Kenneth I. Welsh

IMMUNOGENICITY OF HUMAN AMNIOTIC EPITHELIAL CELLS AFTER TRANSPLANTATION INTO VOLUNTEERS

Human amniotic epithelial cells do not express on their surfaces HLA-A, B, C, and DR antigens, or beta 2-microglobulin. In vitro these cells synthesise the enzymes lacking in patients with selected enzymatic deficiencies: the survival of a transplanted monolayer of human amniotic epithelial cells was therefore investigated in seven volunteers. None of the volunteers showed clinical signs of acute rejection, and amniotic epithelial cells were demonstrated by biopsy up to 7 weeks after implantation. HLA antibodies were not detected in samples of serum from four volunteers thoroughly investigated, and there was no in-vitro lymphocyte reaction to the amniotic cells in two of them. The results suggest that acute immune rejection does not occur after the transplantation of human amniotic epithelial cells.

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 Crohns 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 Crohns disease; in 29 both had ulcerative colitis; in 12 one sibling had Crohns disease, the other ulcerative colitis. For the association study there were 175 patients with ulcerative colitis, 173 with Crohns 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 Crohns 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 Crohns 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 Crohns 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 Crohns disease, important susceptibility genes are likely to exist outside the HLA region.

MBL genotype and risk of invasive pneumococcal disease: a case-control study

BACKGROUND Streptococcus pneumoniae is a major cause of morbidity and mortality in developed and developing countries. No common genetic determinants of susceptibility have been defined. Mannose-binding lectin (MBL) is a key mediator of innate host immunity that activates the complement pathway and directly opsonises some infectious pathogens. Mutations in three codons in the MBL gene have been identified, and individuals homozygous for a mutant genotype have very little or no serum MBL. We did a case-control study in the UK to assess whether these mutant genotypes were associated with invasive pneumococcal disease. METHODS The frequencies of genotypes defined by the three mutations in codons 52, 54, and 57, and a functional promoter polymorphism at -221, were compared in a two-stage study of 337 patients with invasive pneumococcal disease and 1032 controls. All individuals were recruited from an ethnically homogeneous white population in Oxfordshire, UK. Patients had S pneumoniae isolated from a normally sterile site. FINDINGS In our initial set of participants, 28 (12%) of 229 patients and 18 (5%) of 353 controls were homozygotes for MBL codon variants (odds ratio 2.59 [95% CI 1.39-4.83], p=0.002). Neither heterozygosity for these codon variants nor the promoter polymorphism was associated with susceptibility. In a confirmatory study, 11 (10%) of 108 patients were MBL homozygotes compared with 36 (5%) of 679 controls (p=0.046). INTERPRETATION Homozygotes for MBL codon variants, who represent about 5% of north Europeans and north Americans and larger proportions of populations in many developing countries, could be at substantially increased risk of invasive pneumococcal disease.

Vitamin D receptor gene polymorphism: association with Crohn's disease susceptibility.

BACKGROUND The vitamin D receptor (VDR) gene represents a strong positional candidate susceptibility gene for inflammatory bowel disease (IBD). The VDR gene maps to a region on chromosome 12 that has been shown to be linked to IBD by genome screening techniques. It is the cellular receptor for 1,25(OH)2 vitamin D3 (calcitriol) which has a wide range of different regulatory effects on the immune system. IBD is characterised by activation of the mucosal immune system. AIM To determine if polymorphisms in the VDR gene are associated with susceptibility to IBD SUBJECTS European Caucasoids: 158 patients with ulcerative colitis, 245 with Crohns disease, and 164 cadaveric renal allograft donor controls. METHOD Single nucleotide polymorphisms (TaqI,ApaI, and FokI) in VDR were typed in patients with Crohns disease, ulcerative colitis, and controls by polymerase chain reaction with sequence specific primers. RESULTS There were significantly more homozygotes for the TaqI polymorphism at codon 352 of exon 8 (genotype “tt”) among patients with Crohns disease (frequency 0.22) than patients with ulcerative colitis (0.12) or controls (0.12) (odds ratio 1.99; 95% confidence interval 1.14–3.47; p=0.017). CONCLUSION This study provides preliminary evidence for a genetic association between Crohns disease susceptibility and a gene that lies within one of the candidate regions determined by linkage analysis.

