Biomedical subjects

K I Welsh

Publications and source records attributed to K I Welsh.

At least 109 records · Page 6Linked to original sources

PubMed1997

Polymerase chain reaction haplotyping using 3' mismatches in the forward and reverse primers: application to the biallelic polymorphisms of tumor necrosis factor and lymphotoxin alpha.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system that operates under identical conditions to HLA phototyping was devised for characterizing polymorphisms in tumor necrosis factor (TNF) and lymphotoxin alpha (LT-alpha). Mismatches at the 3' end were incorporated into the forward and reverse primers of each PCR so as to unequivocally establish the cis/trans status between the biallelic sites. Three previously described biallelic polymorphisms in TNF and three in LT-alpha were characterized in a 24-reaction PCR-SSP system. The method was used to genotype 20 cell lines and 201 HLA class I and II typed controls from the United Kingdom at the TNF and LT-alpha loci. Population frequencies of TNF haplotypes were determined as was linkage disequilibrium with HLA-A, B, Cw, DRB1 and DQB1 loci. In each gene there were 8 theoretical polymorphic combinations; 4 were observed in TNF and 4 in LT-alpha. A total of 11 TNF-LT-alpha haplotypes were determined from apparent homozygous controls and statistical analysis.

Alleles↗

PubMed1997

High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology.

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed.

Alleles↗

PubMed1997

A rapid molecular method (polymerase chain reaction with sequence-specific primers) to genotype for ABO blood group and secretor status and its potential for organ transplants.

We hypothesize that kidneys from non-secretor blood group A2 donors may be used for transplant into non-A recipients. In addition, we believe that organs from A2 donors may be used in non-A recipients where the anti-A titer is low. In order to reliably identify non-secretor A2 kidneys from cadaver donors, we have developed a rapid molecular method. The PCR-SSP-based method was developed to genotype ABO blood group and secretor status. Samples of known blood group ABO and Lewis phenotype determined by standard serological methods were used to appraise the method. A retrospective renal cadaver donor study was conducted to assess the potential of using A2 non-secretor organs for transplantation into non-A recipients. Phenotype frequencies of blood group A donors were 76% and 24% for A1 and A2 subgroups respectively, whereas 27% of the donor sample population were non-secretors. Three donors were identified as A2 non-secretors, and analysis was performed to theoretically place the organs by considering them as blood group O. These results coupled with a detailed analysis of HLA type and antibody status of our panel suggests that using A2 donors would be a useful adjunct to strategies for transplanting highly sensitized patients and redressing the donor-recipient imbalance in terms of blood group.

ABO Blood-Group System↗

PubMed1997

High-resolution HLA-DQB1 typing using the polymerase chain reaction and sequence-specific primers.

Polymorphism at HLA-DQB1 is known to influence tissue compatibility and disease susceptibility; however, current DQB1 typing methods are unable to distinguish the 32 currently recognized DQB1 alleles. We have developed a 32-reaction PCR-SSP method capable of differentiating all DQB1 alleles that differ in amino acid sequence. This method can resolve all heterozygous combinations of DQB1 alleles, with the exception of several combinations involving alleles not thus far detected in Caucasoid populations.

Base Sequence↗

PubMed1996

Primary alloreactive cytotoxic T-lymphocytes are not commonly restricted by self-HLA class I antigens.

