Study of the inheritance of a polymorphic human trophoblast antigen and its equine equivalent
A thesis submitted by
Abbas Rezai
in partial fulfilment of the requirementsfor the degreeof Doctor of Philosophy
in the University of London
October 1995
Department of Immunopathology
St Mary's Hospital Medical School
1 IN THE NAME OF ALLAH, THE BENEFICENT, THE MERCIFUL
To my family
2 ACKNOWLEDGEMENTS
My main thanks and deep appreciation are due to Professor Mowbray for his invaluableadvice, help, encouragementand helpful criticism throughout the duration of this research. Without his guidance and stimulation this study would not have been completed.
I would like to thank Dr J Underwood for her continuing helpful comments, correction and proof reading.
I would like to thank Professor Allen and S Mathias for their help in collection of the horse placentaeand collaborative studies. This thesiswould not have materialisedwithout their help.
I would also thank to my friend, R Jalali, for all of his kindness over the years.
I wish to thanks Professor's Mowbray family and all of those who volunteered to participate in some of this studies necessaryfor the collection of the blood samples. My thanks is also due to the Labour Ward Staff of St Mary's hospital for their help in collection human placentae.
I would like to extend my gratitude to the Ministry of Health and Medical Education of Iran for it's financial support and scholarship.
I would also like to thank my parents for their patience.
Finally my special tributes are to my wife, Mahnaz, for her patience and moral support which allowed me to focus on my study, despite her sickness.
3 ABSTRACT
It has been shown that a polymorphic antigen, R80K, is expressed on the maternal surface of the human placenta. The antigen is covered by specific maternal IgG. A similar trophoblast antigen is also found on horse placenta, it appears to be analogous to human R80K. The antigen is covered with specific IgG antibody and following trypsin digestion it behavesin the same way as the human R80K. A stallion will cover as many as 30 mares in one breeding season,thus each foal has a common father but different mother. This facilitated the study of distribution of the R80K antigen in the progeny. I have studied the inheritance of the paternal allotypes of this protein in order to investigatethe distribution of allotypes in sibships.
The R80K trophoblast antigen which immunisesthemother is of paternal origin. The inheritance of paternal genes thus in part determines whether the mother can respond to a particular antigen. It has been suggested that antibody to trophoblast antigens may protect from the immune maternattack and subsequent abortion. Women with repeated early pregnancy failure usually do not have detectable antibody to MHC alloantigens; I have found that they do not usually exhibit antibodies to the R80K allotype either. The commonestpattern of recurrent abortion is the loss of all pregnanciesor all but the first. Since the condition is rare and women would be expected to make antibody to nearly all allotypes, it would be difficult to equate lack of antibody to R80K as responsiblefor recurrent abortion, as random inheritance of either of the two paternal allotypes would produce only a 50% loss. A possible explanation might be very unequal transmission of the two paternal R80K allotypes. Thus, instead of a high frequency of losing half the pregnancies,they might show loss of all pregnanciesif they all had only the one allotype to which the mother could not make protective antibody.
IgG antibodies were eluted from the human placenta, 3 families of horses and some unrelated horse controls by acid. The reactions between the eluted IgG antibodies and the antigen on the
4 remaining acid treated microvesicleswere measuredand used to identify the antigenic groupings in the families. The results can be summarisedas follows:
Equine antibodies eluted from placental microvesicles with acid did not react with human
acidified microvesiclesor peripheral blood cellsnor did human antibodiesreact with either horses
cells or acidified horse microvesicles.
The eluted antibody always reacted with its own antigen and with the acidified microvesiclesof
all half siblings in its family. Occasionally the same antigen was detected on unrelated
placentae.
I conclude that there is highly unequaltransmission of the two paternalR80K allelesin horses.
5 CONTENTS
PAGE No 1. Introduction 12
1.1 Mendel's Modes of Inheritance.
1.1.1 Autosomal dominant inheritance. 1.1.2 Autosomal recessiveinheritance. 1.1.3 Sex linked inheritance. 1.1.3.1 Y-linked (Holanderic) inheritance. 1.1.3.2 X-linked recessiveinheritance. 1.1.3.3 X-linked dominant inheritance.
1.2 Transmissionratio distortion.
1.2.1 Transmissionratio distortion of the t- haplotype genesin mice. 1.2.2 Genomic Imprinting. 1.2.2.1 MonoaUelic expression. 1.2.2.2 Monoallelic changesin the genome. 1.2.2.3 DNA Methylation as a mechanismfor imprinting.
1.3 Placentation.
1.3.1 Comparative placentation in Equids and humans. 1.3.2 Placentation in man. 1.3.2.1 Cytotrophoblast and Syncytiotrophoblast. 1.3.2.2 Chorion and Villi. 1.3.3 Placentationin horses.
1.4 Expression of antigensby Trophoblast.
1.4.1 Presenceof an 80kDa antigen on microvillus plasmamembrane. 1.4.2 MHC ClassI and II. 1.4.3 Trophoblast lymphocyte cross reactive antigen. 1.4.4 Placental alkaline phosphatase. 1.4.5 Fc receptors ligands for IgG in the human placenta.
1.5 AUoantibodiesin the Sera of PregnantWomen.
1.5.1 HLA antibodies. 1.5.2 Mixed lymphocyte culture (MLC) blocking antibodies. 1.5.3 Anti-idiotypic antibodies. 1.5.4 Anti-Trophoblast antibodies.
