Geographic variation of the major histocompatibility complex in Eastern Atlantic grey seals (Halichoerus grypus).

Pathogen-driven balancing selection maintains high genetic diversity in many vertebrates, particularly in the major histocompatibility complex (MHC) immune system gene family, which is often associated with disease susceptibility. In large natural populations where subpopulations face different pathogen pressures, the MHC should show greater genetic differentiation within a species than neutral markers. We examined genetic diversity at the MHC-DQB locus and nine putatively neutral microsatellite markers in grey seals (Halichoerus grypus) from eight United Kingdom (UK) colonies, the Faeroe Islands and Sable Island, Canada. Five DQB alleles were identified in grey seals, which varied in prevalence across the grey seal range. Among the seal colonies, significant differences in DQB allele and haplotype frequencies and in average DQB heterozygosity were observed. Additionally, the DQB gene exhibited greater differentiation among colonies compared with neutral markers, yet a weaker pattern of isolation by distance (IBD). After correcting for the underlying IBD pattern, subpopulations breeding in similar habitats were more similar to one another in DQB allele frequencies than populations breeding in different habitats, but the same did not hold true for microsatellites, suggesting that habitat-specific pathogen pressure influences MHC evolution. Overall, the data are consistent with selection at MHC-DQB loci in grey seals with both varying selective pressures and geographic population structure appearing to influence the DQB genetic composition of breeding colonies.

Opposites attract: MHC-associated mate choice in a polygynous primate.

We investigated reproduction in a semi-free-ranging population of a polygynous primate, the mandrill, in relation to genetic relatedness and male genetic characteristics, using neutral microsatellite and major histocompatibility complex (MHC) genotyping. We compared genetic dissimilarity to the mother and genetic characteristics of the sire with all other potential sires present at the conception of each offspring (193 offspring for microsatellite genetics, 180 for MHC). The probability that a given male sired increased as pedigree relatedness with the mother decreased, and overall genetic dissimilarity and MHC dissimilarity with the mother increased. Reproductive success also increased with male microsatellite heterozygosity and MHC diversity. These effects were apparent despite the strong influence of dominance rank on male reproductive success. The closed nature of our study population is comparable to human populations for which MHC-associated mate choice has been reported, suggesting that such mate choice may be especially important in relatively isolated populations with little migration to introduce genetic variation.

Rapid and slow progressors differ by a single MHC class I haplotype in a family of MHC-defined rhesus macaques infected with SIV.

Highly polymorphic HLA class I molecules may influence rates of disease progression of HIV-infected individuals. Recent evidence suggests that individuals who mount vigorous CTL responses to multiple HIV-1 epitopes have reduced viral loads, and survive longer than individuals that make a less robust or less diverse CTL response. It has been difficult, however, to define associations between particular HLA class I alleles and rates of disease progression. This may be due, in part, to the uncontrolled variables associated with naturally acquired HIV infections. Studies using MHC-defined, non-human primates infected with well characterized viral stocks should help to clarify this relationship. To explore the possibility that MHC class I polymorphism can influence disease progression, we infected four Mamu-DRB-identical individuals from a family of MHC-defined rhesus macaques intravenously with 40 TCID50SIVmac239. Two of these macaques developed severe wasting and were euthanized within 80 days of infection, while the other two survived for more than 400 days without showing any symptoms of disease. Since all four of these macaques were Mamu-DRB-identical, we were able to exclude the MHC class II DRB loci as determinant of disease progression. Interestingly, both of the slow progressors made CTL responses to the same three SIV CTL epitopes, which were restricted by two molecules (Mamu-B*03 and B*04) encoded by their common maternal haplotype. The two rapid progressors did not share this haplotype with the slow progressors, and we were unable to detect CTL responses in these two siblings. These observations implicate products of the Mamu-B*03 and B*04 alleles in resistance to disease progression in this family of SIV-infected macaques, and provide additional evidence that certain MHC class I-restricted CTL responses may play a significant role in delaying the onset of AIDS.

Parental MHC antigen sharing and pregnancy wastage in captive pigtailed macaques.

