Biomedical subjects

D Middleton

Publications and source records attributed to D Middleton.

At least 55 records · Page 3Linked to original sources

PubMed1999

Genetic association studies of tumour necrosis factor alpha and beta and tumour necrosis factor receptor 1 and 2 polymorphisms across the clinical spectrum of multiple sclerosis.

Allelic association studies with microsatellite markers around the tumour-necrosis factor (TNF) genes have demonstrated significantly different allele distributions of TNF markers (a and b) between relapsing-remitting/secondary progressive multiple sclerosis (MS) (RR/SPMS) patients and normal controls. Considering the suspected genetic and immunological heterogeneity in MS, we tested this association in primary progressive MS (PPMS) patients. Elevated levels of serum soluble TNF receptors (sTNF-R) are reported in patients with gadolinium enhancing lesions, and animal models suggest a possible therapeutic role of sTNF-RI in MS. Thus we performed similar association studies using markers for the TNF-R genes. Gene association studies were carried out on 199-216 normal controls, 174 RR/SPMS patients and 102 PPMS patients using polymorphic dinucleotide repeat TNF markers (a, b and d), and separate markers for TNF-RI and TNF-RII. Forward primers were fluorescently labelled, polymerase chain reaction (PCR) products were analysed on a fluorescent fragment analyser, and Genescan 672 software was used for allele sizing. Samples were typed for HLA-DR antigens using PCR technology and sequence-specific oligonucleotide probes. TNFa marker allele distributions differed significantly between PPMS patients and controls (P = 0.028), largely attributable to an increase in the 118-bp TNFa allele in PPMS patients (P = 0.00024). Allele distributions were similar in PPMS and RR/SPMS patients (P = 0.91). Logistic regression analysis, however, indicated that these associations were not independent of that with HLA-DRB1*15. For the TNFb marker, the 127-bp allele showed association with both patient categories (PPMS vs. controls, P = 0.010; RR/SPMS vs. controls, P = 0.027), whilst the 128-bp allele occurred more frequently in controls (PPMS vs. controls, P = 0.036: RR/SPMS vs. controls, P = 0.0009). As with the TNFa 118 bp allele, the association with TNFb was not independent of the HLA association. No association occurred with the TNFd marker, and there were also no significant differences in allele frequencies between MS groups and controls regarding the marker for TNF-RI or TNF-RII. In Northern Irish patients the TNF contribution to MS genetic susceptibility is therefore similar across the clinical spectrum of the disease but is not independent of the association with HLA-DRB1*15.

Adult↗

PubMed1999

A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.

Bone Marrow Transplantation↗

PubMed1999

Aberrant splicing of intron 1 creates a novel null HLA-B*1501 allele.

A comparison of serological and DNA HLA class I typing data identified a serological "blank" HLA-B15 antigen in a volunteer donor on the bone marrow registry. Isoelectric focusing and Western blot analysis of a cell line established from this individual confirmed that the HLA-B15 antigen is not expressed at the cell surface. Nucleotide sequence analysis of the HLA-B*15 null allele revealed a 10-bp deletion near the 3' end of intron 1, when compared to the normal HLA-B*1501 sequence. All of the HLA-B*15 specific cDNA clones examined retained the intron 1 sequence. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis demonstrated that the HLA-B*15 mRNA molecule contained the intron 1 sequence, indicating an inability to efficiently splice out intron 1 from the mRNA transcript. The retention of the mutated intron 1 sequence in the mRNA causes a frameshift and premature termination of translation at the start of exon 2, explaining the HLA-B*1501 null phenotype. Our data predicts that the HLA-B*1501 null allele would express a small truncated protein containing the signal sequence fused to an ORF within intron 1 and terminating (out of frame) just within exon 2.

Alleles↗

PubMed1999

A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/ split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.

Hematopoietic Stem Cell Transplantation↗

PubMed1999

Molecular analysis of HLA class I alleles in the Mexican Seri Indians: implications for their origin.