To assess the role of HLA Class I molecules in the indirect presentation of alloantigen, we have investigated the fine specificity and MHC restriction of in vitro primary alloreactive cytotoxic T-lymphocytes (CTL), using limiting dilution analysis of CTL precursor frequencies in HLA-mismatched responder-stimulator pairs. By employing split-well analysis of limiting dilution (LD) microcultures and third-party target cells bearing a stimulatory HLA Class I antigen alone or in combination with a single responder HLA antigen, we demonstrate that self-Class I restriction of HLA-A- or HLA-B-specific CTL precursors is not a common feature of the primary in vitro alloresponse. Higher frequencies of alloantigen-specific CTL precursors in the presence of self-HLA antigens were only detected in 5 of 31 limiting dilution assays established from seven different responder-stimulator pairs. In two cases, the higher precursor frequencies could be explained on the basis of Class II-restricted presentation of Class I-derived antigenic peptide and are supported by flow cytometric analysis of HLA antigen expression on target cells. The remaining 3 assays of this type were suggestive of Class I restriction but revealed only marginally higher frequency estimates. All other LD assays revealed lower CTL precursor frequency estimates in the presence of self-HLA Class I antigens. A higher antigen-specific CTLp frequency was not detected when targets shared three HLA Class I antigens with the responder, demonstrating that we had not biased the responses by selecting single HLA antigen-sharing targets in the other assays. Analysis of reactivity against PHA blast targets at the single cell per well level demonstrated that CTL reactive only with the original stimulator comprised the majority of lytic reactions. Heteroclitic CTL (i.e., CTL that recognize single HLA targets only and not the original stimulator) formed only a small fraction of total reactivity. Our results confirm the role of Class II antigens in the indirect presentation of alloantigen in vitro but suggest that HLA Class I antigens play a limited role in this phenomenon.

Autoantigens↗

PubMed1996

Enumeration of human alloreactive helper T lymphocyte precursor frequencies by limiting dilution analysis of interleukin-2 production.

We describe a limiting dilution assay for the enumeration of alloreactive interleukin-2 (IL-2) producing helper T lymphocyte precursors (HTLp). In place of the commonly used CTLL cell line, we have employed concanvalin A (ConA) stimulated rat thymocytes as IL-2 responsive indicator cells in a proliferation assay to detect IL-2 levels in limiting dilution microculture supernatants. The proliferation of ConA stimulated thymocytes induced by either recombinant IL-2 or culture supernatants could be blocked by co-incubation with a monoclonal antibody against the rat IL-2 receptor alpha chain, demonstrating the specificity of the response. Our investigations of alloantigen-induced IL-2 production show that (i) a minimum stimulator cell irradiation dose of 50-60 Gy is required to prevent backstimulation of microcultures; (ii) frequencies of alloreactive HTLp are significantly associated with HLA-DR antigen matching between responder and stimulator; (iii) HTLp frequencies detected in assays using B lymphoblastoid cell line stimulators are significantly higher than in assays employing peripheral blood lymphocyte stimulators but possibly reflect a degree of non-specific activation; and (iv) allosensitized responders exhibit altered kinetics of IL-2 production which may permit discrimination between sensitized and naive individuals. Our results both confirm and extend previous reports concerning such features of the alloresponse in humans and demonstrate that ConA stimulated thymocytes are a suitable alternative to CTLL as IL-2 responsive indicator cells in limiting dilution assays for HTLp analysis.

Animals↗

PubMed1996

A correlation between HLA-C matching and donor antirecipient CTL precursor frequency in bone marrow transplantation.

A newly developed, reliable, DNA-based method for typing for alleles of the HLA-C locus has been applied in the context of unrelated, volunteer donors for bone marrow transplantation. Some donors matched for HLA-A, -B, -DR, and -DQ have been found to generate in vitro high frequencies of CTL reactive with the recipient's cells. Here we demonstrate that there is a highly significant correlation of the frequencies of CTL precursors and incompatibility at the HLA-C locus. These data indicate that HLA-C locus incompatibility should be avoided in unrelated donor bone marrow transplantation.

Bone Marrow Transplantation↗

PubMed1996

Contribution of genes of the major histocompatibility complex to susceptibility and disease phenotype in inflammatory bowel disease.