Among some human populations, immunogenetic similarity between mates is associated with increased risk of pregnancy loss. To investigate the relationship between histocompatibility and reproductive performance in non-human primates, 128 pigtailed macaque couples were classified as 'reproductively successful' or 'unsuccessful' according to previous breeding performance. These couples were arranged into 64 triads composed of individual females, and a 'successful' and 'unsuccessful' mate. Individuals were typed for class I MHC antigens using a microcytotoxicity technique and species-specific alloantisera. Matched-pair analysis revealed that significantly more 'unsuccessful' couples shared MnLA-A antigens than did the matched 'successful' couples. Conditional matched-pair logistic regression analysis further revealed that parental sharing of MnLA-A antigens is an even more significant predictor of pregnancy wastage than is advanced maternal age. In our study population, sharing of MnLA-A antigens predicted 72% of pregnancy loss among 'unsuccessful' couples (P < 0.009). Identification of histocompatibility-associated factors influencing pregnancy success could have profound clinical implications for chronic spontaneous abortion, intra-uterine growth retardation and birth defects in humans. Among captive primates, identification of MHC or MHC-linked genes affecting reproductive outcome could lead to more efficient colony management strategies as well as development of a model for understanding human immunologically-mediated reproductive failure.

Denaturing gradient gel electrophoresis and its use in the detection of major histocompatibility complex polymorphism.

The major histocompatibility complex (MHC) has been studied extensively in humans and in mice and many methods are available for MHC typing of these well-characterized species. Studies of MHC variation in other species are ever increasing and researchers can choose one of a number of approaches for MHC typing of their species of interest. DNA sequencing is regarded as the 'gold standard' and it is frequently used for MHC typing. However, DNA sequencing is impractical when many individuals must be typed. Denaturing gradient gel electrophoresis (DGGE) offers a flexible and sensitive method for identifying and characterizing MHC alleles in any vertebrate species. This article reviews the theory and the practice of DGGE and examines the use of DGGE for MHC identification in various species. DGGE is compared to other similar techniques for MHC typing, such as single-stranded conformational polymorphism and reference strand-mediated conformational analysis. The advantages, problems, pitfalls and limitations of DGGE are considered and future perspectives on the use of DGGE for MHC typing are discussed.

Genetic diversity and reproductive success in mandrills (Mandrillus sphinx).

Recent studies of wild animal populations have shown that estimators of neutral genetic diversity, such as mean heterozygosity, are often correlated with various fitness traits, such as survival, disease susceptibility, or reproductive success. We used two estimators of genetic diversity to explore the relationship between heterozygosity and reproductive success in male and female mandrills (Mandrillus sphinx) living in a semifree ranging setting in Gabon. Because social rank is known to influence reproductive success in both sexes, we also examined the correlation between genetic diversity and social rank in females, and acquisition of alpha status in males, as well as length of alpha male tenure. We found that heterozygous individuals showed greater reproductive success, with both females and males producing more offspring. However, heterozygosity influenced reproductive success only in dominant males, not in subordinates. Neither the acquisition of alpha status in males, nor social rank in females, was significantly correlated with heterozygosity, although more heterozygous alpha males showed longer tenure than homozygous ones. We also tested whether the benefits of greater genetic diversity were due mainly to a genome-wide effect of inbreeding depression or to heterosis at one or a few loci. Multilocus effects best explained the correlation between heterozygosity and reproductive success and tenure, indicating the occurrence of inbreeding depression in this mandrill colony.

Human reproductive failure II: immunogenetic and interacting factors.

Studies in humans suggest that reproductive failure may be influenced by immunological factors or by genes encoding immunological factors and regulatory mechanisms controlling immunological expression. Using molecular methods, immunological factors can be clearly studied in an immunogenetic context. One example, the major histocompatibility complex (MHC), known as the human leukocyte antigens (HLA) in humans and MHC in other mammals, affects many different stages of reproduction. Studies in some outbred, and in closely related, human populations indicate that HLA, or HLA-linked, genes and HLA regulatory factors affect gamete development, embryo cleavage, blastocyst and trophoblast formation, implantation, fetal development and survival. Studies in non-human mammals indicate that MHC, or MHC-linked, genes such as the grc complex, Ped/Qa-2, t haplotypes and MHC regulatory factors, have similar reproductive effects. Human reproductive failure may also be a consequence of disruption of interacting factors, including interactions between HLA antigens, cytokines and natural killer (NK) cells. In this review, we highlight the importance of immunogenetic and interacting factors in human reproductive failure. We argue that studies in closely related human populations and animal models may contribute to a better understanding of the ways in which immunogenetic and interacting factors are involved in human reproduction.

Human reproductive failure I: immunological factors.