The molecular analysis of HLA class I loci has demonstrated that, although, the genetic profile is restricted in Amerindians, several micropolymorphisms may be important in conferring a biological advantage. We analyzed the HLA-A and B genetic profile of Seris, a Mexican Indian tribe living in northwestern Mexico in the state of Sonora. There are presently only 619 individuals. Our study included 100 Seris belonging to nine families. HLA-A and -B loci typing was performed by polymerase chain reaction using an amplification refractory mutation system (PCR-ARMS) on a select group of samples; all of them were typed by polymerase chain reaction using sequence-specific oliogonuoleotide probes (PCR-SSOP) at a low-intermediate resolution level. The correlation between the techniques was 100%. Only five HLA-A alleles and seven HLA-B alleles were found. A*0201, A*68, A*31, A*24, B*3501, B*40, B*51, B*3512 and B*15 were present in over 5% of the individuals. B*27052 was detected in 2%. B27 is absent in any other Mexican Indian groups previously studied. The presence of B27 may be the result of a founder effect due to different waves of southward migrations. The B-locus is more diverse and the prevalent haplotypes were: A*0201-B*3501, A*0201-B*40, A*0201-B*3512, A*31-B*51, A*68-B*3501 and A*68-B*40. This genetic profile is different from the pattern of other Mexicans. The phylogenetic tree suggests that Seris are more closely related to the Warao Indians from Venezuela, who live in a similar ecosystem, and to some groups of Argentina, than they are to the Mexican Lacandones who live in the jungle. These data emphasize the relevance of the interaction between genes and environment.

Genetics, Population↗

PubMed1999

Allele resolution of HLA-A using oligonucleotide probes in a two-stage typing strategy.

High-resolution polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing methods for HLA-A identification have been established. The four systems, which operate independently of each other, are intended for use as secondary typing systems following HLA-A identification with a medium-resolution PCR-SSOP technique. The systems, all using digoxigenin-labelled probes, are based on group specific amplifications for resolution of: i) HLA-A*29 & -A*33; ii) HLA-A*24 & -A*30; and iii) HLA-A*26, -A*25, -A*11, -A*34, -A*66 and -A*68 alleles, respectively. The fourth system, for the detection of HLA-A*02 alleles, is a modification of a previously reported PCR-SSOP subtyping system. The methods have been applied to individuals from the local bone marrow registry and HLA-A allele frequencies for the Northern Ireland population have been established.

Alleles↗

PubMed1999

Improvements to HLA-B*27 testing.

Explore the source record for details and available documents.

Genetic Testing↗

PubMed1999

Characterisation of a novel HLA-A pseudogene, HLA-BEL, with significant sequence identity with a gorilla MHC class I gene.

During the development of an HLA-A polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) method for the identification of HLA-A*24 and -A*30 alleles, group amplification resulted in the formation of an unusual PCR product in certain individuals. This fragment was approximately 900 bp smaller than the expected product and was also detected in some non-HLA-A*24- and -A*30-positive individuals acting as negative controls for the group specific amplification. Nucleotide sequence analysis of this product identified it as a unique class I gene sequence displaying homology to both primate and human class I A-locus genes. The entire gene was amplified using PCR and the complete DNA sequence information from exon 1 to exon 8, including introns, was determined. A recombination event was identified which results in the fusion of intron 2 with intron 3, causing a deletion of the intervening exon 3 sequence. In addition, there are two cytosine insertions in the poly-cytosine stretch at the start of exon 4 which cause a frameshift and premature termination. The exon 1 and 2 sequences most closely align with the gorilla allele A*0501, displaying only five mismatches. PCR analysis has established that the gene is associated with the following HLA-A types: HLA-A*3001, -A*3301, -A*3303, -A*6802, -A*2901, -A*0203, -A*0205 and -A*31012. Reverse transcription (RT)-PCR analysis of individuals containing this gene failed to detect any mRNA transcription, suggesting that this is a previously undescribed non-expressed class I pseudogene which we have provisionally named HLA-BEL. Its unique gene structure gives a possible insight into the evolutionary pathway that created HLA class I genes.

