David Briggs

Donor ABCB1 Variant Associates with Increased Risk for Kidney Allograft Failure

The impact of variation within genes responsible for the disposition and metabolism of calcineurin inhibitors (CNIs) on clinical outcomes in kidney transplantation is not well understood. Furthermore, the potential influence of donor, rather than recipient, genotypes on clinical endpoints is unknown. Here, we investigated the associations between donor and recipient gene variants with outcome among 4471 white, CNI-treated kidney transplant recipients. We tested for 52 single-nucleotide polymorphisms (SNPs) across five genes: CYP3A4, CYP3A5, ABCB1 (MDR1; encoding P-glycoprotein), NR1I2 (encoding the pregnane X receptor), and PPIA (encoding cyclophilin). In a discovery cohort of 811 patients from Birmingham, United Kingdom, kidney donor CC genotype at C3435T (rs1045642) within ABCB1, a variant known to alter protein expression, was associated with an increased risk for long-term graft failure compared with non-CC genotype (hazard ratio [HR], 1.69; 95% confidence interval [CI], 1.20-2.40; P=0.003). No other donor or recipient SNPs were associated with graft survival or mortality. We validated this association in 675 donors from Belfast, United Kingdom (HR, 1.68; 95% CI, 1.21-2.32; P=0.002), and in 2985 donors from the Collaborative Transplant Study (HR, 1.84; 95% CI, 1.08-3.13; P=0.006). In conclusion, these data suggest that an ABCB1 variant known to alter protein expression represents an attractive candidate for future study and risk stratification in kidney transplantation.

Donor HLA-C Genotype Has a Profound Impact on the Clinical Outcome Following Liver Transplantation

Late allograft dysfunction is a significant problem following liver transplantation and its pathogenesis is uncertain. HLA‐C is the major inhibitory ligand for killer immunoglobulin‐like receptors (KIRs) that regulate the cytotoxic activity of natural killer (NK) cells. HLA‐C alleles can be allocated into two groups, termed HLA‐C1 and HLA‐C2, based on their KIR specificity. HLA‐C2 interactions are more inhibiting to NK cell activation. We studied the clinical importance of HLA‐C genotype in a large liver transplant cohort and found that possession of at least one HLA‐C2 allele by the donor allograft was associated with less histological evidence of chronic rejection and graft cirrhosis, a 16.2% reduction in graft loss (p = 0.003) (hazard ratio: 2.7, 95% CI 1.4–5.3) and a 13.6% improvement in patient survival (p = 0.01) (hazard ratio: 1.9, 95% CI 1.1–3.3) at 10 years. Transplantation of an HLA‐C2 homozygous allograft led to a particularly striking 26.5% reduction in graft loss (p < 0.001) (hazard ratio: 7.2, 95% CI 2.2–23.0) at 10 years when compared to HLA‐C1 homozygous allografts. Donor HLA‐C genotype is therefore a major determinant of clinical outcome after liver transplantation and reveals the importance of NK cells in chronic rejection and graft cirrhosis. Modulation of HLA‐C and KIR interactions represents an important novel approach to promote long‐term graft and patient survival.

Connective tissue disease and autoantibodies in the kindreds of 63 patients with systemic sclerosis

: 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)

Complement polymorphism in herpes gestationis : association with C4 null allele

BACKGROUNDnHerpes 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.nnnOBJECTIVEnOur purpose was to study complement polymorphism in HG.nnnMETHODSnUsing electrophoresis and immunofixation techniques, we determined the allele frequencies of C4A, C4B, C3, and factor B in 42 patients with a history of HG.nnnRESULTSnNinety percent of patients carried a C4 null allele (C4*QO). No statistically significant association with C3 or factor B alleles was seen.nnnCONCLUSIONnHG 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.

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.

