Planning for future demand.
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Blood Banking↗
Biomedical subjects
C Wickenden
Publications and source records attributed to C Wickenden.
Survey of the implementation of the recommendations in the Health Services Circular 1998/224 'Better Blood Transfusion'.
This report describes a questionnaire survey on the implementation of the recommendations of the Health Services Circular (HSC) 'Better Blood Transfusion' 1998/224 for improving transfusion practice. The survey was carried out to inform a second UK Chief Medical Officers' symposium on 'Better Blood Transfusion' in October 2001. Sixty-nine percent of hospitals where blood is transfused in England participated. The results show that, by 2001, most hospitals had established Hospital Transfusion Committees (HTCs), developed protocols for the process of transfusion and were participating in the Serious Hazards of Transfusion (SHOT) scheme. However, there was limited compliance with other recommendations, including the provision of training for staff involved in transfusion and information to patients, the development of protocols for the appropriate use of blood, the performance of audits of transfusion practice and the introduction of peri-operative cell salvage. The survey did not determine the reasons for this limited compliance. New initiatives including the issue of a further HSC on 'Better Blood Transfusion' are aimed at enabling hospitals to improve their transfusion practice in a more systematic way than that was found in the results of this survey.
Blood Transfusion↗
The impact of improved safety on maintaining a sufficient blood supply.
The impact of improved safety on maintaining a sufficient blood supply is becoming an increasingly real issue facing the National Blood Service in England and North Wales. This paper shows the extent of the impact that safety measures can have on reducing collection levels, without making any value judgement on the safety critera themselves. It demonstrates that underlying trends in collection are making it increasingly difficult to meet demand. Further potential restrictions, perhaps associated with variant Creutzfeldt-Jacob disease (vCJD), will require a reassessment of the safety-sufficiency trade-off. The analysis strongly suggests that continual enhancement of safety criteria, for both individual donor and patient benefit, will lead to insufficient supply to meet current levels of hospital demand. New approaches are needed to effectively manage blood throughout the supply chain, thereby ensuring sufficient supply.
Blood Banks↗
Preparation and purification of antisera against different regions or isoforms of beta-amyloid precursor protein.
We describe a procedure for the production and peptide affinity purification of polyclonal antisera against synthetic peptides representing different domains of beta-amyloid precursor protein (APP). Rabbits were immunised with keyhole limpet haemocyanin coupled to synthetic peptides representing the amino-terminal APP18-32, Kunitz-type protease inhibitor (KPI) region APP301-316, the A beta region APP670-686, and the carboxy-terminal APP756-770 of APP770 for the production of antisera anti-AP-1, anti-AP-2, anti-AP-4 and anti-AP-5, respectively. Each antiserum was purified to specific antibody using the respective cognate peptides immobilised on affinity columns as ligand, using the 1-ethyl-3-(dimethylaminopropyl)carbodiimide-diaminodipropylamine method. Purified antibodies of these four antisera were highly specific and in enzyme-linked immunosorbent assays (ELISA) reacted only to the corresponding peptide. These purified antisera have been used in Western blot, immunohistochemical and immunoprecipitation techniques to facilitate the understanding of the regulation of APP and amyloid beta-protein (A beta). The A beta is formed by proteolysis of APP, and its deposition leading to the formation of senile plaques in the brain is considered to be a key step in the pathogenesis of Alzheimer's disease.
Alzheimer Disease↗
Cell-specific expression of beta-amyloid precursor protein isoform mRNAs and proteins in neurons and astrocytes.
The abnormal accumulation of beta-amyloid (A beta) in senile plaques appears to be a central pathological process in Alzheimer's disease. A beta is formed by proteolysis of beta-amyloid precursor protein (APP) with several isoforms generated by alternative splicing of exons 7, 8 and 15. A semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis showed that APP695 mRNA lacking exon 7 and 8 was most abundant in primary cultures of rat neurons, while APP770 and APP751 representing, respectively, the full length and exon 8 lacking isoforms predominated in cultured astroglial cells. Antisera AP-2 and AP-4 were produced by immunizing rabbits with keyhole limpet haemocyanin coupled with synthetic peptides representing KPI region APP301-316 and A beta region APP670-686 of APP770, respectively. These polyclonal antisera were purified against the corresponding peptide using affinity chromatography. Western blot analysis of homogenates of relatively enriched neuronal and astroglial cultures showed that these antibodies discretely stained bands of proteins in a cell-specific manner. Dot-blot analysis using AP-2, AP-4 and 22C11 antibodies indicated that, in comparison with neurons, cultured astrocytes contained 3-fold greater KPI-containing APP isoform proteins. The amount of total APP proteins, which include both KPI-containing and KPI-lacking APP isoforms, was approximately 90% higher in astrocytes than in neurons. Consistent with these in vitro findings in cultured astrocytes, in fimbria-fornix lesioned rat hippocampus, labelling with AP-2 antibody, which specifically reacts with KPI-containing APP proteins, was mainly observed in glial fibrillary acidic protein-positive reactive astrocytes in vivo. The results showed that APP isoforms are expressed in a cell type-specific manner in the brain and, since deposition of A beta is closely associated with the expression of KPI-containing APP isoforms, provide further evidence for the involvement of astrocytes in plaque biogenesis.