1.6 The aim of my study.
2. Materials and methods. 52
2.1 Preparation of microvesicles. 2.1.1 Equine placentae. 2.1.2 Human placentae. 6 2.2 Elution of IgG antibody bound to microvesicles.
2.3 Isolation of IgG from human sera.
2.4 Single radial immunodiffusion (SRID).
2.5 Measurementof equine IgG concentration.
2.5.1 Standard curve. 2.5.2 Concentration of samples.
2.6 Lymphocyte preparation.
2.7 Preparation of B-lymphocyte suspension.
2.7.1 Preparation of 1% sheepred blood cells. 2.7.2 Separationof T and B-lymphocytes.
2.8 Preparation of cells by dextran sedimentation.
2.9 Monocyte preparation.
2.10 Staining of peripheral blood cells.
2.11 Collection of sera.
2.12 Radioiodination of proteins.
2.13 Reaction of acidified vesicleswith acid eluted IgG antibody.
2.13.1 Equine vesicles. 2.13.2 Human placentalmicrovesicles.
2.14 Methods for detection of the R80K antigen on the surfaceof peripheral cells.
2.14.1 Binding of acid eluted IgG antibody to peripheral blood cells. 2.14.2 Detection of antibody using FACS. 2.14.3 Complementdependent cytotoxic antibody test.
2.15 Detection of anti R80K antibody in sera of pregnant women and women with recurrent spontaneousabortion.
2.16 Trypsin digestion of microvesicles.
2.17 SDS PAGE. 2.17.1 Staining and destainingof gels.
2.18 Purification of proteins.
2.18.1 Gel filtration chromatography. 2.18.2 Purification of eluted IgG antiboy from human placentaeby affinity chromatography. 7 2.19 Statistical methods.
3. Results. 69
3.1. Studies of the IgG bound to syncytiotrophoblastmicrovesicles.
3.1.1 Acid elution of antibody from human microvesicles. 3.1.2 Acid elution of antibody from horse placentalmicrovesicles. 3.1.3 Reaction of human eluted antibody with acid treated microvesicles. 3.1.4 Determination of optimal conditions for the detection of binding of cells and antibody. 3.1.5 Eluted human antibody reactswith the father's cells rather than a third party. 3.1.6 Microscopy of dextran and Lymphoprep preparedhuman peripheralblood cells. 3.1.7 Reaction of human antibody with cultured lymphoblastoidB-lymphocytes cell lines homozygousfor IHLA. 3.1.8 Reaction of equine eluted antibody with acid treated vesicles. 3.1.9 Reaction of acid eluted antibody with different preparationsof peripheralblood cells. 3.1.10 Acid eluted IgG antibody is speciesspecific.
3.2 Studies of the antigen detectedwith eluted antibody.
3.2.1 Reaction of the 50KDa fragment with acid eluted antibodies. 3.2.2 Trypsin digestion of horse vesicles.
3.3 Cross reactions of antigen and antibody betweenindividuals of the samespecies.
3.3.1 Cross reactions of humantrophoblast by binding assay. 3.3.2 Human cross reaction of Lymphoprep and dextran prepared cells by a binding assay.
3.4. Familial distribution of antigen allotypes.
3.4.1 Transmissionof a paternal antigen in a human family. 3.4.2 Typing of Nearco family membersusing acid eluted antibodiesand acid treated vesiclesof individual placentae. 3.4.3 Cross reaction within the family shown by eluted antibody to horse PBC. 3.4.4 Distribution of equine allotypes in the family of Nearco.
3.5. Antibodies to paternal allotypes in maternal sera.
3.5.1 Detection of antibodies in sera of women with normal pregnancy by binding to paternal PBC. 3.5.2 Detection of antibodies in sera of women with repeated abortion. 3.5.3. Detection of antibodiesto husbands'PBC in sera of immunisedwomen.
4. Discussion 106
5. Bibliography 122
6. Appendix A 141
7. Appendix B 143 8 List of illustrations.
Figures Page No.
1.16
2.19
3.71
4.73
5.74
6.80
7.81
8.87
9.91
10.99
9 List of Illustrations.
Tables Page No.
I. 28
II. 31
III. 72
IV. 75
V. 78
VI. 79
VII. 82
VIII. 83
IX. 84
X. 85
M. 88
XII. 89
XIII. 92
XIV. 94
Xv. 95
XVI. 97 x vii. 101
XVIII. 103
M. 104
XX 105
Plates
I, 70 II 76 III 77
10 List of abbreviations used
A Abortion Ab Antibody Ae Acid eluted antibody. AMPS Ammonium persulphate APCA Antipaternal cytotoxic antibody. L Live. MAb Monoclonal antibody. NK Natural killer. NSB Never So Bold. PBC peripheral blood mononuclearcell. PBL peripheralblood lymphocyte. PD Primo Domini. PLAP Placentalalkaline phospatase. SDS Sodium Dodecyl Sulphate SS Soviet Star. Te Trypsin treated acidified vesicles. temed N'N'N'N' Tetramethylethylenediamine TRD Transmission ratio distortion. V Placentalsyncytiotrophoblast microvesicles. Va Acid treated microvesiclesvesicles.