Human reproductive failure may be a consequence of aberrant expression of immunological factors during pregnancy. Although the relative importance of immunological factors in human reproduction remains controversial, substantial evidence suggests that human leukocyte antigens (HLA), antisperm antibodies, integrins, the leukaemia inhibitory factor (LIF), cytokines, antiphospholipid antibodies, endometrial adhesion factors, mucins (MUC1) and uterine natural killer cells contribute to reproductive failure. In contrast, fewer data support the roles of anti-trophoblast antibodies, anti-endometrial antibodies, T-cells, peripheral natural killer cells, anti-HLA antibodies, blocking antibodies and suppressor cells in reproductive failure. Although immunological factors involved in reproductive failure have been studied traditionally using assays for antibodies and/or antigens, detailed research on these factors demonstrates conflicting results in humans. Maternal and fetal immunology is also difficult to investigate in humans. For these reasons, molecular assays may serve as a valuable alternative to investigate how the immune system affects reproductive outcome. In Part I of this review, immunological factors involved in human reproductive failure are summarized and critically evaluated. Immunogenetic and interacting factors in human reproductive failure will be summarized and evaluated in Part II.

The MHC-E locus is the most well conserved of all known primate class I histocompatibility genes.

The HLA-E locus is characterized by limited polymorphism and low levels of cell surface expression. However, the function of the products of this nonclassical MHC class I gene remains unknown. To evaluate the conservation of the MHC-E locus throughout anthropoid primate evolution, we identified the homologue of the HLA-E locus in six different New World monkey species. Full-length sequencing of MHC-EcDNAs in four unrelated cotton-top tamarins (Saguinus oedipus) revealed no evidence for polymorphism. Using the PCR, denaturing gradient gel electrophoresis, and direct sequencing, we also identified MHC-E alleles in five other New World monkey species, representing all extant platyrrhine families. In contrast to all other classical and nonclassical MHC class I genes in primates, the rate of synonymous nucleotide substitution is much greater than the rate of nonsynonymous nucleotide substitution within exons 2 and 3 encoding the peptide binding region (PBR) in MHC-E genes. The PBR of the MHC-E molecule, therefore, has evolved under purifying selective pressures, and the very unusual evolutionary history of this ancient gene provides further evidence that the products of the HLA-E locus serve a critical immunological function. Given the remarkable conservation of the PBR during primate evolution, this critical immunological function is probably related to the peptide binding ability of the MHC-E protein.

The effect of 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation induced DNA double strand damage and repair in V79 cells.

Radiation induced DNA double strand breaks are believed to be important lesions involved in processes related to cell killing, induction of chromosome aberrations and carcinogenesis. This paper reports the effects of the radioprotector 2-[(aminopropyl)amino]ethanethiol (WR-1065) on radiation-induced DNA damage and repair in V79 cells using the neutral elution method performed at pH 7.2 or pH 9.6. WR-1065 (4 mM) was added to the culture medium either 30 minutes prior to and during irradiation with Cobalt-60 gamma rays (for dose response experiments) or during the repair times tested (for DNA rejoining experiments). The results indicate that WR-1065 is an effective protector against the formation of radiation-induced double-strand breaks in DNA as measured using a neutral elution technique at either pH. The protector reduced the strand scission factors by 1.44 and 1.77 in experiments run at pH 9.6 and pH 7.2, respectively. The kinetics of DNA double-strand rejoining were dependent upon the pH at which the neutral elution procedure was performed. Unlike the results obtained with alkaline elution, rejoining of DNA breaks was unaffected by the presence of WR-1065 at either pH.

A high frequency of Mamu-A*01 in the rhesus macaque detected by polymerase chain reaction with sequence-specific primers and direct sequencing.

SIV infection of rhesus macaques is an excellent model for HIV infection of humans. Unfortunately, it is has been difficult to identify macaques expressing particular MHC class I alleles. Here we describe the use of PCR-SSP for Mamu-A*01 typing of rhesus macaques. The Mamu-A*01 allele was amplified from genomic DNA using Mamu-A*01-specific primers and positive PCR products were directly sequenced. Our technique identified 15 Mamu-A*01-positive animals of 68 tested. We validated our molecular analysis by showing that lymphocytes from 8 Mamu-A*01-positive animals expressed Mamu-A*01 as determined by immunoprecipitation and 1-D IEF. The technical simplicity and accuracy of this typing method should facilitate selection of Mamu-A*01-positive rhesus macaques for AIDS virus pathogenesis and vaccine studies.

High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing.