Amyloid beta-Protein Precursor↗
Glia-derived cytokines and the biogenesis of amyloid plaques in Alzheimer's disease.
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Alzheimer Disease↗
The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis.
Rapid Ca2+ efflux from intracellular stores during cardiac muscle excitation-contraction coupling is mediated by the ryanodine-sensitive calcium-release channel, a large homotetrameric complex present in the sarcoplasmic reticulum. We report here the identification, primary structure and topological analysis of the ryanodine receptor-calcium release channel from human cardiac muscle (hRyR-2). Consistent with sedimentation and immunoblotting studies on the hRyR-2 protein, sequence analysis of ten overlapping cDNA clones reveals an open reading frame of 14901 nucleotides encoding a protein of 4967 amino acid residues with a predicted molecular mass of 564 569 Da for hRyR-2. In-frame insertions corresponding to eight and ten amino acid residues were found in two of the ten cDNAs isolated, suggesting that novel, alternatively spliced transcripts of the hRyR-2 gene might exist. Six hydrophobic stretches, which are present within the hRyR-2 C-terminal 500 amino acids and are conserved in all RyR sequences, may be involved in forming the transmembrane domain that constitutes the Ca(2+)-conducting pathway, in agreement with competitive ELISA studies with a RyR-2-specific antibody. Sequence alignment of hRyR-2 with other RyR isoforms indicates a high level of overall identity within the RyR family, with the exception of two important regions that exhibit substantial variability. Phylogenetic analysis suggests that the RyR-2 isoform diverged from a single ancestral gene before the RyR-1 and RyR-3 isoforms to form a distinct branch of the RyR family tree.
Amino Acid Sequence↗
Glial cell derived neurotrophic factors and Alzheimer's disease.
In Alzheimer's disease the normal balance of metabolic pathways regulating trophic factors/cytokines is disrupted; local reduction may result in neurons being deprived of neurotrophic factors while an excess may initiate a cascade of interaction between glial cells and beta-amyloid precursor protein metabolism thereby facilitating plaque formation. This paper briefly discusses the findings of our group on aspects ranging from cholinergic humoral and trophic factors to mechanisms underlying amyloidogenesis in Alzheimer's disease.
Alzheimer Disease↗
Expression of a cardiac Ca(2+)-release channel isoform in mammalian brain.
Mammalian brain possesses ryanodine-sensitive Ca2+ channels, which in muscle cells mediate rapid Ca2+ release from intracellular stores during excitation-contraction coupling. Analysis of bovine brain ryanodine receptor (RyR) channels suggests specific expression of the cardiac-muscle RyR isoform in mammalian brain. Localization using cardiac-muscle RyR-specific antibodies and antisense RNA revealed that brain RyRs were present in dendrites, cell bodies and terminals of rat forebrain, and highly enriched in the hippocampus. Activity of skeletal-muscle RyR channels is coupled to sarcolemmal voltage sensors, in contrast with cardiac-muscle RyR channels, which are known to be Ca(2+)-induced Ca(2+)-release channels. Thus Ca(2+)-induced Ca2+ release from intracellular stores mediated by brain RyR channels may be a major Ca(2+)-signalling pathway in specific regions of mammalian brain, and hence may play a fundamental role in neuronal Ca2+ homoeostasis.
Animals↗
Prevalence of human papillomavirus types in the cervices of women before and after laser ablation.
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Cervix Uteri↗
DNA hybridisation of cervical scrapes: comparison with cytological findings in Papanicolaou smears.
One hundred and sixty four cervical scrapes were taken over 26 months from 143 women aged 17 to 53 years, using an Ayre spatula. A smear was prepared from each sample for Papanicolaou staining and for cytological examination for evidence of human papillomavirus (HPV) infection. The cells remaining on the spatula were harvested for DNA analysis. These samples were probed for HPV 6, 16, and 18 and the results of DNA hybridisation and cytology were compared. Cytological changes of HPV (anucleate keratinised and keratinised squamous cells, koilocytic change and dyskeratosis, or multinucleation, or both) were detected in 103 (72%) of the samples where HPV DNA was detected; koilocytic changes were present in only 67 (48%). It is concluded that screening for a range of viral changes is a more sensitive method of detecting HPV infection than screening for koilocytic change alone.