11 1. INTRODUCTION
The process of reproduction in women is a highly complex system and it involves many processesat the molecular and tissue levels. Much attention has been focused on the placenta which is the tissue in closest contact to the maternal circulation. The developing embryo is dependenton this organ for survival and growth. Trophoblast is the fetal cell in direct contact with the mother. It covers the maternal aspect of the placenta and fetal membrane.The trophoblastic layer of the placenta proliferates and forms the chorionic villi which consists of an unbroken layer of syncytiotrophoblast around which the maternal blood flows in the intervillous spaces. The syncytiotrophoblast has a complex branching microvillous plasma membrane,which greatly increasesits surface area for exchangeof nutrients and gases. The isolation and purification of the plasmamembrane from the syncytiotrophoblastof the full term placenta, using procedures based on that of Smith, Brush and Luckett (1974) has enabled investigators to detect various surface components and identify their characteristics. It has been shown (Jalali et al 1989,1995a) that there is an 80kDa antigen on syncytiotrophoblast which has an IgG antibody bound to it in all normal term pregnancies. The antibody is also present in the maternal serum during normal pregnancy. A similar trophoblast antigen is also found in the horse placenta, analogousto the human 80kDa antigen. It has been speculated that the antibody made to this trophoblast antigen may be necessaryfor successfulpregnancy
(Underwood 1993). The trophoblast antigen which immunises the mother is of paternal origin. The inheritance of paternal genesthus determineswhether the mother will respondto the particular alloantigen. It is known that maternal imprinting is the usual finding in trophoblast (Surani et al 1984, Barton et al 1984) and thus the modes of inheritance and the
immune gene expressionmay both play a role in determiningthe maternal response.
12 Women with repeated early pregnancy failure usually do not have antibody to paternal antigens (Mowbray et al 1993). If lack of antibody is responsible for the recurrent failure of pregnancy, and the commonest form involves loss of all pregnancies, then it is difficult to fit this with random inheritance of either of the two paternal allotypes. A possible explanation would, however, be extremely unequal transmission of the two allotypes. Thus, instead of a high frequency of losing half the pregnancies,they might show loss of all. I set out to study the inheritance of the paternal allotypes of the R80K protein in order to investigate their distribution. The study of this would be very difficult in normal human pregnancy. In this thesis I have used thoroughbred horses to demonstrate the mode of inheritance of this polymorphic antigen.
Patterns of Inheritance
1.1 Mendel's Modes of Inheritance.
In family pedigrees most traits follow simple Mendelian modes of transmission. Mendel's fundamentaldiscoveries are explainedby his two laws of inheritance. The first law statesthat each pair of genes segregate,one gene passingto the one gamete and the other of the pair to the other gamete. The second law states that members of different gene pairs assort to gametesindependently of one another. In other words, alleles segregate,non alleles assort.
The following examplesare used to explain these laws. Two alleles may be termed A and a,
in parents with identical genes at the samelocus e.g. AA, produce gametesof the sametype, is this case,the only genotype of the children would be AA. When another genotype present
have AA half Aa. such as Aa in one parent then half of the children will the genotype and will have AA If both mother and father have Aa genotype 25% of their children will the genotype
Aa Mendel's law describes other 25% as genotype and 50% will show the genotype. second inheritanceof more than one gene pair which assort independently.
13 A determined is gene characteristic known as a trait. An autosomal trait, which is determined by be dominant a gene on an autosome,may or recessive. Dominant traits are those expressed in heterozygotes and traits which are expressedonly in homozygotes are termed recessive. A trait is determined by which a gene on one of the sex chromosomesis termed sex-linked and be dominant may either or recessive. Some geneticists,specifically human geneticists,believe that these terms have become redundant and even misleading (Connor & Ferguson Smith
1993), e.g. genes which are subject to imprinting and geneslocated on X-inactivated female. chromosomes are not expressed. In co-dominant traits, the effects of both alleles may be seen in heterozygotes. the These terms would apply at the phenotypic level. In fact, at the level of gene action genes are not recessive or dominant, they are active or inactive. Usually both paternal and maternal genes are expressed, and result in production of their respective products e.g. proteins. If one or both genes,as a result of a mutation or deletion are defective, a normal level of effective products may not be achieved. If both genesare defectivethere will be a zero level of normal product. Defective genes, leading to a 50% level, may or may not produce an abnormality. If 50% of the product is sufficient, the gene is dominant. When
100% of the levels are required, the gene is recessive.
1.1.1 Autosomal Dominant Inheritance.
An autosomal dominant trait affects both male and female alike and will be transmitted to the offspring in the same ratio. If a person possessingan autosomal dominant trait marries a person lacking that dominant trait, then 50% of their children on averagewill have the trait. In this case,the partner having the dominant trait producestwo types of gametesin equal number which pass to one or other offspring, but the other partner produces only one type of gamete.
It should be pointed out that in large samplesthe ratio should be 1:1, but in fact the real number of offspring with a characteristic may not fit with this and factors such as penetration
14 and expressivity affect the situation. Penetrence is a statistical concept and refers to the proportion of individuals who possessthe gene and in whom some manifestationsof the gene can be found (Vogel & Motulsky 1986a). Non-penetrance explains the situation in which a gene may not express itself at all, e.g., normal carrier men for fragile X-syndrome (Brown
1989). Expressivity is a term used when there is a different degree of expression, e.g. in polydactyly where there may be a small wart like appendageor a complete extra finger on the side of the hand of another individual. This is very common in autosomaldominant traits as it is in some diseases. Variable expressivity in familial hypercholesterolaemiais an example.
