Albert Naipal

Biotinylated DRB sequence-specific oligonucleotides : comparison to serologic HLA-DR typing of organ donors in Eurotransplant

A novel HLA-DR typing method was applied using PCR-amplified fragments and biotin-labeled oligonucleotides (PCR-biotin-SSO). The PCR-biotin-SSO method can be used efficiently to perform HLA-DR typing for a large number of individuals when time is not the limiting factor. The reliability of HLA typing of cadaveric organ donors is of vital importance for organ exchange organizations such as ET. Due to lack of time, these typings are usually performed by the complement-dependent cytotoxicity. The individual donor center typings are immediately reported to ET, where the recipient selection procedure is started. DNA isolated from donor spleen material, sent to the ETRL for retyping purposes, was subjected to PCR-biotin-SSO typing. The results were compared with the serological HLA-DR typings as reported to ET. The analysis of 1052 donor samples for the broad DR1-DR10 antigens revealed a concordance rate of over 90% between the donor center and the ETRL. The majority of the discrepancies involved specificities of the HLA-DR5, DR6, and DR8 cross-reacting group, with DR6 as the predominant discordant specificity. The results indicate (a) that PCR-biotin-SSO is a reliable technique for DNA-based HLA-DR typing and (b) that HLA-DR serology is still a useful technique when time is limited, such as for cadaveric donor typing.

Polymorphism of HLA-DRB, -DQA1, and -DQB1 in rheumatoid arthritis in Asian Indians: Association with DRB1 *0405 and DRB1 *1001

We investigated the DRB, DQA1, and DQB 1 polymorphism and haplotypes in sporadic and familial RA subjects of Asian Indian origin by PCR oligotyping using biotinylated SSOPs. Molecular subtyping of DRB 1*04 in RA patients showed strongest association with highest relative risk with DRB 1*0405, followed by DRBI*0401. A significant decreased frequency of DRBI*1502 was observed in patients compared to controls (chi 2 = 4.5). Among other alleles, DRBI*1001 was found to be significantly increased. A total of 73.3% of patients carried the shared sequence of the third HVR (67-74) of DRB1 domain compared to its presence in only 37.6% of controls. A significant number of patients carried DR4 haplotypes on DQBI*0302 (58%) as against DQBI*0301 which was present only on 10.5% of the haplotypes. When compared to controls, the difference was significant for the latter allele only. Few unique DRDQ haplotypes were observed in Asian Indians. Among DR-DQ haplotypes, DRB1*0401-DQB1*0302 gave the highest risk whereas DRB1*0403-DQB1*O301 was negatively associated. Alleles with negative charge at position 70 confer protection or are negatively associated with RA whereas among the associated alleles, glycine at position 86 resulted in higher risk than those with valine at this position. A heterogenous association of DQB1 alleles with DR4 subtypes, influencing susceptibility to RA, suggests the DQB locus is not primarily associated with RA and susceptibility lies in the sequence 67-74 of the DRB1 loci.

Tumor necrosis factor α-308 gene variants in relation to major histocompatibility complex alleles and Felty's syndrome

Abstract The location of the human TNF genes within the MHC complex has prompted much speculation about the role of TNF alleles in the etiology of MHC-associated autoimmune diseases. On sequencing the 5′ regulatory region of the human TNFA gene a G ( TNFA −308G ) to A ( TNFA −308A ) transition polymorphism at position −308 was discovered. We have developed a simple PCR assay to facilitate the screening of the −308 polymorphism at the DNA level. In view of the possible linkage between the TNFA −308A allele and a certain MHC type, TNFA −308 genotypes in HLA-typed healthy individuals ( n = 88) were determined. A statistically significant association between the TNFA −308A allele and HLA-DR3, DQB1 ∗ 0201, DQA1 ∗ 0501, A1, B8, and the Ncol 5.5-kb RFLP of the TNFB gene was observed. In addition, we determined the frequency of the TNFA −308A allele in patients with FS ( n = 13), an HLA-DR4-associated disease. In this study, no association was found of Feltys syndrome with the TNFA −308A allele, indicating that this allele does not appear to be a susceptibility factor for FS. Human Immunology 41, 259–266 (1994) .

Improved fusion technique. II: Stability and purity of hybrid clones

Optimal conditions are defined for hybridoma formation between mouse spleen cells and mouse myeloma cells. The results of using different numbers of spleen cells in the fusion process is reported in 2 parts. Part I deals with the number of spleen cells in relation to hybridoma formation and antibody production. Part II treats of the purity of hybridoma clones and the loss of antibody production following fusion. Part I. Two series of experiments show that when cell fusion is performed properly the total number of antibody producing clones is greater than in non-standard conditions. The yield of hybridomas obtained with a ratio of mouse myeloma to mouse spleen cells of 1:10 did not differ from that reported by De Blas et al. (1981). The number of hybridomas formed seems to depend mainly on the number of mouse spleen cells available. The most satisfactory yield of monoclonal antibodies is obtained under conditions producing growth in approximately 100% of the wells. Part II. Two weeks after fusion a number of antibody producing clones were cultured in limiting dilution. Analysis of the hybridomas indicated that at least 40% of the antibody producing clones disappear during the first 3 weeks. Antibody producing hybridomas were as a rule not outgrown by non-antibody producing clones.