CYTOKINE (TNF ALPHA, LT ALPHA AND IL-10) POLYMORPHISMS IN INFLAMMATORY BOWEL DISEASES AND NORMAL CONTROLS: DIFFERENTIAL EFFECTS ON PRODUCTION AND ALLELE FREQUENCIES

The influence of biallelic polymorphisms in the tumour necrosis factor-alpha (TNFα), lymphotoxin-alpha (LTα) and interleukin-10 (IL-10) genes on stimulated TNFα and IL-10 production was studied in ulcerative colitis (UC) patients, Crohn’s disease (CD) patients and in healthy controls. A polymerase chain reaction sequence-specific primer (PCR-SSP) system was developed to type nine biallelic polymorphisms, three in each of the TNFα, LTα and IL-10 genes. Production of the TNFα and IL-10 was measured by ELISA in lipopolysaccharide (LPS) stimulated whole blood. Four haplotypes of the TNFα gene, three haplotypes of LTα and three haplotypes of IL-10 were identified. No significant differences in haplotype frequencies were found between patients and controls overall. On subgroup analysis however, haplotype TNF-2 was more frequent in women with extensive colitis compared to distal colitis (31% vs 12%; P = 0.028). This difference was even greater for the combined TNF-2-LTα-2 haplotype (56% vs 21%; P = 0.0007). The TNF-2 and LTα-2 haplotypes were associated with higher TNFα production in CD patients, and the TNF-4 haplotype was associated with lower TNFα production in UC patients. The A allele in the IL-10 promoter region at position −1082 was associated with decreased IL-10 production in CD patients and controls (P = 0.005, P = 0.015 respectively). These data provide evidence that the effect of TNFα, LTα and IL-10 gene polymorphisms on cytokine production differ in CD, UC patients and controls.

The Influence of HLA Class I Alleles and Heterozygosity on the Outcome of Human T Cell Lymphotropic Virus Type I Infection

The inflammatory disease human T cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM/TSP) occurs in only 1–2% of HTLV-I-infected individuals and is associated with a high provirus load of HTLV-I. We hypothesize that a person’s risk of developing HAM/TSP depends upon the efficiency of their immune response to the virus, which differs between individuals because of polymorphism in genes that influence this response. Previously we showed that the possession of HLA-A*02 was associated with a lower risk of HAM/TSP, and with a lower provirus load in healthy carriers of HTLV-I. However, HLA-A*02 did not account for all the observed difference in the risk of HAM/TSP. Here we present evidence, in the same study population in Japan, that HLA-Cw*08 was also associated with disease protection (probability value, two-tailed test = 0.002) and with a lower proviral load in healthy carriers. Possession of the A*02 and/or Cw*08 genes prevented 36% of potential HAM/TSP cases. In contrast, HLA-B*5401 was associated with a higher susceptibility to HAM/TSP (probability value, two-tailed test = 0.0003) and with a higher provirus load in HAM/TSP patients. At a given provirus load, B*5401 appeared to increase the risk of disease. The fraction of HAM/TSP cases attributable to B*5401 was 17%. Furthermore, individuals who were heterozygous at all three HLA class I loci have a lower HTLV-I provirus load than those who were homozygous at one or more loci. These results are consistent with the proposal that a strong class I-restricted CTL response to HTLV-I reduces the proviral load and hence the risk of disease.

Clinical phenotype is related to HLA genotype in the peripheral arthropathies of inflammatory bowel disease

BACKGROUND & AIMS The detection of phenotype-determining genes as opposed to disease susceptibility genes requires precise phenotypic characterization of patients. Peripheral arthropathies in inflammatory bowel disease (IBD) are well recognized and are classified with the HLA-B*27-related spondyloarthropathies by the European Spondyloarthropathy Study Group. However, previous HLA studies in IBD have only shown this association with axial disease rather than peripheral arthropathy. We recently reported a clinical classification that describes 2 types of peripheral arthropathy, distinguished by their natural history and articular distribution. We now report the results of immunogenetic studies in these patients and compare them with other spondyloarthropathies. METHODS IBD patients with type 1 (n = 57) and type 2 (n = 45) peripheral arthropathy were identified by case note review and questionnaire. Patients and 603 controls from Oxfordshire were assigned HLA-A, -B, -C, -DR, and -DQ genotypes by sequence-specific primer polymerase chain reaction. Patient results were compared with controls (corrected for multiple comparisons), then with each other in light of existing hypotheses. The results were compared with those of a cohort of 30 patients with postenteric reactive arthritis (ReA) and 16 patients with IBD-associated ankylosing spondylitis (IBD-AS). RESULTS Type 1 arthropathy was associated with HLA-DRB1*0103 (DR103; a rare subtype of DR1) in 33% (P < 0.0001; relative risk [RR], 12.1), B*35 in 30% (P = 0.01; RR, 2.2), and B*27 in 26% (P = 0. 001; RR, 4.0). In contrast, type 2 was associated with HLA-B*44 in 62% (P = 0.01; RR, 2.1). Similar significant associations to type 1 arthropathy were found in ReA, except that the HLA-B*27 association was significantly stronger and an association was found with DRB1*0101 (DR1) in 43% (P = 0.001; RR, 2.2). IBD-AS was associated only with HLA-B*27 and DRB1*0101. CONCLUSIONS These data suggest that the clinical classification into type 1 and type 2 arthropathies describes immunogenetically distinct entities and establish that in polygenic disorders, genes may determine clinical phenotype without conferring overall disease susceptibility (in this case, HLA genes). Type 1 arthropathy is clinically and immunogenetically similar to the spondyloarthropathies, but different HLA associations may define phenotypically distinct groups. Type 2 arthropathy has different HLA associations and may have a different etiology. Further studies are now required to confirm these associations and to elucidate the different pathogenetic mechanisms.

