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Biomedical subjects

K I Welsh

Publications and source records attributed to K I Welsh.

At least 127 records · Page 7Linked to original sources
PubMed1995

Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP).

Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.

Alleles↗
PubMed1995

Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP).

We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.

Base Sequence↗
PubMed1995

An analysis of HLA class II gene polymorphism in British and Greek idiopathic membranous nephropathy patients.

Fifty-two British and 29 Greek idiopathic membranous nephropathy (IMN) patients were analysed for DRB, DQA1, DQB1 and DPB1 gene polymorphism using second exon amplification and sequence-specific oligonucleotides (SSO). In addition 100 British and 92 Greek controls were analysed. A highly significant increased frequency of the DRB1*0301 allele was found in IMN patients from Britain (80%), when compared to controls (27%, OR 10.6, P = 0.000004). A lower frequency of DRB1*0301 was observed in Greek IMN patients (33%), but this was just significant before correction, when compared to Greek controls (15%, OR 3, P = 0.02). The DRB3 allele most often associated with DRB1*0301 was DRB3*0101 (OR 4.2, P = 0.00025) in British patients and DRB3*0201/2 (OR 11, P = 0.006) in Greek patients. In Greek IMN patients a decrease in DR16 was found (OR 0.08, P = 0.004), and the overall incidence of DR2 was significantly lowered when both sets of IMN patients were combined (OR 0.21, chi 2 17.6, P = 0.00013). The incidence of DQA1*0501 was raised in both Greek (96% vs. 66%, OR 9.7, chi 2 6.9, P = 0.009) and British IMN (85% vs. 45%, OR 7.4, chi 2 20, P = 0.00007) patients. This gives some support to a proposal for a major role for this allele in IMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1995

Human CD4+ T-cell repertoire of responses to influenza A virus hemagglutinin after recent natural infection.

The human CD4+ T-cell repertoire of responses to hemagglutinin (HA) of influenza virus A/Beijing/32/92 was examined 3 to 6 months after natural infection by using a panel of 16-mer peptides overlapping by 11 residues. Short-term CD4+ T-cell lines were derived by using full-length HAs of virus A/Beijing/32/92 from 12 unrelated, major histocompatibility complex (MHC) class I and II haplotyped adults with a history of influenza in November and December 1993 and from 6 adults with no history of influenza during the preceding 4 years but who responded to HA. In contrast to recent murine studies, the human CD4+ T-cell repertoire of responses was dominated by the recognition of highly conserved epitopes. The HA2 subunit, widely regarded as nonimmunogenic, induced strong responses in every donor. This resulted in functional cross-reactivity among influenza A viruses. Our study included one pair of unrelated donors expressing identical HLA DRB1 and DQB1 alleles and two pairs of donors sharing low-resolution MHC class II types. These pairs responded to identical peptides; furthermore, clearly identifiable patterns of response were seen in donors sharing single class II haplotypes, irrespective of the presence of other alleles and exposure history. Two conserved regions which induced responses in 17 of 18 donors were identified (residues 295 to 328 and 407 to 442). Possible implications for cross-reactive T-cell vaccines are discussed.

Adult↗
PubMed1994

Immediate destruction of xenogeneic islets in a primate model.

Transplantation of pancreatic islets from other species to man has the potential to cure diabetes, but whether such islet grafts will be subject to damage due to natural antibody-mediated hyperacute rejection is unknown. We have examined the fate of islet xenografts in a recipient with direct relevance to man, the cynomolgus monkey. Rabbit islets were prepared by an intraductal collagenase technique and incubated in neat rabbit, human, or cynomolgus serum, with and without heat inactivation, for up to 6 days. Islets were analyzed by flow cytometry for IGG and IGM binding, and scored for viability by supravital staining. For in vivo studies, isolated islets were prepared from 4 New Zealand White rabbits (15-34 x 10(3) islets 70-85% purity) and transplanted beneath the kidney capsule of normal cynomolgus monkeys after aggregation in either a rabbit or monkey blood clot. The tissue was retrieved at various times up to 4 days after transplantation and processed for light and electron microscopy. The results showed that rabbit islets bind heterophile antibody of both IGG and IGM subtypes. There was slow loss of islet viability in vitro over 3 days of culture in neat human or cynomolgus serum. Destruction of islets in vivo was more rapid with visible damage within 6 hr associated with neutrophil infiltration. Subsequently, there was heavy mononuclear cell infiltration leading to total destruction within 4 days. The results suggest that immediate mechanisms of graft rejection, possibly compliment and neutrophil mediated, represent a major barrier to islet xenotransplantation in humans.