High-resolution HLA-DRB typing is required for bone marrow transplantation between unrelated donors and recipients and also for identification of novel HLA-DRB alleles. Here we describe a method for the unambiguous identification of HLA-DRB alleles using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The highly variable second exon of all HLA-DRB1, -DRB3, -DRB4, -DRB5, -DRB6 and -DRB7 alleles was amplified using a single pair of generic DRB-specific primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and the sequences of these alleles were determined using fluorescent-based sequencing and allele-assignment software. The validity of this typing procedure was confirmed by identification of HLA-DRB alleles for 17 individuals previously characterized by PCR-SSP and/or cloning and sequencing techniques. We identified 34 different HLA-DRB alleles in these 17 unrelated individuals. Importantly, our analysis revealed HLA-DRB1 alleles which had not been identified using the PCR-SSP typing technique. Additionally, alleles from the HLA-DRB3, -DRB4 and -DRB5 loci were identified. Whereas traditional HLA-DRB typing methods provide limited information or require the use of multiple oligonucleotide primers or probes, our technique provides a reliable, specific and relatively rapid way of identifying all HLA-DRB alleles for high-resolution tissue typing.

The major histocompatibility complex and mate choice: inbreeding avoidance and selection of good genes.

It has been known for decades that MHC genes play a critical role in the cellular immune response, but only recent research has provided a better understanding of how these molecules might affect mate choice. Original studies in inbred mouse strains revealed that mate choice was influenced by MHC dissimilarity. Detection of MHC differences between individuals in these experiments was related to olfactory cues, primarily in urine. Recent studies in humans have shown an analogous picture of MHC-based mating. Taken together, these findings could support either the hypothesis of MHC-based inbreeding avoidance or the hypothesis of MHC-related avoidance of reproductive failure, since studies in mice, humans and pigtailed macaques have shown that parental sharing of certain MHC alleles correlates with frequent spontaneous abortion or prolonged intergestational intervals. Data from many mammalian species clearly demonstrate that reproductive failure occurs as a result of inbreeding. Therefore, MHC similarity might serve as an indicator of genome-wide relatedness. In contrast, increased fitness due to the presence of individual MHC alleles in a pathogenic environment could explain MHC-based selection of currently good genes. Specifically, the physical condition of long-living animals depends on the ability to respond to immunological challenge and an individual's MHC alleles determine the response, since, unlike the T cell receptors, MHC alleles are not somatically recombined. Therefore, sexual selection of condition-dependent traits during mate choice could be used to select successful MHC alleles, thereby providing offspring with a higher relative immunity in their pathogenic environment.

HLA-B typing by allele separation followed by direct sequencing.

Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous molecular typing method for the alleles at this locus. Here we describe a technique for the direct sequencing of HLA-B alleles. Initially, HLA-B alleles were PCR-amplified after locus-specific reverse transcription of RNA. Alleles were then separated using denaturing gradient gel electrophoresis (DGGE), which separates DNA fragments based on their sequence composition. Amplification products were excised from the gel and eluted DNA was reamplified and directly sequenced. The derived sequences were aligned to a database of published HLA-B sequences, and an initial allele assignment was made. This approach was theoretically sufficient to type 92 of the 118 known HLA-B alleles. The majority of the remaining 26 alleles contain differences at the beginning of exon 2, a region outside the DGGE-separated PCR products. Therefore, we used heterozygous sequencing of this region to identify 19 of these 26 alleles, raising the resolution power to 111 alleles. Using this technique, we analyzed immortalized cell lines and blood samples from several different sources. Nine immortalized cell lines were obtained from the 10th International Histocompatibility Workshop (IHWS) and nine were derived from aboriginal peoples. Additionally, 25 blood samples were acquired from a panel of donors previously shown to be difficult to type using serological techniques. Altogether, using this new method of allele separation by DGGE followed by direct sequencing, we typed 52 different alleles from 57 individuals, covering 40 serological specificities.

Identification of new mamu-DRB alleles using DGGE and direct sequencing.

Rhesus macaques represent important animal models for biomedical research. The ability to identify macaque major histocompatibility complex (Mhc) alleles is crucial for fully understanding these models of autoimmune and infectious disease. Here we describe a rapid and unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. Importantly, our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different Mhc-DRB lineages. Interestingly, two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. This new technique should also contribute to ongoing studies of Mhc function and evolution in many different species of nonhuman primates.

Denaturing gradient gel electrophoresis: a rapid method for differentiating BoLA-DRB3 alleles.