This is due to a gene located on a short arm of chromosome 19 (19P) which codes for a protein that acts as a receptor for somelipoproteins. Defective clearanceof cholesterol and an elevated level is a result of defects of this receptor (Larricke & Burck 1991).
1.1.2 Autosomal Recessive Inheritance.
This type of inheritanceis manifestedonly when the gene is presentin a double dose, i. e. in the homozygotes. In these circumstances,people who manifest recessivetraits, are usually the offspring of parents who are both heterozygotesfor the gene in question. Most recessive disorders, however, are rare and offspring of an affected person will be normal, except when the affected person marries someonecarrying the samegene. Thus, the pedigreepattern of an
autosomal recessivetrait differs from the autosomaldominant traits.
15 Figure 1. Pedigree of autosomal recessiveinheritance.
)13 Hetavrygotes
0 Affected female
consanguineous marriage
If two heterozygotes carrying the same recessive gene marry, about one quarter of the offspring will inherit a double dose of the recessive gene, one quarter will be normal homozygotes and one half will be heterozygoteslike their parents. These are averagefigures, as rarely will a carrier be recognised (Connor & Ferguson Smith 1993). Therefore, the proportion of each offspring in a family carrying the trait must be calculated using mathematical devices formulated for this purpose. A great variety of disorders are inherited in this way. Human sickle cell diseaseand phenylketonuria are classicalexamples of this type of inheritance (Harper 1993a, Emery & Rimoin 1983).
In sickle cell disease,a point mutation which occurs on chromosome 11 causessubstitution of valine for glutamic acid in the 0 chain of haemoglobin S (HbS) (Harper 1993b). Normal adult human haemoglobin consists of four globin chains (o:202) each of which carries a haem group (Perutz 1976). This is HbA and carries oxygen from lungs to the tissues. HbS carries
16 less oxygen and causesaffected cells to clump together and thereby obstruct the small arteries.
Heterozygotes are clinically normal, their normal 0 chain gene product is enough to prevent
symptoms, but homozygotes show characteristic sickle shaped red cells with low oxygen
tension.
Phenylketonuria is causedby a mutation affecting the enzyme phenylalaninehydrolase which
converts phenylalanineinto tyrosine. The result is accumulation of phenylalanineand a toxic
metabolite, phenylpyruvic acid, which causes damage to the developing brain and leads to
profound mental retardation (Lewin 1994a).
1.1.3 Sex-Linked Inheritance.
Sex-linked inheritance, either X-linked or Y-linked, is the type of inheritancepattern shown by
genescarried on a sex chromosome.
1.1.3.1 Y-linked (Holandric Inheritance).
In this type of inheritance only males receive a trait as the genesinvolved are carried on the Y
chromosome. They transmit the gene to all of their male offspring, but to none of their
daughters. Apparently there are just a few genes located on the Y-chromosome other than
male determinant genes, however, no human Y-linked diseaseshave been described (Connor
& Ferguson Smith 1993).
1.1.3.2 X-linked RecessiveInheritance.
The Males only have one X chromosomeand any particular gene carried on it is unpaired. man is determined is said to be hemizygous for that particular factor. An X-linked recessivetrait in homozygous by a gene carried on the X-chromosome. The trait will be only manifested the
17 female where the gene is in double dose. Males always manifest the trait becausethere is no
other gene to counteract the effect(s) of the gene in question, even though it is a recessiveone
(Emery 1983). Thus, the traits are inherited in families by both female carriers and affected
males.
Lyonisation is a process by which one of the two X- chromosomesis inactivated in every
female mammalian cell. Inactivation occurs only in somatic cells during early embryonic
development. In humans it is a random process and either the paternal or maternal X-
chromosome is inactivated. Once established,the same chromosome is inactivated in all the
cells of the progeny (Lyon 1972). Therefore, there are two types of cells in a heterozygote
female, one with the inactivated gene and one with the normal gene (mosaic status). The
molecular basis of this inactivation is only now being revealed. DNA modification by
methylation of cytosine residuesin CpG dinucleotideshas been consideredin the maintenance
and production of X-chromosome inactivation (Garther & Riggs 1983). The mechanismis
similar to that found in other kinds of gene imprinting (see 1.2.2). Methylation patterns of X-
linked housekeepinggenes are different on the active and inactive X-chromosome. The CpG
islands of these geneson the inactive chromosomeare hypermethylatedwhereas expressionof
genes on the active X-chromosome is associatedwith low methylation (Wolf et al 1984, Keith
et al 1986)
An X-linked trait is never passedfrom father to son, but carrier females can transmit to sons
or half the daughters also become carriers:
18 Male Female
Parents xY Xx
Gametes XYX X
xx Xx xY Offspring xY
Figure 2. Pedigree pattern of X- linked recessiveinheritance. Xx is a female unaffected carrier of a defective gene(x) and xY is an affected male, where there is no compensationby a normal paternal X chromosome.