Variation in Iron Homeostasis Genes Between Patients With ARDS and Healthy Control Subjects

BACKGROUNDnAbnormal plasma and lung iron mobilization is associated with the onset and progression of ARDS and is detectable in specific at-risk populations. Patients with ARDS also have pronounced oxidative and nitrosative stress that can be catalyzed and thereby aggravated by the bioavailability of redox active iron. ARDS of pulmonary and extrapulmonary origin may differ pathophysiologically and require different ventilatory strategies. Evidence suggests that genetic predisposition is relevant to the pathogenesis of ARDS. We therefore explored the hypothesis that polymorphisms from a panel of genes encoding iron-metabolizing proteins determine susceptibility to ARDS.nnnMETHODSnRetrospective case-control study conducted at the adult ICUs of two university hospitals. Patients with ARDS (n = 122) and healthy control subjects (n = 193) were genotyped. Sequence-specific primer polymerase chain reaction was used to genotype selected biallelic single-nucleotide polymorphisms. An audit of the patient database was conducted, and 104 of the 122 ARDS patients were eligible for the final data analysis.nnnRESULTSnPreliminary analysis indicated differences between ARDS and healthy control subjects in the incidence of polymorphism of the gene encoding ferritin light chain. Subgroup analysis indicated the prevalence of ferritin light-chain gene -3381GG homozygotes was increased in patients with ARDS of extrapulmonary origin compared to healthy control subjects. Secondly, a common haplotype in the heme oxygenase 2 gene was reduced in patients with ARDS compared to healthy control subjects and was more evident in those with ARDS of direct or pulmonary etiology.nnnCONCLUSIONSnThese results provide preliminary evidence to suggest a distinction in the genetic background of the subpopulations studied, inferring that the ferritin light-chain gene genotype confers susceptibility to ARDS, while the heme oxygenase 2 haplotype is protective against the onset of the syndrome. Such data support further previous findings that suggest abnormalities in iron handling resulting in redox imbalance are implicated in the pathogenesis of ARDS.

Indirect Recognition of T-Cell Epitopes Derived from the α3 and Transmembrane Domain of HLA-A2

Indirect allorecognition has been implicated in the mechanism of chronic rejection and alloantibody formation but precise definition of the epitopes involved has been limited. We have undertaken a detailed assessment of the antigenic properties of peptides derived from HLA‐A2. Candidate epitopes were identified in vitro by assessment of MHC class II binding. The immune response to these epitopes was determined in patients awaiting a renal transplant by the assessment of PBMC activation using γ‐interferon ELISPOT. Twenty‐two of fifty‐five patients responded to peptides from HLA‐A2 and this was associated with but not confined to those who had made antibody to HLA‐A2 (14/18). Nineteen of twenty‐two patients responded to peptides derived from the hypervariable α1 and α2 domains and 18/22 responded to peptides from the α3 and transmembrane domain, the sequences of which show little polymorphism. In six patients, the sequence of these peptides was identical to self, that is, the response was autoimmune. The finding of indirect epitopes derived from regions of MHC class I that exhibit little polymorphism provides a novel perspective on the immune response to alloantigen and has potential implications for the development of specific therapies.

A polymorphism in the promoter region of the CD86 (B7.2) gene is associated with systemic sclerosis

Systemic sclerosis (SSc) is a connective tissue disease of unknown aetiology characterized by fibrosis of the skin and internal organs, vascular abnormalities and humoral autoimmunity. Strong T‐cell‐dependent autoantibody and HLA associations are found in SSc subsets. The co‐stimulatory molecule, CD86, expressed by antigen‐presenting cells, plays a crucial role in priming naïve lymphocytes. We hypothesized that SSc, or one of the disease subsets, could be associated with single‐nucleotide polymorphisms of the CD86 gene. Using sequence specific primer‐polymerase chain reaction (SSP‐PCR) methodology, we assessed four CD86 polymorphisms in 221 patients with SSc and 227 healthy control subjects from the UK. Haplotypes were constructed by inference and confirmed using PHASE algorithm. We found a strong association between SSc and a specific haplotype (haplotype 5), which was more prevalent in patients than in controls (29% vs 15%, OR = 2.3, χ2 = 12, P = 0.0005). This association could be attributed to the novel −3479 promoter polymorphism; a significant difference was observed in the distribution of the CD86 −3479 G allele in patients with SSc compared to controls (43.7% vs. 32.4%, OR = 1.7, χ2 = 12.1, P = 0.0005). TRANSFAC analyses suggest that the CD86‐3479T allele contains putative GATA and TBP sites, whereas G allele does not. We assessed the relative DNA protein‐binding activity of the −3479 polymorphism in vitro using electromobility gel shift assays (EMSA), which showed that the −3479G allele has less binding affinity compared to the T allele for nuclear proteins. These findings highlight the importance of co‐stimulatory pathways in SSc pathogenesis.