The products of the BoLA-DRB3 locus are important molecules in the bovine immune response. Several techniques have been used to study and define this locus but they are generally time consuming and limited in their ability to detect novel alleles. In this study we used denaturing gradient gel electrophoresis (DGGE), and direct sequencing, for BoLA-DRB3-typing. First, modified locus-specific primers were used in polymerase chain reaction (PCR) to amplify a 240 bp fragment of exon 2 of BoLA-DRB3 from the genomic DNA of 22 cattle and one pair of twin calves. The reverse primer included a GC-rich clamp to improve the physical separation of the BoLA-DRB3 alleles by DGGE. The denaturing gradient needed to produce separation of alleles was determined using perpendicular DGGE, and this gradient was then applied to parallel denaturing gels. The optimal time for producing allele separation was determined using a time-series analysis. The bands representing individual BoLA-DRB3 alleles were excised from the gels, reamplified, and the nucleotide sequence determined using fluorescent-based automated cycle sequencing. The nucleotide sequences of the separated bands were then compared to published BoLA-DRB3 alleles. A gradient of 10-15% acrylamide combined with a 15-50% ureaformamide gradient was successfully used to separate BoLA-DRB3 alleles in all individuals examined. Nucleotide sequencing showed that the 24 animals possessed 13 BoLA-DRB3 alleles, all of which have been previously described. The BoLA-DRB3 genotypes included 20 heterozygotes and two homozygotes. Three BoLA-DRB3 alleles were seen in each of the twin calves, possibly due to leukochimerism. The technique is reliable and rapid, and avoids cloning alleles prior to nucleotide sequencing and therefore offers distinct advantages over previous techniques for BoLA-DRB3-typing.

Three different MHC class I molecules bind the same CTL epitope of the influenza virus in a primate species with limited MHC class I diversity.

One of the most remarkable features of the MHC class I loci of most outbred mammalian populations is their exceptional diversity, yet the functional importance of this diversity remains to be fully understood. The cotton-top tamarin (Saguinus oedipus) is unusual in having MHC class I loci that exhibit both limited polymorphism and sequence variation. To investigate the functional implications of limited MHC class I diversity in this outbred primate species, we infected five tamarins with influenza virus and defined the CTL epitopes recognized by each individual. In addition to an immunodominant epitope of the viral nucleoprotein (NP) that was recognized by all individuals, two tamarins also made a response to the same epitope of the matrix (M1) protein. Surprisingly, these two tamarins used different MHC class I molecules, Saoe-G*02 and -G*04, to present the M1 epitope. In addition, CTLs from one of the tamarins recognized target cells that expressed neither Saoe-G*02 nor -G*04, but, rather, a third MHC class I molecule, Saoe-G*12. Sequence analysis revealed that Saoe-G*12 differs from both Saoe-G*02 and -G*04 by only two nucleotides and was probably generated by recombination between these two alleles. These results demonstrate that at least three of the tamarin's MHC class I molecules can present the same epitope to virus-specific CTLs. Thus, four of the tamarin's 12 MHC class I molecules bound only two influenza virus CTL epitopes. Therefore, the functional diversity of cotton-top tamarin's MHC class I loci may be even more limited than their genetic diversity suggests.

The MHC class I genes of the rhesus monkey. Different evolutionary histories of MHC class I and II genes in primates.

Homologues of the human HLA-A and -B MHC class I loci have been found in great apes and Old World primates suggesting that these two loci have existed for at least 30 million years. The C locus, however, shows some sequence similarity to the B locus and has been found only in gorillas, chimpanzees, and humans. To determine the age of the MHC class I C locus and to examine the evolution of the A and B loci we have cloned, sequenced, and in vitro translated 16 MHC class I cDNAs from two unrelated rhesus monkeys (Macaca mulatta) using both cDNA library screening and PCR amplification. Analyses of these sequences suggest that the C locus is not present in the rhesus monkey, indicating that this locus may be of recent origin in gorillas, chimpanzees, and humans. The rhesus monkey's complement of MHC class I genes includes the products of at least one expressed A locus and at least two expressed B loci, indicating that a duplication of the B locus has taken place in the lineage leading to these Old World primates. Comparison of rhesus monkey MHC class I cDNAs to their primate counterparts reveals fundamental differences between MHC class I and class II evolution in primates. Although MHC class II allelic lineages are shared between humans and Old World primates, no such trans-species sharing of allelic lineages is seen at the MHC class I loci.