Very rarely, a heterozygousfemale may show some features of an X-linked recessivedisorder
(Emery 1983). The reasonsmight be : a) Lyonisation: Inactivation by chance happens to the normal gene carried on the unimprinted X chromosomein most of her cells. b). The second explanation is that such femalesmay have a 45,X (Turner's Syndrome) or
XY(testicular feminisation syndrome) sex chromosome constitution. Females with Turner's
Syndrome (45.X) have only one sex chromosomeand usually will be aborted. Individuals who survive are sterile, and have a number of congenital abnormalities. Affected individuals with testicular feminisation syndrome are femaleswith a normal phenotype, but they are invariably
(Vogel Motulsky 1986b). sterile. They have a male karyotype and male gonads and
1.1.3.3 X-linked Dominant Inheritance.
An X-linked dominant condition manifests itself in the hemizygous males as well as in heterozygous females. Males with an X linked dominant trait will all be affected, but in the
19 females it is variable due to Lyonisation, so that, on averagemales are more severelyaffected than females. All daughters inherit the trait from an affected father, but if the father marries a normal woman all sons are free of the trait. The segregation ratio among the children of affected women is 1:1 regardlessof the child's sex, similar to an autosomal dominant trait. A male transmits the trait to all his daughters,and there is an excessof affected females(Connor
& Ferguson Smith 1993). A common example of X-linked dominant inheritancein humansis vitamin D-resistant rickets (McKusick 1983), Xp21-22, with growth retardation and low serum phosphate.
1.2 Transmission ratio distortion.
Transmission ratio distortion (TRD) is a phenomenonin which the inheritance of a gene does not follow normal Mendelian inheritance. As stated in the abstract such a phenomenonhas been seen in the study of inheritance of the paternal R80K alleles where there is a highly unequal transmission of the two paternal alleles in horses studied i. e. one allele is transferred and the other is somehow eliminated. The following section explainsa well known exampleof
TRD. Possible mechanismswhich might be involved in transmission of only one paternal
R80K allele are then discussed.
1.2.1 Transmission ratio distortion of the t haplotypes genesin mice.
There are two variant forms of the proximal region of mouse chromosome 17 in nature, a wild type (+) and the other form which is defined as at haplotype (t) (Fox et al 1985). A male
bearing mouse heterozygous for a complex t- haplotype can transmit the t chromosometo more than 95% of his offspring. At haplotype is not a single locus mutation or a mutant gene family. It consists of many genes, some of these genesfunction normally, the products of the
development(Lyon other genes cause sterility, reduced fertility and abnormality in embryonic
20 1979 Artzt et al ; et al 1974). Meiotic recombination is strongly suppressed along the t haplotype in heterozygous DNA mice for any t haplotype (+/t) (Lyon et al 1979). A complete
haplotype t is one in which the recombination is suppressed along the entire 12cM region of chromosome 17. This region of mouse chromosome 17 has been called the t complex (Fox et al 1985). Thus t haplotypes are variant forms of the t complex. Rare crossing over, however, produces partial haplotypes. Different partial t haplotypes have different extents of t haplotype DNA (Fox et al 1985).
Males heterozygous for at haplotype will transmit the t-bearing chromosometo nearly all of their offspring. This is due to TRD and is mediatedby production of functionally inequivalent gametes. Thus TRD of the t haplotypes genes in mice is the phenomenonin which a male heterozygous for a complex t haplotype can transmit the t bearing chromosome to more than
95% of his offspring. Genetic studies have confirmed that at least five t haplotype encoded loci combine to cause TRD. Lyon (1984) and Silver & Remis (1987) have shown that TRD occurs through the action of at least four t complex distorter (Tcd) loci upon a single t complex responder (Tcr) locus. They showed that various distorters act on a responder to increase its transmission. A heterozygotesmale for Tcr which carries multiple t- complex distorters (Tcdl, 2,3 and 4 loci) transmits the Tcr containing chromosome 17 homologue to nearly all of his progeny. The presenceof at least two distorters are necessaryto give more than 50% transmissionratio. These frequenciesbecome <20% in the absenceof Tcd loci for a
depend, however, male carrying the Tcr locus (Dunn & Bennett 1968). These all on the
locus, be Tcr has presenceof the Tcr locus, in the absenceof this there would a normal ratio. Recently been mapped to a small region in the centre of the t haplotypes called T66B. the
family, the molecular cloning of the four t members of the T66 gene the genetic mapping of
21 haplotype loci adjacent t and comparative restriction enzymeanalysis of these genes,has been described (Bullard & Schimenti 1990).
