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Derek Middleton

Publications and source records attributed to Derek Middleton.

32 records · Page 2Linked to original sources
PubMed2003

A multi-laboratory characterization of the KIR genotypes of 10th International Histocompatibility Workshop cell lines.

Killer immunoglobulinlike receptors (KIRs) are expressed on natural killer and T cells. Both inhibitory and noninhibitory forms have been described, leading to inhibition or continuation of cellular killing activity. The natural ligands identified so far of KIRs are class I human leukocyte antigens (HLA). In particular, the interaction of some KIRs with HLA-Cw has been well characterized. Recent work has implicated KIRs in affecting the outcome of hematopoietic stem-cell transplant (HSCT). This may well lead to a requirement for prospective KIR typing of donor and recipient. We have utilized different typing systems (two using polymerase chain reaction-sequence-specific primers, and one using polymerase chain reaction-sequence-specific oligonucleotide probes) in three separate laboratories to characterize the KIR gene complement of 25 cell lines from the 10th International Histocompatibility Workshop. There were consistent results in 22, and minor differences in 3. When compared with previous results for some of these cell lines, no further differences were found. The differences are due to typing of KIRs KIR2DL1 and KIR2DS5, and may be explained by technical differences or the inability to type new variants. Further improvements in typing may be required if population and clinical studies are to produce accurate results.

Cell Line↗
PubMed2003

Multiple copies of KIR 3DL/S1 and KIR 2DL4 genes identified in a number of individuals.

Multiple copies of the killer immunoglobulin-like receptor gene, 3DL/S1, have been identified in certain individuals. Additionally, allele determination of the killer immunoglobulin-like receptor gene (KIR), 2DL4, has identified three alleles of this gene present in these same individuals. This event has been confirmed by isolating three distinct KIR2DL4 allele clones in each individual, which sequenced as the alleles identified by the allele identification technique. It is our assumption that an unequal crossover event has occurred between differing KIR haplotypes resulting in the duplication of the 2DL4, 3DS1/3DL1 genes on the newly formed haplotype(s).

Alleles↗
PubMed2003

mt4216C variant in linkage with the mtDNA TJ cluster may confer a susceptibility to mitochondrial dysfunction resulting in an increased risk of Parkinson's disease in the Irish.

Polymorphism of the mtDNA genome has been implicated as playing a role in the development and pathogenesis of Parkinson's disease (PD). A PCR-RFLP methodology was employed to generate genetic haplotypes for a cohort of 90 PD sufferers. No association was observed between the various mtDNA haplotypes observed and PD in comparison to healthy aged controls. The longevity-associated European J haplogroup and T haplogroup were identified and were both found to be in tight linkage with the mt4216C polymorphism. The mt4216C variant was observed at a significantly increased frequency in the PD cases (28%) in comparison to the healthy aged controls (15%; p=0.014). However, when the frequency of the mt4216C variant was examined in a cohort of 200 young controls (18-45 years) a similar frequency to the PD cases (25%) was observed. The frequencies obtained for the two branches of the J haplogroup (J1 and J2) and the T haplogroup in the cohort of PD subjects also reflected those observed for the young controls used in the previous longevity study. These findings lead one to postulate that the mt4216C variant, in linkage with the mtDNA TJ cluster, may influence mitochondrial dysfunction, resulting in an increased risk of PD.

Adult↗
PubMed2003

Increased frequency of the 2437T allele of the heat shock protein 70-Hom gene in an aged Irish population.

The frequency of the functional polymorphism, T2437C transversion (Met-->Thr), in the HSP 70-Hom gene was investigated within a healthy aged Irish population using oligonucleotide probes. The 2437T polymorphic nucleotide was observed to increase in the elderly, although not attaining statistical significance. The TT genotype was observed to be significantly increased within the Irish aged population (p=0.03), while conversely the TC genotype was significantly decreased in the aged subjects (p=0.01). These findings would support the theory that the change from a Met (non-polar and hydrophobic) residue to a Thr (polar and neutral) residue may disrupt the peptide-binding specificity of HSP 70-Hom and have an effect on its functional efficiency. One postulates that the highly significant p-value obtained for the TC genotype may infer that the presence of both the T and the C allele (heterozygosity) resulting in the generation of two different HSP 70-Hom protein species may negatively influence longevity.

