a 680 base-pair sequence from within the first amplified fragment. A restriction enzyme digest of the DNA amplified from a single oocyte with the endonuclease Dde I confirmed the identity of the amplified beta-haemoglobin fragment; this technique provides a diagnostic test for the genetic defect responsible for sickle cell anaemia. Analysis of the DNA from the first polar body may enable detection of such defects in unfertilised eggs from carrier women. Selection of eggs without the defect for fertilisation may therefore obviate the need for diagnostic procedures on embryos.

Amino Acid Sequence↗
PubMed1989

Diagnosis of beta-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos.

Mouse preimplantation embryos were accurately diagnosed as normal or mutant at the beta-major haemoglobin locus by amplification of specific DNA sequences in a single cell. A DNA sequence containing the whole of exon 3 and some 3' untranslated sequences within the beta-major haemoglobin gene was amplified in single blastomeres by means of the polymerase chain reaction (PCR). Blastomeres were removed from embryos of four to eight cells from normal BALB/c mice and from mutant (thalassaemic) BALB/c mice homozygous for a deletion of the whole beta-major haemoglobin gene. The sensitivity of the amplification procedure was enhanced by the sequential use of two sets of oligonucleotide primers for 30 cycles of amplification each, the second pair being located within the segment amplified by the first pair. The product (204 base-pairs) could be easily visualised in ethidium bromide-stained agarose gels. Stringent precautions to prevent contamination were taken, and with these precautions the PCR amplification procedure could be carried out under normal laboratory conditions. These procedures for diagnosis of genetic disease before implantation should be applicable to preimplantation diagnosis of any monogenic disorder in man for which the affected DNA sequence is known.

Alleles↗