1.2.2 Genomic Imprinting.
In is mammals the genome inherited equally from both parents and thus two copies of every autosomal gene are present. Recently investigators have tried to identify and explain conditions where the inheritance of certain traits does not follow normal Mendelian inheritance. The concept of genomic imprinting has become important and refers to differential expression of the paternally or maternally inherited genetic material. The term
`imprinting" explains a modification in which a gene is not expressed. Thus paternal imprinting of a gene means that expression is from the maternal gene and maternal imprinting infers that the phenotypic expression or more noticeable expression is transmitted from the father. Most of our knowledge of imprinting comes from studies of pronuclear transplantation in mice. In these experimentstransgenes have been shown to be differentially modified and expressed in an allele specific manner (Surani et al 1986, Chaillet et al 1991). Other observations that have confirmed the existenceof genomic imprinting are when specific genes, known to be involved in a certain disorder appearto be inherited from one parent, or when a more severe form of the disorder is seenif the genesare inherited from one parent rather than the other. An example of this is seen in Huntington's Disease(Ridley et al 1988, Reik 1988) where, if inherited from the father the diseaseis more serious. Transgenicuniparental disomy
(Cattanach mice in which both intact chromosomesof the pair come from the only one parent is by and Kirk 1985) have provided further evidence. If a gene silencedwhen transmitted one be parent, then if both chromosomes come from the parent of the same sex, there will no has been in fibrosis. active allele. Uniparental disomy in the human also recognisede. g. cystic
This is a maternal uniparental disomy (Voss et al 1989). The phenotypes of triploids in the
22 human and the expression of certain chromosomal disomies in mice and humans are further for evidence mammalian genomic imprinting. The most dramatic evidence for parental imprinting in human is the complete hydatidiform mole, in which fertilised oocytes lack the maternal genome and have two paternal copies (Lawler & Fisher 1993). The following properties for imprinting have been suggestedby studiesin mammals:
a) The mechanismmust be reversible following passagethrough the gremlin of the opposite sex. b) Imprinting most likely changestranscription e.g. H19 gene on mouse chromosome7 and insulin growth factor 2 receptor (Igf2r) gene on chromosome 17 are repressedat the paternal locus (Bartolomei et al 1991, Barlow et al 1991). c) Imprinting is set during gametogenesis(the only period that maternal and paternal genomes are separatedand can be subjectedto any differences). d) Imprinting must be the result following DNA replication and must remain on only one chromosome.
Four classesof imprinting have been described(Varmuza & Mann 1994). Mechanismswhich might produce TRD would be those which resulted in deletion of one allele from any offspring. The two paternal alleles are separatedin the sperm and in the embryos resulting from fertilisation of an egg by the sperm. If one allele is presentvery rarely one would assume that either the sperm or the embryos containing that allele have been selectively killed or removed. Imprinting of the R80K gene might, by resulting in differential expressionof the gene product in different spermatozoa or embryos allow of such a selection process. by Monoallelic expression would then be the name of the process, probably occurring methylation of one of the two paternal genespresent.
23 1.2.2.1 Monoallelic Expression.
Expression of a few genes in the mouse embryo may dependupon the parental origin, so that one locus is expressedand the other is silent. So far, in heterozygous mice, the following endogenousgenes have been recognisedto be imprinted and expressedmonoallelically. These are the Ig 2 gene (insulin like growth factor II), SNRpN gene (small nuclear RNA associated polypeptide N), Igf2r gene (insulin like growth factor II receptor), H19 (coding for a polyadenylated RNA transcript whose function is unknown), Insulin 1 and 2 genes in the mouse yolk sac and finally the U2af binding protein related sequence(U2afbp-rs).
Igf2 is a polypeptide hormone present in fetal plasma and tissue. In rats, the plasma level of
1gf7 declines progressively in the postnatal period. Its major physiological role is as a embryonic growth factor (DeChiara et at 1990). These genes are located on the mouse chromosome 7 (Laley and Chirguin 1984). Mouse chromosome 7 is homologous to the human chromosome 11p 15.5. DeChiara et at (1991) demonstratedthe pattern of expression of the Igf 2 gene. The Igf 2 gene is subject to parental imprinting, the paternal allele is expressedin embryos and the maternal allele is silent. They also showed that the maternalIgf2 allele is only expressedin the choriod plexus and leptomeninges. Ferguson-Smithet al (1991) investigated genes expressedon distal chromosome7 and found that one of these geneswas
1gf7. Rotwein & Hal (1990) also reported that one of these genes is Ig 2. All these data
7, suggest parental imprinting for some genes located on mouse chromosome with paternal expression and silence of maternal genes.
SNRpN (small nuclear ribonucleic protein N) encodes part of a ribonuclear protein which
brain. It is the and is a catalyses RNA splicing in the also expressed on paternal allele
in (Leff 1992). SNRpN human homologuesmay play a maternally imprinted gene mouse et al
24 part in the aetiology of the imprinting disorder called Prader-Willi Syndrome (Cattanachet al
1992 Reed & Leef 1994 ). ,
Igf2r Insulin-like growth factor H receptor is identical to the mannose6-phosphate receptor
(Roth 1988, Morgan et al 1987). Kiess et al (1988) showed that the mannose-6-phosphate receptor has a separatebinding site for Ig 2. Barlow et al (1991) showed that the Ig 2r gene maps to the same genetic location as Tme (T associated-maternaleffect), is imprinted and expressed only from the maternal chromosome, whereas neighbouring loci are transcribed from both chromosomes. These data are good explanationsfor heterozygousmice which have the deletions of the locus. They grow normally if the paternal allele is missing but, Arlmaternal -if allele is missing they die during early development.
H19 the murine H19 gene, maps to the distal segmentof mouse chromosome7 and encodesa
2.5 kilobare mRNA which is highly expressedin large variety of fetal tissues during cell
differentiation (Pachnis et al 1984). Bartolomei et al (1991), detected that the H19 gene is
subject to imprinting. The maternal copy is expressedand paternal copy is silent.
