Jeffrey L. Bidwell

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.

DNA-RFLP analysis and genotyping of HLA-DR and DQ antigens

Abstract The remarkable correlation between DNA restriction fragment length polymorphism (RFLP) analysis and conventional tissue typing has confirmed and reinforced previously held concepts of the genetic basis for HLA class II allospecificity. As Jeffrey Bidwell reviews here, RFLP analysis has developed into a powerful genotyping tool for DR and DQ specificities and also provides valuable insight into the molecular basis for recognition of Dw epitopes by T cells. Furthermore DNA-RFLP typing in genotypic selection of histocompatible donor-recipient pairs could potentially reduce many logistic problems in live-donor transplantation.

Vascular endothelial growth factor gene polymorphism and acute respiratory distress syndrome.

Background: Non-cardiogenic pulmonary oedema is a characteristic feature of the acute respiratory distress syndrome (ARDS). The properties of vascular endothelial growth factor (VEGF) as a potent vascular permogen and mitogen have led to investigation of its potential role in this condition. Lower VEGF plasma levels have been linked to the presence of the T allele in the +936 CT polymorphism. We hypothesised that the presence of the T allele would be associated with the development and severity of ARDS. Methods: A cohort of 137 normal subjects, 117 ventilated patients with ARDS, and 103 “at risk” of ARDS were genotyped for the VEGF+936 CT polymorphism. The severity of physiological disturbance and mortality was determined in the ventilated cohorts. Results: The CT and TT genotype frequencies were increased in ARDS patients compared with both normal subjects (OR 2.01, 95% CI 1.13 to 3.58, p = 0.02) and those “at risk” (OR 2.05, 95% CI 1.02 to 2.20, p = 0.03). In patients with ARDS but not those “at risk”, CT and TT genotypes were associated with a higher mean APACHE III score (80.9 (4.3) v 69.3 (2.9), p<0.05). Conclusion: These data support a role for VEGF in the pathogenesis of ARDS and its associated physiological derangement.

In vitro development of human monoclonal antibody-secreting plasmacytomas

A polyclonal human lymphoblastoid cell line transformed in vitro with Epstein-Barr virus produced specific anti-Rhesus D antibody. It was repeatedly enriched by rosetting procedures and subsequently cloned. The cloning conditions employed a combination of mouse macrophage feeder layers, antimycoplasma agents, and low density passage. Formal evidence of monoclonality was obtained in one case which was of human IgG1 isotype and was secreted at the level of 15-20 micrograms/ml. All clones showed long-term stability in culture after 10 months of continuous passage. Both polyclonal and monoclonal cell lines possessed antigens characteristic of highly differentiated B cells, yet they also expressed Epstein-Barr virus nuclear antigen (EBNA). This study exemplifies a simple method for obtaining monoclonal antibody secreting plasmacytomas of human origin.

Simultaneous genotyping for all three known structural mutations in the human mannose-binding lectin gene.

We describe a rapid and simple method for genotyping the three known structural mutations within exon 1 of the mannan‐binding lectin (MBL) gene. A PCR‐amplifiable synthetic DNA (Universal Heteroduplex Generator) was annealed to genomic PCR product from exon 1 to generate unique DNA heteroduplexes for each mutation. Heteroduplexes were then resolved by non‐denaturing polyacrylamide gel electrophoresis. The technique was initially validated with previously typed samples and then applied to previously untyped samples with the results confirmed by DNA sequencing.

Advances in DNA-based HLA-typing methods

The last five years has witnessed spectacular progress in the developments of PCR-based HLA-typing methods. Here, Jeffrey Bidwell charts a course through the huge array of methodologies now available both for MHC class I and class II analysis.