BACKGROUND: Despite strong evidence implicating immune dysfunction and genetic predisposition in the pathogenesis of the chronic inflammatory bowel diseases Crohn's disease and ulcerative colitis, the importance of the genes of the major histocompatibility complex remains uncertain. We have investigated the contribution of HLA DRB1 and DQB genes by the strategies of non-parametric linkage analysis (affected sibling pair method) as well as association study. The relation between genotype and phenotype was examined in detail. METHODS: For linkage analysis 74 families in whom two or more siblings had inflammatory bowel disease were studied. A total of 83 affected sibling pairs were involved: in 42 pairs both siblings had Crohn's disease; in 29 both had ulcerative colitis; in 12 one sibling had Crohn's disease, the other ulcerative colitis. For the association study there were 175 patients with ulcerative colitis, 173 with Crohn's disease, and 472 controls. Details of sex, age of onset, disease extent, and family history were analysed. 24 patients with ulcerative colitis and 92 with Crohn's disease required surgery for refractory disease. HLA DRB1 and DQB1 gene-typing was performed by polymerase chain reaction with sequence-specific primers. FINDINGS: In ulcerative colitis, the sharing of alleles among affected sibling pairs provided evidence for linkage with DRB1 locus (p = 0.017, chi2 = 5.32). Of 29 affected sibling pairs studied, only one pair shared no DRB1 DQB haplotypes. 15 shared two DRB DQB haplotypes. In contrast, no linkage was noted for Crohn's disease (42 sibling pairs; p = 0.30, chi2 = 0.16) or for inflammatory bowel disease overall (83 sibling pairs, p = 0.16, chi2 = 2.28). In the association study the rare DRB1*103 (8.3% vs 3.2% in controls) and DRB1*12 (8.6% vs 2.1% in controls) alleles were associated with ulcerative colitis (p = 0.0074, chi2 = 7.22, odds ratio OR = 2.9 [95% CI 1.3-6.4] and p = 0.0056, chi2 = 12.63, OR = 4.33 [1.8-11.0] respectively). No association with alleles representing DR2 (p = 0.55, chi2 = 0.34) was noted. No overall association was seen in Crohn's disease. In ulcerative colitis, the frequency of DRB1*0301 DQB*0201 (DR3 DQ2) was reduced in females (9.8% vs 26.3% in controls, p = 0.037, chi2 = 8.39 OR = 0.34 [0.15-0.71]), particularly in those with distal disease (2.3%, p = 0.001 vs controls, chi2 = 11.35, OR = 0.07 [0.00-0.39]). In both males and females, the DR3 DQ2 haplotype was predictive of extensive ulcerative colitis (32.9% vs 10.7% in distal disease, p < 0.01, chi2 = 10.94, OR 4.09 [1.70-10.6]) but not of need for surgery (p = 0.93, chi2 = 0.01). INTERPRETATION: These data provide strong evidence for genetic heterogeneity in inflammatory bowel disease. Genes of the major histocompatibility complex are implicated as important inherited determinants of susceptibility to ulcerative colitis and may also influence the pattern of disease. In Crohn's disease, important susceptibility genes are likely to exist outside the HLA region.

Adult↗

PubMed1996

Human cytomegalovirus infection is not increased in common variable immunodeficiency.

It has been postulated that human cytomegalovirus (HCMV) infection may have a role in the pathogenesis of common variable immunodeficiency (CVID). Many patients have a lymphocyte phenotype similar to that seen in HCMV infection, HCMV mononucleosis may precipitate hypogammaglobulinaemia, and a previous small study of common variable immunodeficient patients reported a high rate of active HCMV infection. This study investigated the presence and activity of HCMV infection in 102 CVID patients. Buffy coats were examined for the presence of HCMV IE and glycoprotein B genes using highly sensitive nested PCR. 30 blood donors of known HCMV serologic status were used as controls. There was no significant difference in HCMV positivity by PCR between patients and controls. Enrichment for mononuclear cells prior to PCR had no effect on sensitivity. Twenty-five patients were also examined for HCMV antigenaemia by staining buffy coat cytospins with monoclonal antibodies directed against the HCMV pp65 lower matrix protein, a technique widely used for diagnosis of active HCMV disease. Only one patient was positive (and also positive by PCR). Whilst these results do not exclude prior infection contributing to antibody deficiency in a small proportion of CVID patients, this study refutes the previously reported increase in active HCMV infection in CVID.

Adolescent↗

PubMed1996

DNA allelic alterations within VNTR loci of scleroderma families.