Alleles↗

PubMed1999

Histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors: report from The World Marrow Donor Association. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

Adult↗

PubMed1999

Influence of the mu-chain C-terminal sequence on polymerization of immunoglobulin M.

Immunoglobulin (IgM) is found in various states of covalent polymerization (microL)n, where n is typically 8, 10, or 12. The usual form of IgM of bony fish is tetrameric (8 microL units) as compared to the pentameric form (10 microL units) observed in cartilaginous fish and mammals. Two hypotheses were tested in this study. First, that the length of the mu-chain C terminus following Cys575 determines whether an IgM polymerizes as a tetramer or as a pentamer. This was tested by examining the covalent polymerization state of mouse IgM mutated to contain a series of mu-chain C-termini from bony and cartilaginous fish. The results proved this hypothesis wrong: mouse IgM bearing the C-terminal sequence of shark, salmon and cod mu-chain behaved identically to native mouse IgM, forming predominantly (microL)10 and (microL)12 forms. The second hypothesis was that an additional Cys residue near the C terminus of the mu-chain is responsible for the multiple covalent structures seen in IgM of the channel catfish. The addition of a catfish C terminus to the mouse mu-chain resulted, as predicted, in the production of a series of covalently bonded forms, with the major species being (microL)4. When a Ser-Cys unit was removed from the catfish C terminus added to the mouse mu-chain, this resulted in production of IgM indistinguishable in structure from that of wild-type mouse IgM.

Animals↗

PubMed1999

Diagnostic PCR with Leishmania donovani specificity using sequences from the variable region of kinetoplast minicircle DNA.

Kala azar or visceral leishmaniasis, a parasitic disease caused by Leishmania donovani, is presently causing an epidemic in the eastern region of India. Diagnosis of kala azar is often complicated. We developed a pair of oligonucleotides suitable as primers from the variable region of a predominant sequence class of minicircles of L. donovani. These primers were used in a nonisotopic polymerase chain reaction and found to be highly specific for the parasites of L. donovani complex. Using these primers, amplification of L. donovani kinetoplast DNA minicircle from the peripheral blood of kala azar patients results in a product of 204 bp. The patient group was comprised of individuals from a highly endemic region of India. We feel that PCR could assess the efficacy of new leishmanicidal drugs under investigation in these patients. PCR could also predict response to therapy which would be useful for both clinical and research applications.

Adolescent↗

PubMed1999

Paraoxonase polymorphisms are not associated with cardiovascular risk in renal transplant recipients.

BACKGROUND: Paraoxonase (PON1) gene variants have been identified as risk factors for cardiovascular disease (CVD). There are two common PON1 polymorphisms at position 55 (Leu-Met change) and 192 (Gln-Arg change) of the amino acid chain. Leucine at position 55 and arginine at position 192 have been associated with increased cardiovascular risk. The increased prevalence of CVD in renal transplant recipients can be only partly explained by the increased prevalence of conventional risk factors. METHODS: We therefore investigated PON1 polymorphisms in renal transplant recipients (N = 491) with (N = 103) and without CVD (N = 388) using polymerase chain reaction-restriction fragment length analysis. PON1 polymorphisms and their associated PON1/arylesterase activities were also assessed in a subgroup of patients (N = 165). RESULTS: The genotype distribution and allele frequencies for both polymorphisms were similar in both groups. The frequencies for LL, LM, and MM genotypes for the 55 position in patients with CVD were 0.39, 0.51, and 0.10, respectively, compared with 0.43, 0.43, and 0.14 in patients without CVD (P = 0.31). The distribution for the QQ, QR, and RR genotypes at the 192 position were 0.48, 0.43, and 0.09, respectively, in patients with CVD compared with 0.46, 0.46, and 0.08 in patients without CVD (P = 0.8). There were highly significant differences in serum activities of PON1/arylesterase between genotypes defined by 55 and 192 polymorphisms. Leucine at position 55 and arginine at position 192 were associated with higher activities. CONCLUSION: These data indicate that there is no association between the PON1 gene variants, conferring higher enzyme activity, and the increased cardiovascular risk in renal transplant recipients.