Delayed graft function: risk factors and the relative effects of early function and acute rejection on long‐term survival in cadaveric renal transplantation

Delayed graft function (DGF) and acute rejection have both been associated with reduced renal allograft survival. In some studies, they have been shown to have an interactive effect. We studied the risk factors for DGF and the relative impact of DGF and rejection on both short‐ and long‐term survival in recipients of cadaveric renal transplants.Data from the Oxford Transplant Centre Database were assessed on 710 cadaver allografts over a 10‐yr period, during which time all recipients received cyclosporin‐based immunosuppressive protocols. The interaction between DGF and acute rejection was examined using logistic and Cox multivariate regression.Long cold ischaemia time (CIT), sensitisation and older donor age were found to be independent predictors of DGF. The occurrence of DGF resulted in a reduced 5‐yr survival (56 vs. 75%). However, the effect of DGF was confined to the first year post‐transplant, as there was no significant difference in survival, as measured by half‐life (t1/2) of grafts functioning at 1 yr, with DGF alone and a group with good early function (t1/2=21.3 vs. 20.0 yr). There was no increase in acute rejection in grafts with DGF. However, the combination of DGF and acute rejection resulted in the worst short‐term graft survival (68% at 1 yr, compared to 92.3% in those grafts with no DGF or acute rejection) and this continued over the long term (t1/2=10.5 yr).These data suggest that early function is critical to the success of renal transplantation. The effects of DGF are limited to the first year post‐transplant. Long‐term graft survival may be improved by efforts to limit CITs, particularly for grafts from older donors and sensitised recipients.

REMOVAL OF ANTI-HLA ANTIBODIES BY EXTRACORPOREAL IMMUNOADSORPTION TO ENABLE RENAL TRANSPLANTATION

10 highly and persistently sensitised patients awaiting renal transplantation underwent extracorporeal immunoadsorption to remove anti-HLA antibodies. 7 patients have since received transplants. Only 1 allograft has been lost because of rejection, and there have been no serious side-effects attributable to treatment. Extracorporeal immunoadsorption may therefore be of considerable value in the management of highly sensitised patients.

Genotype-phenotype analysis of the Crohn’s disease susceptibility haplotype on chromosome 5q31

Background and aims: Recent molecular data suggest that genetic factors may underlie the disease heterogeneity observed in both ulcerative colitis (UC) and Crohn’s disease (CD). A locus on chromosome 5q has been implicated in susceptibility to CD, and recently refined by linkage disequilibrium mapping to a conserved 250 kb haplotype (5q31). No data regarding the contribution of this locus to clinical phenotype exist. In this case control study, we investigated the contribution of this haplotype to both susceptibility and phenotype of CD and UC. Patients and methods: We studied 330 Caucasian CD and 457 UC patients recruited from a single UK centre. Association with disease susceptibility and phenotype was analysed with haplotypes reconstructed from three single nucleotide polymorphisms chosen to span this susceptibility region. Evidence for possible genetic epistasis between IBD5 and NOD2/CARD15 was sought. Results: Linkage disequilibrium across this region was confirmed, with two haplotypes comprising 88% of all chromosomes. Susceptibility to CD, but not to UC, was associated with homozygosity for a common haplotype, H2 (pc=0.002; relative risk (RR) 2.0). Genotype-phenotype analyses demonstrated that this association was particularly strong in patients with perianal disease (pc=0.0005; RR 1.7), especially in individuals homozygous for this haplotype (pc=0.0005; RR 3.0). Importantly, no association with H2 was found in 186 patients without perianal disease. No evidence of epistasis between IBD5 and NOD2/CARD15 was demonstrated. Conclusions: The IBD5 risk haplotype is associated with CD only. Genotype-phenotype analysis reveals that the strongest association is observed in patients with perianal CD. While the precise gene involved is unclear, these data provide further molecular evidence for a genetic basis of the clinical heterogeneity of CD.