Benjamin A. Bradley

A DNA-RFLP typing system that positively identifies serologically well-defined and ill-defined HLA-DR and DQ alleles, including DRw10.

A single enzyme/multiple probe system of HLA-DR and DQ typing using restriction fragment—length polymorphism (RFLP) analysis is presented. TaqI-digested genomic DNAs are hybridized sequentially with short DRβ, DQβ, and DQα cDNA probes. The DRβ probe discriminates between the DR allelic specificities DR1 to DRwl4, with the two exceptions of some DR3/DRwl3 and some DR7/DRw9 combinations. We describe the positive identification of a DRw10-specific RFLP and demonstrate its segregation in families. The DQβ probe defines an allelic system that identifies the alleles DQw1, DQw2, and DQw3. This permits the resolution of DR3/DRw13 and DR7/DRw9 alleles by defining the DR/DQ association caused by linkage disequilibrium. The DQα probe defines another allelic series interrelated with, but independent from, the DQβ series. Specific DQβ/DQα RFLP combinations correlate with known Dw splits of DR2, DRw6, and DR7. Combined use of the three probes permits the identification of HLA-DR, DQ, and certain Dw specificities and provides an effective and easily interpretable system for major histocompatibility complex class II allogenotyping.

Conclusions of the corneal transplant follow-up study

AIM On the basis of finalised data from the Corneal Transplant Follow up Study to identify and quantify factors influencing corneal graft outcome in terms of graft survival, rejection, visual acuity, and astigmatism. METHODS Multifactorial analysis of 2777 grafts registered by the UK Transplant Support Service from July 1987 to June 1991. RESULTS Several recipient factors influencing graft survival, rejection, and visual acuity were identified, but no donor factors. Of the operative factors amenable to change, mixed suturing was associated with reduced graft survival, and larger grafts with increased risk of rejection but better visual acuity when surviving. There was increased risk of rejection with poor matching at HLA class I antigens, but mismatched HLA-DR grafts suffered less rejection than those with zero HLA-DR mismatches. Recipient age below 10 years was associated with increased risk of both rejection and graft failure. However, whereas increasing age above 10 years was not associated with differential graft survival, it was significantly associated with decreasing risk of rejection. CONCLUSIONS While confirming possible benefits of HLA-A and B matching, the expense and delay involved in awaiting matched HLA-DR tissue is unlikely to be justified. Other donor factors are unrelated to graft outcome following screening of tissue by eye banks. The highest rates of graft failure and rejection happen in the early postoperative period, and factors influencing visual outcome are also apparent at this stage.

Influence of donor and histocompatibility factors on corneal graft outcome.

The Corneal Transplant Follow-up Study has followed 2311 penetrating keratoplasties for up to 450 days after transplant. A total of 207 failures were observed, including 65 classical rejections and 35 endothelial decompensations. At 12 months, graft survival was 89%, and survival free from rejection was 87%. For surviving grafts, risk of failure reduced from 4.8% in the first 75 days and stabilized after 5 months at 1.2% in each 75-day interval. Risk of rejection initially followed a similar pattern, but then increased after 12 months. Multifactorial analyses accounted for differences in recipient characteristics and interrelationships of donor factors. Donor age, sex, cause of death, and method of corneal storage were not found to influence significantly either time to graft failure or time to first rejection. Grafts in prospectively tissue-typed donor-recipient pairs were generally considered before surgery to be at increased risk of either graft failure or rejection. With due allowance, increasing risk of rejection was associated with increasing numbers of mismatches at HLA-A and HLA-B broad antigens. The opposite was true at HLA-DR broad antigens, where increased risk of rejection was observed with no mismatches.