Animals↗
PubMed1994

Familial scleroderma--evidence for environmental versus genetic trigger.

Families with more than one case of scleroderma are unusual. Four families each with two members (in one case monozygotic twins) with scleroderma (systemic sclerosis, SSc) were identified. Clinical, immunogenetic and autoantibody studies were carried out. Multicase SSc families cited in the literature were reviewed. Each family pair shared cutaneous subset of disease severity, and SSc-associated autoantibody. HLA typing showed two pairs shared an HLA-DR allele associated with scleroderma (DR3 or DR5), while one also had alleles reported in association with their SSc-specific autoantibody. Review of dates and ages of onset suggested that the timing of onset of scleroderma is more likely to have an environmental trigger than to be encoded genetically.

Adult↗
PubMed1994

Rapid DNA typing for HLA-C using sequence-specific primers (PCR-SSP): identification of serological and non-serologically defined HLA-C alleles including several new alleles.

Detection of HLA-C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undetectable HLA-C locus antigens and 9 out of the 29 sequenced HLA-C alleles so far published encode serologically undetected antigens. In addition, HLA-C molecules are expressed at the cell surface at about 10% of the levels of HLA-A and HLA-B. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable and rapid method for typing HLA-DR, HLA-DQA and HLA-DQB genes. PCR-SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA-C alleles corresponding to the serologically defined series HLA-Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA-C PCR-SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA-C antigens can be readily identified. DNA typing for HLA-Cw by PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

Alleles↗
PubMed1993

A highly sensitive, rapid screening method for the detection of antibodies directed against HLA class I and II antigens.

Screening of potential transplant recipients for antibodies that can cause graft rejection is an essential part of the pre-transplant monitoring carried out by tissue typing laboratories. This is a time-consuming process and the rapid reporting of results is dependent on the maintenance of frozen cell panels. The usual procedure of screening against a panel of random cells takes up to 6 weeks. In this study we have used flow cytometric analysis of pooled chronic lymphatic leukaemia (CLL) cells to detect antibodies directed against HLA antigens. We show that FACS screening of pooled cells can accurately and rapidly detect these antibodies and that the method is suitable for routine use. An estimate of the degree of patient panel reactivity can be determined within a few hours. In addition, the technique is more sensitive than those conventionally used, an advantage that may be of importance in preventing graft damage.

Autoantibodies↗
PubMed1993

An analysis with sequence-specific oligonucleotide probes of the association between aspirin-induced asthma and antigens of the HLA system.

BACKGROUND: Previous work has suggested that in addition to environmental influences, there is a genetic predisposition for the development of both asthma and atopy. A subset of asthmatic subjects who are intolerant to aspirin has been identified. A previous study has suggested that there is an association between aspirin sensitivity and HLA-DQw2. METHODS: To further assess this association, we studied two populations of aspirin-sensitive subjects and aspirin-tolerant subjects with asthma. Genomic DNA was amplified by polymerase chain reaction and hybridized with radiolabeled oligonucleotide probes specific to the second exon of the DRB1, DQA1, DQB1, and DPB1 chains. RESULTS: Using polymerase chain reaction amplification of genomic DNA and sequence-specific oligonucleotide probes, we have been unable to confirm a significant association between aspirin sensitivity and HLA-DQw2. CONCLUSION: We have shown, however, that there is a significant decrease in the incidence of DPB1*0401 in both aspirin-tolerant and aspirin-intolerant subjects with asthma in both populations studied.

Adolescent↗
PubMed1993

Complement polymorphism in herpes gestationis: association with C4 null allele.