We have characterized genetic alterations at the molecular level in 49 scleroderma and 45 control families using variable number tandem repeats (VNTRs). Additionally, paired fibroblast cell lines from the 'affected' and 'unaffected' skin and peripheral blood leucocytes of 30 patients were also examined. All families in this study were typed for Class I Cw alleles and Class II-DRB, -DQA and -DQB to confirm family membership. There were significant rises in the level of VNTR mutations in scleroderma patients (36.7%, n = 18), their siblings (16.3%, n = 13) and offspring (21.7%, n = 15). The level of VNTR mutations in the control group was 0.6% (n = 5). These mutations did not correlate with the presence of autoantibodies and no patient was taking a known clastogenic drug. The most common VNTR site for mutation was pYNZ22 (17p13.4). Differences were also seen in the VNTR alleles between fibroblast and lymphocyte DNA from the same patient, as measured by size alteration of one of the alleles. We have found that VNTRs are unstable in scleroderma patients, relatives and offspring. The reason for the genomic changes remains unknown, but previous studies have implicated the presence of a clastogen.

Alleles↗

PubMed1996

Telomere reduction in scleroderma patients: a possible cause for chromosomal instability.

We have hypothesized that the chromosomal instability observed in scleroderma patients and their family members may result from the loss of long stretches of the telomeric repeat which is found at the ends of all linear chromosomes. We examined the telomere lengths in scleroderma (SSc) patients (n = 43), their family members (n = 182) and in age-matched controls (n = 96) using restriction fragment length polymorphism (RFLP) and chemiluminescent labelled probes. The average loss of telomeric DNA in SSc patients and family members was found to be 3 kb when compared to the controls. This loss was not related to age or the duration of the disease. These results may reflect a genetic predisposition for chromosomal instability in these families, or exposure to a common environmental agent. A wide variety of common environmental agents are known to produce chromosomal aberrations: these include fungicides, pesticides, air pollutants and drugs. Scleroderma-like syndromes may be induced by some of these agents.

Adolescent↗

PubMed1996

Detection of the DRB4 null gene, DRB4*0101102N, by PCR-SSP and its distinction from other DRB4 genes.

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Genes, MHC Class II↗

PubMed1996

High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology.

Alleles↗

PubMed1996

HLA-DPB polymorphisms: Glu 69 association with sarcoidosis.

Sarcoidosis is a chronic granulomatous disorder, which is characterized by the accumulation of activated CD4+ T lymphocytes (T cells) at disease sites. There is up-regulation of cell surface expression of MHC molecules in sarcoidosis, and it has been suggested that specific MHC class II alleles are associated with the disease. A study of chronic beryllium disease (CBD), a granulomatous disorder which is pathologically similar to sarcoidosis, has identified an association between this disease and the presence of a glutamine residue at position 69 (Glu 69+) of the B1 chain of the HLA-DPB molecule. A further study also suggested the importance of Glu at position 55 of the same chain. The aims of the present study were to attempt to define MHC class II alleles associated with sarcoidosis by comparison of their frequency in two groups of subjects and to compare the frequency of HLA-DPB1 Glu 69+/- and Glu 55+/-alleles in the same subjects. Forty-one subjects with sarcoidosis and 76 normal subjects were studied. The polymorphic regions of the class II MHC were identified by PCR in association with sequence-specific oligonucleotide probes. There were no significant differences in the phenotype frequencies of MHC class II or Glu 55+ alleles between the two groups of subjects. However, there was a significant increase (P = 0.02) in the frequency of HLA-DPB1* Glu 69+ alleles compared with the control population. We therefore suggest that the presence of a Glu residue at position 69 on the DPB1 chain may play an important role in antigen presentation and recognition in chronic granulomatous diseases.

Alleles↗

PubMed1996

Scleroderma fibroblast phenotype is modulated by endothelial cell co-culture.

OBJECTIVE: We established a co-culture system to investigate endothelial cell-fibroblast interaction in scleroderma (systemic sclerosis, SSc). Such a system allows reciprocal interaction between these cells. The pattern of phenotypic modulation for normal and SSc fibroblasts in co-culture was compared. METHODS: A virally transformed human umbilical vein endothelial cell (HUVEC) derived cell line (1E-7) was cultured on nitrocellulose membrane inserts above dermal fibroblast monolayers. The effect of co-culture on fibroblast number, [3H]-thymidine ([3H]-TdR) incorporation, and collagen (type I) production were compared for 10 SSc and 5 control cell lines. Co-culture with the epithelial lines A549 and A431, and nontransformed HUVEC, was also investigated. RESULTS: We observed a statistically significant increase in cell number and a reduction in collagen production for SSc, but not control, fibroblasts co-cultured with endothelial cells. This co-culture also promoted [3H]-TdR incorporation in both SSc and control fibroblasts. While epithelial cell lines did not influence fibroblast cell number, collagen production by SSc fibroblasts was diminished by A549. CONCLUSIONS: Endothelial cell derived soluble factors modulated fibroblast properties in co-culture, and the different response of SSc compared with normal fibroblasts provides further evidence for a link between endothelial and fibroblast dysfunction in this disease. However, similar effects on SSc fibroblast collagen production were also observed for some epithelial cells, suggesting that modulation of fibroblast properties is not restricted to cells of endothelial origin.