Corneal Graft Survival and Visual Outcome: A Multicenter Study

PURPOSE The Corneal Transplant Follow-up Study has followed 2385 corneal transplants performed in the United Kingdom and the Republic of Ireland for up to 450 days to quantify factors influencing corneal graft survival and visual outcome 3 and 12 months postoperatively. METHODS Multifactorial analyses of grafts registered by United Kingdom Transplant Support Service from July 1987 to June 1990 were used. Corrected visual acuity of functioning grafts was assessed at 3 and 12 months. RESULTS Of 2385 corneal transplants followed, 214 failures were observed: graft survival was 95% at 3 months and 89% at 1 year. Similar factors affected outcome at each time. Decreased risk of failure was associated with surgeons reporting most grafts, and increased risk was associated with regrafts, patients younger than 10 years of age, nonvisual reasons for grafting, endothelial failure, and deep vascularization. Visual outcome was worse in older patients and was associated with cosmetic reasons for grafting, superficial vascularization preoperatively, and secondary endothelial failure. Visual acuity was better when the other eye had been grafted previously, or when the diagnosis was keratoconus or stromal dystrophy. CONCLUSIONS Primary endothelial failure was associated with high failure rates but good visual results when functioning. Most other factors had similar effects on both outcome measures. Improved outcome under highest-reporting surgeons was slight, and indicated possible differences in postoperative care.

REJECTION AND RECIPIENT AGE

In transplantation the risk of acute rejection decreases with recipient age. This is clearly illustrated in transplantation of a non-vascularised tissue, such as the cornea. In vascularised transplants, such as kidneys, acute rejection decreases with recipient age, but the phenomenon is obscured by the fact that chronic allograft nephropathy increases with age, and is further confounded by increased death from infectious disease and drug-related causes. The underlying cellular mechanisms responsible for this weakening of rejection are discussed, as is defective signal transduction leading to decreased activation of cells and decreased resistance to immunosuppressive drugs. This supports a view that less intensive immunosuppressive drug therapy is desirable in elderly recipients. Although pharmacokinetic studies are documented, there are no routine assays to measure efficacy of these drugs in individual patients. In summary, the decline in acute rejection with increasing recipient age may be due both to immunosenescence and decreased resistance to immunosuppressive drugs. Future assays to test these mechanisms could be used to tailor therapy to individual needs.

Effect of autologous salvaged blood on postoperative natural killer cell precursor frequency

BACKGROUND Immunosuppression after major surgery increases the risk of infections. Natural killer cells play a pivotal part in defence against infection. We aimed to investigate the immunomodulatory effects of different types of postoperative blood transfusion by use of a new assay for measuring the frequency of peripheral blood natural killer precursor cells (NKpf assay). METHODS We measured the natural killer cell precursor (NKp) frequency before and 5 days after surgery in 120 patients undergoing joint replacement surgery. The patients were assigned to one of five groups according to the type of transfusion received: non-transfused (n=32), allogeneic non-leukodepleted blood (eight), allogeneic leukodepleted blood (30), autologous predeposited blood (ten), and autologous salvaged blood collected within the first 24 h after surgery (40). We also measured interferon gamma and interleukin 10 concentrations before and after surgery. FINDINGS The mean postoperative NKp frequency for all patients was lower than the preoperative values, except in patients receiving autologous salvaged blood, which was higher than all other groups (p<0.0001). Postoperative NKp frequencies for patients receiving allogeneic or autologous predeposited blood responded similarly (p=0.99), but these patients had lower NKp frequencies than did the non-transfused group (p<0.0001). Postoperative interferon gamma concentrations were higher in the autologous salvaged blood group (p<0.0001) than in other groups, which did not differ from each other. Interleukin 10 concentrations were similar across all groups (p=0.49). INTERPRETATION Immunosuppression associated with surgery and blood loss was reflected in a reduced frequency of NKp and decreased interferon gamma. This immunosuppression was reversed by transfusion of autologous salvaged blood, suggesting that this fluid contained immunostimulants.

Substantial benefits of tissue matching in renal transplantation

The purpose of this study was to perform a rigorous statistical analysis of the benefits of HLA-A,B, and DR matching in renal transplantation. Graft survival in 2282 first cadaver kidney transplants, recorded and followed up by the United Kingdom Transplant Service (UKTS), was analyzed using the piecewise proportional hazards regression method. The results show that substantial improvements in graft survival are obtained when there is DR compatibility and at most one A or B mismatch, but that there is little advantage in tissue matching unless this degree of matching can be attained. So far, few graft recipients have benefited substantially through tissue matching (24% of kidneys exchanged through UKTS in 1984). This is partly attributable to unresolved technical problems in DR typing. However simulations show that under ideal conditions, with a pool of 3000 patients awaiting transplantation, considerable improvements in graft survival can be obtained in over 60% of recipients.