U2afbp-rs U2af binding protein related sequence(U2aJbp-rs) gene is located on mouse
is chromosome 11. It is a recently discovered imprinted. gene and only the paternal allele from homology expressed. The amino acid sequencededuced the cDNA showed significant (Hayashizaki to the U2 small nuclear ribonucleoprotein auxiliary factor 35kDa subunit et al
1994).
(Ins 1& 2) Insulin 1&2 Giddings et al (1994) evaluatedexpression of insulin 1 and 2 genes
Their indicated that the in the mouse yolk sac and in the embryonic pancreas. result maternal
25 is inactive allele and only paternal alleles were expressedfor both genes in the yolk sac, but both alleles were active in the embryonic pancreas.
Mas The Mas gene on mouse chromosome 17 encodes for, a protein. This protein is structurally similar to a class of receptor peptides that interact with aG protein to stimulate both DNA synthesis and increase intracellular calcium. Villar and Pedersen(1994) analysed the allele specific expression of the Mas gene during mouse development. Their results indicated that during early stages of fetal development, expressionwas exclusively from the paternal allele in all embryonic tissues. By 13.5 day of gestation expressionwas restricted to tongue, skeletal muscle, heart and visceral yolk sac. The maternally inherited allele was repressedin a developmentaland tissue specific manner.
In mammals other than the mouse, naturally occurring examples of genomic imprinting are beginning to be recognised. Kanbour-Shakir et al (1990) showed that expression of the maternal major histocompatibility complex (MHC) Class I antigen is suppressedin the rat placenta.
Monoallelic expression in the human also has been recognised. Zhang and Tycko (1992) showed that the human H19 gene located at chromosome 11p 15 is expressedmonoallelically.
Then in 1993 Zhang et al showed that the active H19 allele is a maternal allele and showed
is loss of this allele in a case of the nephroblastomaWilm's Tumour(WT). WT assumedto
11 (Mannens result from a homozygous mutation of a gene located on chromosome p et al
1988). It is characterised by aniridia, genitourinary abnormalities and mental retardation. inactivation Rainier et al (1993) showed that H19 and Igf2 displayed imprinting with of
They focused kidney WT becauseboth H19 paternal and maternal alleles, respectively. on and
26 Ig f2 in and are expressed the developing kidney. Their result showed that there was no loss of heterozygocity (LOH) at 11p in 69% of WT and this locus showed biallelic expressionof one both or genes. With respect to these data they suggesteda new mechanismin carcinogenesis
`relaxation or loss of imprinting" Ogawa et al (1993) showed that Igf1 in human kidney tissue was subject to imprinting and only paternal alleles were expressed,whereas in WT the expression was biahelic. Henry et al (1991) studied patients with the Beckwith Wiedemann
Syndrome (BWS). They demonstrated that both copies of 11p 15.5 were of paternal origin.
Having considered that transgenic mice lacking a functional paternal Igf-II gene undergo growth retardation (DeChiara et al 1991) together the results of the studies of Scott et al
(1985) indicate an increase of IgC mRNA in tumours of BWS and the location of Igf2 gene on mouse chromosome 7 and human homologous 11p 15.5. It makes sense that Ig/7 is probably the candidate gene for BWS. Ohlsson et al (1993) demonstrated that only the paternally inherited Igf2 allele is active in both the embryonic and the extra embryonic compartment of the human conceptus. Giannoukakis et al (1993) designed a direct demonstration for imprinting of 1gf7 gene in human placenta. They studied six term and two pre-term heterozygote placentaeand showed that humanIgf 2_is subjectto imprinting.
In addition to H19 and Igf1, the SNRpN gene is also subject to imprinting in humans(Reed &
Leff 1994). This gene is located at 15g12 chromosome. They showed only the paternal allele is expressedin fetal brain and heart. This supports the hypothesisthat paternal absenceof the
SNRpN gene is possibly associated with Prader Willi Syndrome (PWS), a human
feet, flat faces neuroendocrine genetic disorder characterisedby obesity and small hands and disomy is and delay in mental and physical development. Another example of uniparental
Angelman's Syndrome(AS), in which both copies of chromosome 15 come from the father,
AS is by defective with a maternal deletion at 15q11-13. characterised muscular control
27 resulting in jerky movement, very poor speechand mental retardation. In contrast to PWS, the deleted 15 is invariably of maternal origin in AS. Therefore PWS and AS are reciprocal deletion syndromes involving imprinted loci caused by two very closely linked but distinct genes (or gene clusters) which are oppositely imprinted (Wagstaff et al 1992). In conclusionit is highly likely that an associationbetween the SNRpN gene and PWS, and paternal absenceof
SNRpN is responsiblefor the PWS phenotype(Reed & Leff 1994).
Table L Current list of known imprinted genesin mice and men.
Imprinted genes in mouse and Man
Gene Expressed Mouse Human Comments Allele Chromosome Chromosome
Igel paternal 7 11p 15 mouse:LoI in chorioid plexus human: Imprinting in all tissue except adult liver
H19 maternal 7 lip 15 ----
Igf2r maternal 17 6q 15 Human homologue unimprinted
SNRpN paternal 7 15g12 ----
Imprinted in Insl&2 paternal ------the mouse yolk sac but not in the embryonic pancreas
U2afbp-rs paternal 11 ------is Mas paternal 17 differential expression manifestedin developmentand tissue specific manner
28 1.2.2.2 Monoallelic Changes in the Genome.