Aged↗
PubMed2002

A novel polymorphic triplet repeat in intron five of the alpha-synuclein gene: no evidence of expansion or allelic association with idiopathic Parkinson's disease in the Irish.

The recent discovery of two mutations associated with autosomal dominant Parkinson's disease (PD) has led to the hypothesis that the alpha-synuclein gene plays a role in the pathogenesis of PD. Here we report a novel triplet CAA repeat within the unusually large intron 5 sequence of the alpha-synuclein gene. Microsatellite analysis revealed a high degree of polymorphism within the Irish population with seven alleles detected, ranging from eight to 17 CAA repeats. Analysis of the allele/genotype frequency differences observed between an Irish idiopathic PD cohort (eta = 98) and a healthy aged control group ( eta= 92) revealed no strong association with either group. All PD subjects displaying homozygous profiles were examined for expansion of the trinucleotide repeat, but no expansion was observed. These results would suggest that polymorphism of the alpha-synuclein gene may not play as significant a role in the pathogenesis of idiopathic PD as previously hypothesised.

Adult↗
PubMed2002

Current and emerging technology for HLA typing.

Many techniques based on polymerase chain reaction amplification have replaced the traditional serological technique in the Histocompatibility laboratory. In some tests the specificity is provided in the PCR amplification whereas in others all alleles will be amplified and the specificity is obtained in a second step. The choice of method will depend on a number of factors including time needed to provide result, resolution required and number of samples being processed. New methods or variations of existing methods are continually being developed. However the cost of equipment makes it difficult for a laboratory to decide on which methods to use.

Histocompatibility Testing↗
PubMed2002

Molecular diversity of the HLA-C gene identified in a caucasian population.

A DNA typing procedure, based on a two stage polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) typing strategy, has been developed and applied to DNA from 1000 healthy individuals from the Northern Ireland region. The two-stage procedure involves human leukocyte antigen (HLA-C) identification through the use of a medium resolution PCR-SSOP system, followed by four secondary group specific PCR-SSOP systems, to enable allele resolution. The PCR-SSOP systems were designed for the identification of HLA-Cw alleles with possible discrimination within exons 2 and 3 of the HLA-C gene, i.e., HLA-Cw*01-Cw*16. PCR-SSP tests were designed for the resolution of HLA-Cw*17 and -Cw*18 alleles. The systems can also be used independently of each other if selective allele resolution is required. HLA-Cw allele frequencies occurring within the Northern Ireland population have been compiled, along with estimations of HLA-B/Cw haplotype frequencies.

Genetic Variation↗
PubMed2002

Frequency of cytokine polymorphisms in populations from western Europe, Africa, Asia, the Middle East and South America.

PCR-SSOP identification procedures for IL-2, IL-6, IL-10, TNF-alpha and TNF-beta cytokine polymorphisms have been developed. Application of the procedures to a range of diverse geographically distributed populations has identified ethnic differences within the groups studied. Five populations were investigated, Northern Ireland, South African Zulu, Omani, Singapore Chinese and Mexican Mestizos.

Africa↗
PubMed2002

Natural killer cells and their receptors.

Natural killer (NK) cells have been known for a long time to be a very important component of the innate immune system. However, it is only during the last 10 years that knowledge of their receptors has emerged. Described in the present review are those receptor families killer inhibitory receptor (KIR) (belonging to the immunoglobulin superfamily), and killer lectin like receptor (KLR) CD94/NKG2, that both use HLA as a ligand and have inhibiting and activating types of receptors, and natural cytotoxic receptors (NCR) which do not associate with HLA. Association of the receptor gives rise to either an inhibiting or activating signal leading to either failure or success in lysing a target cell. The KIR receptors are very polymorphic both in the number of genes expressed in an individual and the alleles present for a gene. They would appear to have had a rapid evolution compared to the CD94/NKG2 receptors. The roles that NK cells and their receptors have with various facets of transplantation, disease, pregnancy and control of virus infection in humans are described.

Animals↗
PubMed2002

The outcome of pediatric cadaveric renal transplantation in the UK and Eire.