BACKGROUND: Herpes gestationis (HG) is a rare, pregnancy-related skin disease characterized by the production of an autoantibody to a component of the hemidesmosome. It is associated with the class II antigens HLA-DR3 and HLA-DR4, but its potential association with the "class III antigens" C2, C4, and factor B has not previously been studied. OBJECTIVE: Our purpose was to study complement polymorphism in HG. METHODS: Using electrophoresis and immunofixation techniques, we determined the allele frequencies of C4A, C4B, C3, and factor B in 42 patients with a history of HG. RESULTS: Ninety percent of patients carried a C4 null allele (C4*QO). No statistically significant association with C3 or factor B alleles was seen. CONCLUSION: HG is associated with the presence of a C4*QO. Whether the C4*QO is the primary genetic association, or whether the C4*QO is related to its linkage disequilibrium with DR3 and DR4 has yet to be determined.

Adult↗
PubMed1993

Rapid HLA-DQB typing by eight polymerase chain reaction amplifications with sequence-specific primers (PCR-SSP).

Molecular genotyping of HLA class II genes using group-specific DNA amplification by the PCR followed by probing with (PCR-SSO) probes is too time consuming for the typing of cadaveric organ donors. Recently, amplification of DNA using PCR-SSP has proved a reliable and rapid method for typing HLA-DRB1 genes. PCR-SSP takes 2 hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers that in eight PCR reactions will positively identify the HLA-DQB1 alleles corresponding to the serologically defined series HLA-DQ2, DQ4, DQ5, DQ6, DQ7, DQ8, and DQ9. Presently, 30 homozygous cell lines and 138 individuals have been typed by the DQB1 PCR-SSP technique and compared with a combination of serology and RFLP with 100% concordance. No false-negative or false-positive amplifications were recorded. All combinations of DQB1 can be readily identified. DQB1 PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

Alleles↗
PubMed1993

HLA-DQ2 second-domain polymorphisms may explain increased trans-associated risk in celiac disease and dermatitis herpetiformis.

Sequence polymorphism in the DQ2 beta chain was investigated in 80 Caucasoid patients with CD, 23 patients with DH, and 64 healthy controls. A set of amplification primers were designed to amplify a 281-bp region between amino acids 95 and 135 encoding the second domain of the DQ beta chain gene. A polymorphism at amino acid 135 was shown to distinguish DR3 and DR7 haplotypes. Two SSO probes were designed to identify amino acid sequences (133-135) RND (DR3-DQ2) and RNG (DR7-DQ2). To establish whether polymorphism existed elsewhere in the second-domain sequence, which could explain the migratory characteristics of the CD-associated DR3-DQ2 beta-chain reported by Roep et al. DQB1 second-domain PCR products were sequenced from the genomic DNA of three CD patients. The results showed that the polymorphism at amino acid 135 distinguishing DR3 and DR7 haplotypes was present in CD, DH patients, or normal controls of the appropriate DR and DQ genotypes by oligonucleotide hybridization. Cloning and sequencing of DQB1 second domains of three CD patients (two DR3,3 and one DR3,7) gave normal sequences expected from their genotypes. No specific polymorphism of DQB1 second domains on CD-associated DR3 haplotypes distinguishes them from normal DR3 haplotypes. We conclude that individuals positive for the DR3,7 genotype have the potential to express a unique trans-encoded heterodimer with enhanced ability to predispose people to CD.

Amino Acid Sequence↗
PubMed1993

Connective tissue disease and autoantibodies in the kindreds of 63 patients with systemic sclerosis. The United Kingdom Systemic Sclerosis Study Group.