Adult↗

PubMed1999

Genetic, morphometric and immunohistochemical markers of latent coeliac disease.

BACKGROUND: It is recognized that coeliac disease may exist in a latent form characterized by HLA-DR3 and increased counts of intra-epithelial lymphocytes (IELs) and gamma/delta T cells in jejunal biopsies. To determine whether subjects with persistent serological markers 4 and 13 years after a population screening survey have the HLA constitution of coeliac disease and/or minor morphometric abnormalities of the small intestine, including raised gamma/delta T-cell counts, as possible indicators of latent coeliac disease. SUBJECTS: Participants with positive serology detected by the Belfast MONICA Project surveys (1983 and 1991) were subdivided into those with persistently positive serology (persistent serology), negative serology at follow-up (transient serology) and those with enteropathy (coeliac disease). Morphometric features were compared with MONICA controls who had negative serology and HLA antigen frequencies were compared with blood donor controls. METHODS: Subjects were followed up in 1994-1996 and were re-tested for IgA antibodies to gliadin, endomysium and reticulin. HLA typing was carried out and IELs and gamma/delta T-cell counts were assessed in jejunal biopsies in subjects who gave consent. RESULTS: Persistent serology mainly concerned antigliadin (AGA) and antireticulin (ARA) antibodies but one patient had positive antiendomysial antibody (EMA) and ARA in 1983, which became negative at follow-up, at which time they were positive for AGA. No significant differences were observed between IELs or gamma/delta T-cell counts when the persistent and transient groups were compared in turn with the MONICA controls. HLA-DR2 was expressed in 11 of 16 in the persistent group compared to 47 of 150 blood donor controls (P = 0.013). HLA-DR3 occurred in 15 of 17 coeliac patients compared to 37 of 150 blood donors (P = 0.00001). CONCLUSIONS: Persistent serological markers following population screening do not appear to indicate latent coeliac disease.

Adult↗

PubMed1999

Cyclosporin associated gingival overgrowth in renal transplant recipients.

OBJECTIVES: To investigate the prevalence and severity of gingival overgrowth in a group of renal transplant recipients treated in one centre in Northern Ireland. STUDY DESIGN: A consecutive group of patients who had had a renal transplant for at least 6 months and were attending the Renal Unit in Belfast City Hospital took part in the study. These were divided into a group of 84 subjects treated with cyclosporin since their transplant who were compared with a control group of 36 transplant recipients who had never received cyclosporin. Each subject had a periodontal examination and completed a questionnaire. The severity of gingival overgrowth was scored from plaster models. OUTCOME MEASURES: Clinically significant gingival overgrowth was equated with a score of > or = 30 using the index developed by Seymour et al (1985). RESULTS: 41 (49%) of the cyclosporin group had clinically significant gingival overgrowth compared with none of the controls. A significantly higher proportion of males had overgrowth than females. There were significant correlations between age at transplant, plaque, bleeding, pocketing and the severity of gingival overgrowth. Many patients with clinically significant gingival overgrowth were apparently unconcerned about this condition. CONCLUSIONS: It is concluded that gingival overgrowth is a significant problem for renal transplant patients treated with cyclosporin, particularly if they are also treated with a calcium channel blocker. None of the factors measured, in isolation, explained the variable expression of gingival overgrowth in those at risk.

Adult↗

PubMed1999

Distribution of life cycle stages of Sarcoptes scabiei var wombati and effects of severe mange on common wombats in Victoria.