An analysis of all pediatric cadaveric renal transplant recipients in the UK and Eire was undertaken to review the outcomes of pediatric cadaveric renal transplantation and to consider the implications for organ allocation procedures for pediatric recipients. Factors influencing the outcome of 1,252 pediatric cadaveric renal transplants in the UK and Eire in the 10-yr period from 1 January 1986 to 31 December 1995 were analyzed by Cox proportional hazards regression, including analysis of four distinct post-transplant epochs (0-3 months, 3-12 months, 12-36 months, and beyond 36 months). At the time of analysis (December 2000), 113 (11%) recipients had died and 47% of grafts had failed. In the multi-factorial modelling, the factors significantly affecting transplant outcome were cold ischaemia time, donor and recipient age and human leucocyte antigen (HLA) matching. Epoch analysis demonstrated that these factors operated at different times post-transplant. Cold ischaemia time had a strong influence on outcome at 3 months. A highly significant increased risk of graft failure was associated with donors under 5 yr of age. Young recipients had an increased risk of failure in the short term, but beyond 1 yr post-transplant there were few failures in young recipients while a steady rate of graft loss persisted in the older children. In terms of HLA matching, the worst outcome was observed for two HLA-DR mismatched grafts, while 000 and favorably matched kidneys (100, 010, 110 HLA-A, -B, -DR mismatches) survived longest. Hence, a policy of exchanging organs on the basis of HLA matching is justified for 000 mismatched and favorably matched kidneys. The poor outcome associated with very young donors should discourage pediatric units from transplanting kidneys from such young donors. The reasons for late losses in older recipients need investigation.

Cause of Death↗
PubMed2002

Identification of medically important pathogenic fungi by reference strand-mediated conformational analysis (RSCA).

This report describes the application of reference strand-mediated conformational analysis (RSCA), a novel DNA typing technique, for the identification of clinically significant fungal pathogens. RSCA is a heteroduplex-based conformational method which relies on detecting differences in the DNA conformation of heteroduplexes generated in this study by the annealing of different fungal 18S rRNA amplicons to a common fluorescent-labelled reference (FLR). These heteroduplexes are then observed with laser-based instrumentation and computer software to detect differences in the DNA conformation reproducibly. This technique was shown to generate unique and reproducible profiles for the 18S rRNA gene sequences of a number of medically important fungi, distinguishing different Candida species (C. albicans, C. kefyr, C. dubliniensis, C. lusitaniae, C. guilliermondii, C. tropicalis, C. krusei, C. glabrata, C. sake and C. parapsilosis), and in some cases detecting single nucleotide differences between 18S rRNA sequences. The RSCA technique was further evaluated with 50 human clinical isolates of Candida spp., previously identified by culture techniques, and was shown to identify the isolates correctly. This technique displays enormous potential as an alternative to DNA sequence determination and has the potential to become an automated technique that can be implemented in the routine setting.

Candida↗
PubMedDate not supplied

Mitochondrial DNA damage in lymphocytes: a role in immunosenescence?

An age-related increase of DNA damage/mutation has been previously reported in human lymphocytes. The high copy number and mutation rate make the mtDNA genome an ideal candidate for assessing damage and to act as a potential biomarker of ageing. In the present study, two assays were developed to evaluate the level of mtDNA(4977) and the accumulation of point mutations with age. A competitive polymerase chain reaction (PCR) methodology incorporating three primers was used to detect and quantify the levels of mtDNA(4977) and a novel heteroduplex reference strand conformational analysis (RSCA) technique was used to analyse the accumulation of point mutations. The assays were applied to an in vitro model of T cell ageing and ex vivo DNA samples from an elderly cohort of subjects and a younger control group. The mtDNA(4977) was detected in all the DNA samples examined but only a very low concentration was observed and no age-related increase or accumulation was observed. No accumulation of point mutations was identified using RSCA within the T cell clones as they were aged or the ex vivo lymphocytes from the elderly cohort. A higher level of variation was observed within the ex vivo DNA samples, verifying the high resolution of RSCA and its ability to identify different mtDNA species, although no correlation with age was observed. The low level of mtDNA damage observed with respect to the ex vivo lymphocyte DNA samples within this study may be due in part to the high turnover of blood cells/mtDNA, which may inhibit the accumulation of genetically abnormal mtDNA that may play a role in immunosenescence. A similar explanation may also apply to the in vitro model of T cell ageing if the vast majority of the cells are replicating rather than entering senescence.

Adult↗