Systemic sclerosis (SSc) tends to occur in a sporadic fashion and familial occurrence of the disease is unusual. Nevertheless, reports of related connective tissue diseases (CTD), autoantibodies, or both in family members together with associations of certain HLA class II phenotypes with SSc suggest that genetic factors might play a part in susceptibility to the disease. Because of the relative rarity of SSc only a small number of family pedigrees have been studied previously. This report represents the largest study to date, to our knowledge, of family members of patients with scleroderma, and provides the opportunity to investigate the relative importance of genetic and environmental factors operating in the disease. The family pedigrees of 63 patients with systemic sclerosis were examined with respect to clinical, serologic, and immunogenetic features. Multiple cases of SSc were seen only in 1 family, in which the disease affected a father and daughter. Disease expression in these 2 individuals was very similar both clinically and serologically; relatives with other connective tissue diseases were found in 9 families, and nonspecific features of CTD such as Raynaud phenomenon, and arthralgia or arthritis, occurred commonly, especially in female relatives. Antinuclear antibodies (ANA) were also detected more frequently in relatives than in controls. However, antibodies previously demonstrated to have a high degree of specificity for SSc were confined to patients with this disease. Probands had an increase in the frequency of HLA-DR3, DR5, and C4AQO. Patients with diffuse scleroderma had an increased frequency of HLA-DR3, while those with the limited form of the disease had an increased frequency of HLA-DR5.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Anti-HLA antibodies in pemphigoid gestationis (herpes gestationis).

Pemphigoid gestationis (PG; herpes gestationis) is a rare autoimmune disease associated with pregnancy, currently defined by the presence of complement deposition along the cutaneous basement membrane zone. It is known to be associated with HLA-DR3 and DR4, and an increase in anti-HLA antibodies in those with a history of PG has been reported. We have studied 39 patients with an immunofluorescence-confirmed diagnosis of PG for the presence and specificity of anti-HLA antibodies. Anti-HLA antibodies were found in all 39 patients. Specificity was against class I antigens in 98% (controls 10%; P < 0.001) and class II antigens in 25% (controls 8.5%; P < 0.001). Almost all anti-HLA antibodies were cytotoxic. The universal presence of anti-HLA antibodies in PG suggests that they may develop coincidently with antibasement membrane antibodies, and may reflect a common immunological event.

Adult↗
PubMed1993

Gold induced nephropathy in rheumatoid arthritis and HLA class II genes.

OBJECTIVES: To elucidate the role of HLA-DRB, -DQA, and -DQB genes in patients with rheumatoid arthritis (RA) who developed gold induced nephropathy. METHODS: Southern blot analysis of HLA-DRB, -DQA, and -DQB genes was performed on DNA from 27 patients with RA with gold induced nephropathy, 37 patients with RA who were treated with gold but did not develop nephropathy, and 122 ethnically matched normal subjects. RESULTS: The 4.7 kb DQA1/Taq I band associated with DQA1*0501 and DR3 and DR5 was found in 16 (59%) patients with gold induced nephropathy compared with five (14%) patients without gold induced nephropathy. CONCLUSION: It is concluded that HLA-DQA region genes may be an important susceptibility factor for the development of gold induced nephropathy in patients with RA.

Arthritis, Rheumatoid↗
PubMed1993

Clinical and socioeconomic benefits of serological HLA-DR matching for renal transplantation over three eras of immunosuppression regimens at a single unit.

The efficacy of HLA-DR matching in cadaveric renal transplantation is controversial in the cyclosporine (CsA) era. Reports have questioned both the reliability of serological HLA-DR typing as well as the benefit of matching in terms of improved graft survival. Analysis of 1,000 consecutive cadaver donor transplants performed at Oxford between 1975 and 1992 has shown that with improved immunosuppressive regimens and increased transplant success there has been a steadily diminishing influence of HLA-DR matching measured in terms of first graft outcome. For patients treated with azathioprine and prednisolone (n = 278) overall one-year first graft survival was 65%, but there was a 20% improvement associated with HLA-DR matching which has been maintained for up to 15 years. With the introduction of CsA, used either alone or in conjunction with low dose steroids (n = 96), one-year first graft survival was 69% and the difference between HLA-DR-matched and -mismatched transplants was 14%. Our current maintenance immunosuppressive protocol is triple therapy (N = 425) with an 81% one-year first graft survival for both matched and mismatched transplants. However, we do continue to find a marked correlation between HLA-DR matching and clinical course. HLA-DR-mismatched patients suffer more rejection episodes, spend a longer time in the hospital, and have higher creatinine levels at 3 months. This costs, on average, an extra 1,500 pounds for each mismatched transplant. The effect is most apparent in unsensitized males. For cadaveric regrafts, one-year graft survival for patients on triple therapy is 80% (n = 116) which does not differ from first graft survival rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Graft Rejection↗