Adult↗

PubMed1995

HLA type as a predictor of mixed connective tissue disease differentiation. Ten-year clinical and immunogenetic followup of 46 patients.

OBJECTIVE: To determine any clinical or genetic markers of differentiation and outcome in a previously described cohort of 46 patients with mixed connective tissue disease (MCTD). METHODS: Patients were clinically evaluated, chart notes reviewed, and HLA subtyping and immunology profiles performed where possible. Eleven had died and 7 were lost to followup. RESULTS: MCTD had differentiated into systemic lupus erythematosus in 12 patients and into systemic sclerosis in 13. The latter was associated with HLA-DR5 (P = 0.038), and nondifferentiation was associated with HLA-DR2 or DR4 (P = 0.007). Three HLA-DR4 positive patients had MCTD that evolved into rheumatoid arthritis. Erosive and/or deforming arthritis was associated with HLA-DR1 or DR4 (P = 0.015). HLA-DR3 was associated with interstitial lung fibrosis (P = 0.044) and keratoconjunctivitis sicca (0.001 < P < 0.01). Severe Raynaud's phenomenon predicted higher mortality (0.01 < P < 0.05). CONCLUSION: We suggest that MCTD is, for most patients, an intermediate stage in a genetically determined progression to a recognized connective tissue disease. Those whose disease remains undifferentiated might be considered a distinct subset.

Adolescent↗

PubMed1995

The flow cytometric detection of alloantibodies in screening for renal transplantation.

Flow cytometric screening of sera using pooled chronic lymphocytic leukaemia (CLL) cells has previously been reported as a quick method for detecting HLA antibodies of the IgG class. In this study we investigated the sensitivity of this method in the detection of IgG and IgM alloantibodies, and its performance in serum screening when compared to conventional microlymphocytotoxic screening. Results indicate that flow cytometric screening is more sensitive in the measurement of IgG alloantibodies by up to five doubling dilutions, whereas the converse is true for IgM. IgM autoantibodies were found not to be detectable by flow cytometry. By testing a large number of sera by both methods in parallel, we have found that a significant proportion of sera exhibiting no activity of IgM activity alone on cytotoxic screening contain IgG antibodies detectable with a pool of CLL cells on the flow cytometer.

Cytotoxicity Tests, Immunologic↗

PubMed1995

Class II MHC typing in pemphigoid gestationis.

Pemphigoid gestationis (PG) is a rare, autoimmune skin disease associated with pregnancy or the immediate post-partum period, previously shown to be associated with the HLA class II antigens DR3 and DR4. Advances in molecular analytical techniques now allow the identification of HLA alleles previously difficult to define by serological assays. Unsuspected polymorphism within the HLA-DR3 and DR4 classes can, therefore, be identified. The aim of our study was to apply these newer techniques to the question of genetic predisposition in PG by re-evaluating the association with DR3 and by studying a possible link with DQ. We have investigated by restriction fragment length polymorphism, the DQA, and by sequence specified oligonucleotide probing the DQB and DRB1 (HLA DR) specificities of 41 women with immunofluorescence-confirmed PG. The principal finding of this study is that there is an association between PG and DRB1*0301 (DR3) and DRB1*0401/040X (DR4). Although there is also an increase (P = 0.06) in the concurrent presence of both antigens, this appears to be due to the association with either antigen alone. We also found an increase in the frequency of DQA1*2 (P = 0.016 vs. control) and a decrease in frequency of DQB1*0201 (P = 0.022 vs. controls) and DQB1*0602 (P = 0.026 vs. controls).

Case-Control Studies↗