Seven female and three male common wombats (Vombatus ursinus) collected from forested areas of Victoria (Australia) over a 10 mo period, 10 April 1997 to 22 February 1998 had at least 30% of their skin affected by severe hyperkeratotic sarcoptic mange. Mangy wombats were grazing during the day, could be readily approached, were in poor body condition, and lacked subcutaneous fat. The anterolateral surface of the body was most heavily parasitised with Sarcoptes scabiei var wombati followed by the posterolateral surface, the dorsal region between the ears, the ears, ventral abdomen, medial aspect of the legs, axillary and inguinal areas, and the dorsal midline. Larvae were the most prevalent life-cycle stage followed by eggs, nymphs, females, and males. Mite numbers and the severity of clinical signs, namely thickness of scale crust and the degree of alopecia, were correlated and were symmetrical on each side of the body. Fissuring of crust and skin only occurred when scale crust was present. Bacterial infections occurred in three of 10 wombats within lymph nodes or the pleural cavity. Lymphoid depletion did not occur in lymph nodes or spleens and prescapular lymph nodes contained a greater amount of nuclear debris in germinal centres than non-mangy wombats. Seven wombats had fatty change in their livers. Gonads of mature wombats were not active or had minimal activity. Significant histopathological changes were not seen in the gastrointestinal tract, kidney, brain, myocardium, spleen, thyroid, reproductive tract, and gonads. Hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and concentrations of hemoglobin, lymphocytes, calcium, glucose, creatinine, total solids, total protein, albumin determined both colormetrically and electrophoretically, and globulins were significantly lower and concentrations of neutrophils, monocytes, phosphorus, urea, glutamate dehydrogenase, aspartate aminotransferase and creatine kinase were significantly higher in mangy versus captive wombats. Concentrations of erythrocytes, mean corpuscular hemoglobin, leucocytes, band neutrophils, eosinophils, nucleated erythrocytes, sodium, potassium, chloride, total bilirubin, alkaline phosphatase, and gamma glutamyltransferase for mangy wombats were not significantly different from that reported for captive wombats. Hematological and pathological changes in mangy wombats were consistent with anemia, inflammation, and changes seen with starvation.

Alopecia↗

PubMed1999

History of DNA typing for the human MHC.

The last twenty years have seen an exponential growth in the application of DNA technology to the field of Histocompatibility and Immunogenetics (H&I). Initially, this was confined to a few research laboratories. However, the development and application of several different DNA methods by many laboratories has led to the situation whereby nearly every H&I laboratory performs some DNA typing for the detection of HLA alleles. It would not be unfair to say that H&I has shown diagnostic laboratories in other disciplines how useful the DNA techniques can be. This review attempts to summarise the history and application of DNA methods in the field of Histocompatibility (Table 1).

DNA↗

PubMed1998

Mutation screening in exons 3 and 4 of alpha-synuclein in sporadic Parkinson's and sporadic and familial dementia with Lewy bodies cases.

Recently it has been reported that a missense G(88)C mutation within exon 3 and a missense G(209)A mutation within exon 4 of the alpha-synuclein gene were linked to familial Parkinson's Disease (PD). We decided to investigate if these and any other mutations in exons 3 and 4 of the alpha-synuclein gene could be detected in sixty two sporadic PD and dementia with Lewy bodies (DLB) patients. Four cases of familial DLB were also studied, two of which were from the same family. Single stranded conformational polymorphism, DNA sequencing analyses and PCR-RFLP of exons 3 and 4 failed to reveal any nucleotide changes. However, three nucleotide differences occurred in the intron 4 sequence compared to the published sequence. This study adds further support to the idea that these particular mutation in the alpha-synuclein gene are a rare case of PD and now, as we have shown here, also of DLB.

Dementia↗

PubMed1998

Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments.

Alpha-synuclein (alpha-syn) protein and a fragment of it, called NAC, have been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the alpha-syn gene have been reported in families susceptible to an inherited form of Parkinson's disease. We have shown that human wild-type alpha-syn, mutant alpha-syn(Ala30Pro) and mutant alpha-syn(Ala53Thr) proteins can self-aggregate and form amyloid-like filaments. Here we report that aggregates of NAC and alpha-syn proteins induced apoptotic cell death in human neuroblastoma SH-SY5Y cells. These findings indicate that accumulation of alpha-syn and its degradation products may play a major role in the development of the pathogenesis of these neurodegenerative diseases.

Amyloid↗