Clinical and Surgical Factors Influencing Corneal Graft Survival, Visual Acuity, and Astigmatism

Purpose: To quantify clinical and operative factors that influence corneal graft outcome. Methods: A multifactorial analysis was done on 2242 corneal grafts registered by the United Kingdom Transplant Service from July 1987, to June 1991. Results: There was an increased risk of graft failure in patients with preoperative diffuse and other noncentral stromal edema, less-common eye diseases, small trephine size, difference in donor and recipient sizes greater than 0.25 mm, and use of mixed continuous and interrupted sutures. Visual acuity 3 months after surgery was poorer in patients who had glaucoma and low visual acuity preoperatively, small trephine size, and combined vitreous surgery. Use of interrupted sutures resulted in higher astigmatism at 3 months. Conclusions: After allowing for the effects of recipient factors, surgical factors significantly affected corneal graft outcome. No factors that showed significant benefits for graft survival also adversely affected visual performance. Details of medical history, clinical condition, and surgical method failed to predict more than a small proportion of observed variability in visual performance of functioning grafts.

The predictive value of epitope analysis in highly sensitized patients awaiting renal transplantation

The accumulation of highly sensitized patients (HSP) on renal transplant waiting lists is a problem faced by all transplant registries. We have studied the HLA class I serological reactivity of 20 random HSP and have related antibody specificity to primary amino acid sequence. In six patients we identified significant correlations (x test, r ≥ O.93) between panel reactivity and specific amino acid substitutions characteristic of HLA-A, -B, and -C public epitopes. Antibody reactivity was associated with up to three public epitopes in each patient. The 12 separate antibody specificities identified were associated with 10 residues. Seven correlated with HLA-A locus substitutions (Glu-62/Gly-65, Lys-66, Arg-114, His-114/Tyr-116/Lys-127, Thr-142/His-145 [X2], and Thr-149), two with HLA-B locus substitutions (Thr-24, Ser-24) and three with interlocus antibodies associated with either HLA-A and B (Leu-82/Arg-83 [X2]) or with HLA-B and -C substitutions (Leu-163). This information allowed us to predict HLA class I allelic products of known primary sequence that would react negatively with each HSP serum. Windows of acceptable mismatches (WAMMs) can thus be delineated with a view to cross-match negative transplantation without the need for exhaustive serological analysis. Surprisingly we found that WAMMs for these patients included up to 80% of the 10 commonest HLA class I haplotypes in the British population with four patients being crossmatch compatible with Al,3; B7,8. These observations lead us to propose a more intelligent approach to transplanting HSP based on epitope analysis and definition of WAMMs.

Molecular characterization of a recombinant HLA-DR1/DR2 haplotype

Serologic analysis of two families identified an HLA-DR haplotype in which DR1 and DR2 cosegregated. DNA-RFLP analysis of these families with an HLA-DRB probe revealed a pattern of hybridization suggestive of a recombination between DR1 and DR15. Following amplification, cloning, and nucleotide sequencing of HLA-DRB-gene second-exon DNA sequences, three DRB amplification products associated with the novel haplotype were identified: these corresponded to DRB1*0101, DR2 pseudogene, and DRB5*0101. Clones representing the DRB1*1501 and DR1 pseudogenes were not identified: oligonucleotide typing with DRB1*1501-specific probes confirmed the absence of this gene within the DR1/DR2 haplotype. We postulate that the DR1/DR2 haplotype represents a recombinant between those of DR1-Dw1 and DR15-Dw2, and that the crossing-over may have been between the DRB1*0101 gene and the DR2 pseudogene. This is further supported by DNA-RFLP analysis with HLA-DQB and DQA CDNA probes, which revealed conserved linkage genes between the DQB1*0501, DQA1*0101, and DRB1*0101 genes.