Haplotypes in the tumour necrosis factor region and myeloma

This study described the haplotypic structure across a region of chromosome 6 including the tumour necrosis factor (TNF) gene, and investigated its influence on the aetiology of myeloma. A total of 181 myeloma cases from the Medical Research Council Myeloma VII trial and 233 controls from the Leukaemia Research Fund Case Control Study of Adult Acute Leukaemia were included in the analysis. Genotyping by induced heteroduplex generator analysis was carried out for single nucleotide polymorphisms (SNP) located at positions −1031, −863, −857, −308 and −238 of the 5′ promoter region of TNF‐α gene, and 252 in the LT‐α gene; and five microsatellites, TNFa, b, c, d and e. Haplotypes were inferred statistically using the phase algorithm. A limited diversity of haplotypes was observed, with the majority of variation described by 12 frequent haplotypes. Detailed characterization of the haplotype did not provide greater determination of disease risk beyond that described by the TNF‐α−308 SNP. Some evidence was provided for a decreased risk of myeloma associated with the TNF‐α−308 variant allele A, odds ratio, 0·57; 95% confidence interval, 0·38–0·86. The results of this study did not support our starting hypothesis; that high producer haplotypes at the TNF locus are associated with an increased risk of developing myeloma.

Patient interleukin-18 GCG haplotype associates with improved survival and decreased transplant-related mortality after unrelated-donor bone marrow transplantation.

Interleukin‐18 (IL‐18), a proinflammatory cytokine, is elevated in patients with acute graft‐versus‐host disease (aGVHD). IL‐18 induces Th1 differentiation and cytotoxic T‐lymphocyte function, both of which have been implicated in the pathogenesis of aGVHD. However, recent studies have shown that neutralization of IL‐18 by antibodies leads to an increased risk of aGVHD‐related mortality while administration of IL‐18 significantly improved survival. We have genotyped a cohort of 157 patient/donor pairs undergoing unrelated donor bone marrow transplantation (BMT) for three polymorphisms recently identified in the promoter of the IL‐18 gene: G‐137C, C‐607A and G‐656T. Using phase software, three main haplotypes were reconstructed: GCG, CAT and GAT. We found no association between the occurrence of aGVHD and patient/donor haplotypes. The presence of the GCG haplotype in patients was associated with significantly decreased risk of transplant‐related mortality at 100 d (23% in patients with GCG vs. 48% in patients without GCG, P < 0·01) and at 1 year (36% vs. 65%, P < 0·01). The presence of the GCG haplotype in patients was also associated with improved survival (57% vs. 32%, P < 0·01). Cox regression analysis showed that the presence of the GCG haplotype was associated with a twofold increased probability of survival. These data suggest that the IL‐18 promoter GCG haplotype may influence survival after unrelated donor BMT without altering the risk of aGVHD.

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.

Relationship Between Vascular Endothelial Growth Factor + 936 Genotype and Plasma/Epithelial Lining Fluid Vascular Endothelial Growth Factor Protein Levels in Patients With and at Risk for ARDS

BACKGROUND Vascular endothelial growth factor (VEGF) is postulated to have a role in ARDS. The functional VEGF + 936 polymorphic T allele is associated with an increased susceptibility to and severity of ARDS. The reasons for this are unclear. We hypothesized that the T allele would be associated with an alteration in the relation between epithelial lining fluid (ELF) and plasma VEGF levels as a potential explanation for its association with susceptibility to and severity of ARDS. METHODS Plasma and ELF VEGF protein levels were measured by enzyme-linked immunosorbent assay from 10 at-risk patients receiving mechanical ventilation and 16 ARDS patients with the T allele, as well as 18 at-risk patients receiving mechanical ventilation and 26 ARDS patients without the T allele (wild-type CC genotype). RESULTS The T allele was associated with a significantly lower mean ELF VEGF level in ARDS patients (2,090 +/- 758 pg/mL vs 3,292 +/- 865 pg/mL, p < 0.05) and mean ELF/plasma VEGF level ratio (13.7 +/- 4.6 pg/mL vs 94.7 +/- 51.2 pg/mL, p < 0.01). There was no relation between the T allele and plasma VEGF level, oxygenation, or acute physiology score in at-risk and ARDS patients. ELF VEGF levels were significantly higher than plasma levels in both cohorts except for at-risk patients without the T allele (wild-type CC genotype). CONCLUSION The T allele is associated with a significant decrease in ELF levels and the ELF/plasma ratio in ARDS patients. This may explain the increased susceptibility and physiologic derangement in ARDS patients with the T allele. We speculate VEGF has a protective function in the lung. Further studies are necessary to clarify the underlying mechanisms.