KIR and HLA-C Interactions Promote Differential Dendritic Cell Maturation and Is a Major Determinant of Graft Failure following Kidney Transplantation

Background HLA-C is an important ligand for killer immunoglobulin like receptors (KIR) that regulate natural killer (NK) cell function. Based on KIR specificity HLA-C molecules are allocated into two groups, HLA-C1 or HLA-C2; HLA-C2 is more inhibiting to NK cell function than HLA-C1. We studied the clinical importance of HLA-C genotypes on the long-term graft survival of 760 kidney transplants performed at our centre utilising a population based genetic study and cell culture model to define putative mechanisms. Methods and Findings Genotyping was performed using conventional DNA PCR techniques and correlations made to clinical outcomes. We found that transplant recipients with HLA-C2 had significantly better long-term graft survival than transplant recipients with HLA-C1 (66% versus 44% at 10 years, log-rank pu200a=u200a0.002, HRu200a=u200a1.51, 95%CIu200a=u200a1.16–1.97). In in-vitro NK and dendritic cell (DC) co-culture model we made several key observations that correlated with the population based genetic study. We observed that donor derived NK cells, on activation with IL-15, promoted differential HLA-C genotype dependent DC maturation. In NK-DC co-culture, the possession of HLA-C2 by DC was associated with anti-inflammatory cytokine production (IL-1RA/IL-6), diminished DC maturation (CD86, HLA-DR), and absent CCR7 expression. Conversely, possession of HLA-C1 by DC was associated with pro-inflammatory cytokine synthesis (TNF-α, IL-12p40/p70), enhanced DC maturation and up-regulation of CCR7 expression. By immunohistochemistry the presence of donor NK cells was confirmed in pre-transplant kidneys. Conclusions We propose that after kidney transplantation IL-15 activated donor derived NK cells interact with recipient DC with less activation of indirect allo-reactivity in HLA-C2 positive recipients than HLA-C1 positive recipients; this has implications for long-term graft survival. Early events following kidney transplantation involving NK-DC interaction via KIR and HLA-C immune synapse may have a central role in long-term kidney transplant outcomes.

Soluble CD30 and Cd27 levels in patients undergoing HLA antibody-incompatible renal transplantation

HLA antibody-incompatible transplantation has a higher risk of rejection when compared to standard renal transplantation. Soluble CD30 (sCD30) has been shown in many, but not all, studies to be a biomarker for risk of rejection in standard renal transplant recipients. We sought to define the value of sCD30 and soluble CD27 (sCD27) in patients receiving HLA antibody-incompatible transplants. Serum taken at different time points from 32 HLA antibody-incompatible transplant recipients was retrospectively assessed for sCD30 and sCD27 levels by enzyme-linked immunosorbent assay (ELISA). This was compared to episodes of acute rejection, post-transplant donor-specific antibody (DSA) levels and 12 month serum creatinine levels. No association was found between sCD27 and sCD30 levels and risk of acute rejection or DSA levels. Higher sCD30 levels at 4-6 weeks post-transplantation were associated with a higher serum creatinine at 12 months. Conclusion patients undergoing HLA antibody-incompatible transplantation are at a high risk of rejection but neither sCD30 (unlike in standard transplantation) nor sCD27 was found to be a risk factor. High sCD30 levels measured at 4-6 weeks post-transplantation was associated with poorer graft function at one year.