Asian Indian HLA-DR2-, DR4-, and DR52-related DR-DQ genotypes analyzed by polymerase chain reaction based nonradioactive oligonucleotide typing unique haplotypes and a novel DR4 subtype

We have employed a PCR-based nonradioactive technique using biotinylated SSOPs to define HLA-DR2-, 4-, DR51-, and DR52-associated DR-DQ genotypes in Asian Indian families. In the DR2 group, most haplotypes described by us in a previous study were confirmed by family analysis. Evidence for one additional haplotype was available in this study. The classic DRB1*1501- and DRB1*1502-associated caucasoid haplotypes occurred with an appreciable frequency in Asian Indians, but two of the DRB1*1601-associated Caucasoid haplotypes were absent. At least six unique and unusual DR2-associated genotypes were encountered. In the DR52 group, the three most common alleles are DRB1*0301, DRB1*1404, and DRB1*1101. The DR6-associated alleles were DRB1*1301, 1302, 1401, and 1404. A few unique haplotypes occurred with low frequency in this group. In the DR4 group, at least three unusual patterns of hybridization were noticed by family analysis. One of these appears to be a novel DR4 subtype upon sequencing. These results demonstrate that, besides HLA-DR2, appreciable complexity occurs in the DR4- and DR52-associated alleles among Asian Indians. The presence of unique DR-DQ haplotypes in addition to those found characteristically among Western Caucasians suggests that the Indian population provides valuable source of many HLA class II haplotypes.

Improved fusion technique. I. Human umbilical cord serum, a new and potent growth promoter, compared with other B cell and hybridoma activators

Accelerated proliferation of hybridoma cells was observed in the presence of human umbilical cord serum (HUCS). This had very strong growth-promoting activity, even at a concentration of 2%. A comparison was made between HUCS and other B cell growth promoters, such as lipopolysaccharide (LPS) and dextran sulfate (DxS), macrophage supernatant, and human endothelial culture supernatant (HECS). The growth-promoting effect of HUCS was superior. Using a microcytotoxicity assay, we found no significant differences in the number of antibody producing clones with the various culture media, except for fetal calf serum.

Improved fusion technique: III. The growth promoting activity of lipopolysaccharide, dextran sulfate, and red cell lysate added to Hy-clone calf serum

A new high quality young-calf serum, Hy-clone calf serum (HcCS), was tested for use in hybridoma culture. This calf serum alone had little growth promoting activity and was much inferior to fetal calf serum (FCS). Red cell lysate (RCL) used in combination with the young-calf serum showed very good growth promoting activity. Growth was increased about threefold over that in the presence of FCS. However, HcCS and RCL could not substitute for feeder cells when hybridomas were cultured as single cells under conditions of limiting dilution. It is thought likely that the potent growth promoting factor in red cell lysate is hemoglobin.

D6STNFa Microsatellite Locus Correlates with CTLp Frequency in Unrelated Bone Marrow Donor-Recipient Pairs

The use of unrelated donors for bone marrow transplantation is associated with an increased morbidity and mortality when compared with HLA identical siblings, primarily due to an increased rate of graft-versus-host-disease. HLA matching for donors and recipients is the most important factor influencing the outcome of BMT. However, unrelated donor selection generally relies on matching only for HLA antigens without considering potential incompatibility for other MHC loci. Cellular assays have been developed to predict incompatibility that cannot be detected by current typing methods. The CTLp frequencies correlate with the degree of incompatibility of patient/donor and the clinical grade of GVHD. Since the CTLp assay is expensive and time consuming, an alternative is wanted. We studied the means of matching for microsatellites in determining MHC identity and possible correlation with CTLp frequencies. Therefore, 26 recipient/donor pairs were analysed for eleven microsatellite loci within and around the MHC region. Our study provides evidence that the D6STNFa locus correlates with CTLp frequency. The D6STNFa locus provides an additional marker that may help to improve the matching of unrelated donors and bone marrow recipients.

Selection by Typhoid and Yellow Fever Epidemics Witnessed by the HLA-DR Locus

Several years ago we presented evidence for an HLAlinked control of survival from typhoid fever and yellow fever epidemics (1). At that time we were only able to type for HLA-A, -B, and -C antigens. Because we were interested to know whether these epidemics might have left even stronger marks on -DR or -DQ genes, we recently revisited the survivors and also performed HLA-DR and -DQ typing. The results indicate that most of the observed heterogeneity may be explained by a primary association of HLA-DR2 with mortality and DR4 + DRw13 with survival from typhoid and/or yellow fever. We cannot exclude one or two primary associations with HLA-B alleles, but no primary association was observed with HLA-A, -DRw52/53, or -DQ alleles. These data suggest that gene products on which the typhoid bacillus and/or the yellow fever virus could exert their apparent selective pressure are coded by genes located on or very near to the DRs1 locus.