Adolescent↗
Sexual transmission of human papillomaviruses in heterosexual and male homosexual couples, studied by DNA hybridisation.
The prevalence of human papillomavirus (HPV) 6, 11, 16, and 18 in 36 heterosexual couples and seven male homosexual couples with genital warts was investigated for evidence of sexual transmission of genital HPV. The prevalence of virus type and number of copies of viral genome equivalents/cell in the lesions were assessed, and the factors influencing transmission analysed. Our results show that HPV 6 and, to a lesser extent, HPV 11 were the types most readily transmitted, and that transmission appears to depend on the copy number and the duration and frequency of exposure.
DNA, Viral↗
Prevalence of HPV DNA and viral copy numbers in cervical scrapes from women with normal and abnormal cervices.
Cervical scrapes were obtained from 215 women with cytologically normal cervices and 74 women with cervical intraepithelial neoplasia (CIN) and probed for HPV 6, 11, 16, and 18 by dot-blot hybridization. Viral copy numbers were determined by densitometric scanning of autoradiographs. The prevalence of HPV DNA in women with normal smears was as follows: 23 per cent of women attending a Family Planning Clinic (FPC), 16 per cent of women attending a Sexually Transmitted Diseases (STD) clinic, and 48 per cent of women attending a laser follow-up clinic after treatment of CIN. Ten per cent of women with normal cervices harboured HPV 16. Viral copy numbers ranged from 1300 to 52,800 genome equivalents per cell. Twenty-seven HPV-positive women with normal smears (including four patients infected with HPV 16) were followed for up to 3 years. None developed CIN irrespective of copy number. Our results show that HPV is present in normal cervices in the absence of CIN. Copy number has no relation to the development of CIN and factors other than the amount of HPV DNA may trigger the neoplastic process.
Adolescent↗
Cross-hybridisation of human papillomavirus DNA on filters.
Accurate type assignment of the different HPV types which infect the female genital tract, is essential in view of the differing pathological potential of the common virus types present in the cervix. We have developed hybridisation, washing and autoradiography conditions that minimise cross-hybridisation on filters and so allow clear-cut type assignment. We describe the conditions in this paper and have used this method to screen for HPV infection in clinical populations.
Autoradiography↗
Use of hybridot assay to screen for BK and JC polyomaviruses in non-immunosuppressed patients.
Urine samples from 50 patients attending a genitourinary outpatient clinic and from 13 renal allograft recipients were investigated for evidence of infection with human BK and JC polyomaviruses using cytology and a new DNA hybridot assay. Forty four per cent of samples from the renal allograft recipients were positive by cytology and 75% by DNA hybridisation, indicating that hybridot assay is more sensitive than cytological screening. BK and JC viral DNA was found in 20% of the patients attending the genitourinary clinic, showing infection with BK virus and JC virus in a group of patients with clinical conditions not normally associated with immunological deficiency-a finding that has not been reported before.
Adult↗
DNA hybridization of cervical tissues.
The increasing frequency of cervical neoplasia among younger women and the increased invasiveness of these tumors has led to a considerable growth in research into this disease. Conventional methods (epidemiology, cytology, and immunology), while being extremely useful, also have significant limitations. Recent advances in techniques for the manipulation of DNA now make it possible to analyze tissues for the presence of viral genomes. This review introduces these techniques and describes their application to the search for herpes simplex virus and human papillomavirus sequences in cervical tissue. The significance of the findings both for the mechanism of transmission of the disease, and also the consequences for early detection and hence more successful treatment, are also discussed.
Cloning, Molecular↗
DNA probes in human disease.
Nucleic acid probes are able to detect the presence of particular sequences in a sample down to the level of a few hundred molecules. They can discriminate between similar sequences to a resolution of better than one part in 10(9). They are capable of detecting inherited defects in tissues where the phenotype is not being expressed, and in cases where the biochemical aberration is not understood. They can characterize acquired diseases in somatic cells (both tumours and infectious agents). Additionally, they can be used to characterize multifactorial (either polygenic or requiring an environmental stimulus to interact with a genetic predisposition) diseases. Nucleic acid 'fingerprints' provide an unequivocal identification of the origin of cells which may be applied in criminal law, civil law, and in the follow up to bone marrow transplants. In spite of this tremendous potential, there is still a large gap between their use in research laboratories and their widespread application in pathology laboratories. There are two basic reasons for this. The first is the number of labour-intensive steps involved in the various 'blotting' techniques which greatly reduces the rate at which assays may be performed. The second is the need to use probes labelled with isotopes which are short-lived and may require stringent safety measures to be employed. Recent work both in this laboratory and elsewhere is designed to circumvent both these problems.
Biotin↗