Preferential loss of heterozygosity (LOH) of specific parental alleles in several childhood tumours (Caron et al 1993) and expansion of trinucleotide repeat in the fragile X syndrome
(Fra(X)) are examples of monoallelic changes. Fra(X) mental retardation is a `triplet repeat
disease"which results from the presenceof extensive expansion of a (CGG)n repeat in the fragile X-gene (FMR-1) located at Xq27. When the repeat sequencesbecome longer than a
critical length, they become unstable so that during DNA replication they undergo massive
length expansionsand functionally inactivate their associatedgenes. Fragile X mutations can
be subdivided into premutations and full mutations. A small increase in the length of triplet
repeat is referred to a`ýremutation" while massive expansions have been called `full
mutations" The premutation becomesa full mutation only when it is transmitted by a female
and this is a postzygotic event (Reyneirs et al 1993).
1.2.2.3 DNA methylation as a mechanism for imprinting.
Having considered the properties of imprinting, its reversibility and the imposition of
suppressionof transcription during gametogenesis,heritable in a chromosomespecific manner, individual DNA methylation seemsto be the best candidate for preventing reading of genes
(Razin & Cedar 1991). DNA methylation occurs in mammaliancells only at cytosine residues
in have in the context of CpG dinucleotides. Gene transfer experiments mice provided
data indicate that molecular evidence for an imprinting process and the most straightforward (Reik 1987). Several differential expressionof the transgeneinvolves DNA - methylation et al
DNA between It groups have reported examplesof differencesof methylation parental alleles. inhibits by the interaction is suggested that methylation probably gene expression affecting Fully between the protein and DNA required for transcription (Cedar 1988). methylated
29 sequences, resistant to DNAase I are structurally similar to the inactive genes of cells.
Conversely unmethylatedDNA has a DNAse I sensitivestructure (Keshet et al 1986).
Ferguson-Smith et al (1993) showed that hyper-methylation is consistent with repressionof the paternally inherited mouse H19 gene. They observed that the H19 promoter is semi- methylated in normal embryos and unmethylatedin the maternally disomic for H19 (mat D 17) embryo, this indicated that the maternal allele is normally methylated. Zhang et al (1993) described an absolute correlation between allele specific DNA methylation and monoallelic expression at the H19 locus. Two other genesfor Igf7 and Igf2r, however, have been shown to have some degree of allele specific methylation which is different from the H19 gene.
Stager et al (1993) investigated the role of methylation in imprinting by examining parental- specific patterns of methylation at the Ig 2r locus in male and female gametes,in the embryo and in the adult. Their results suggestedthat methylation is necessaryfor expressionof the
Igf2r gene.
in Igf2 but in Allele specific methylation is also present at severalCpG sites the gene of mice,
hypermethylated in late this case, the active Ig f2 allele was found to be only the embryo and it is believed if the adult liver tissues (Brandeis et al 1993). In conclusion that methylation fertilisation. In the regulates imprinting it takes place after allele specific methylation, parental (Brandeis et al 1993, allele can be fully methylated, partially methylated or unmethylated is or Stöger et al 1993 and Bartolomei et al 1993) and this associatedwith either repression expression of a defined gene.
30 1.3 Placentation.
The develop mammalian embryo cannot without the placenta. It exhibits a wide variety of structural differencesbetween modifications, so major placentaemay be found even in closely
related species (Amoroso 1952). Therefore it is important that these differences be
considered.
1.3.1 Comparative Placentation in Equids and Humans.
The process of placentation is highly variable among even closely related mammals. Several different types of placentae within the mammalian kingdom have been recognised (Amoroso
1952). Although mammalian placentae can be classified by several factors; classification
involving both shapeand structure is most commonly used.
Table II. Some differencesbetween horse and human placentae
MAN Horse
Shape Discoid Diffuse
Fine Layer H -.mochorial Epitheliochorial
Decidual Deciduate Non-Deciduate
Placental Villi Long with many branches Short with relatively few branches
Amoroso (1952) classified placentaeby shapeinto diffuse, cotyledonary, zonary and discoid. the On diffuse placentae, e.g., imare, the chorionic viii are distributed quite uniformly on the outer
found in surface of the chorion. With discoid placentaesuch as man and rodents, the placenta
is disc shaped.The disc is either double e.g. rhesusmonkey or single as in man. Cotyledonous
31 the placentae found in are the goat, sheep and cow. In thes llous area is restricted to a number of circular forming areas cotyledons. In the zonary placentae,the aggregatedchorionic villi be band can seenas a which encirclesthe chorionic sac e.g. dog and cat placentae.
Structural is based classification on the number of layers of tissue which separate maternal and fetal blood circulation. This varies from only one layer where the trophoblast is bathed in
blood maternal to a maximum of six layers. According to the absenceor presenceof these layers four groups of placentae can be classified. These are syndesmochorial,haemochorial, epitheliochoria] and endotheliochorial (Steven 1975).
The placenta of the mare is classified as epitheliochorial, but is not a completely true representative of this group since a group of trophoblast cells, the girdle, invades into the maternal endometrium ( Allen et al 1973 ). The invasive trophoblast, however, has a short duration within the 11 months gestation period of equids. 'The remaining majority of the allantochorion is non-invasive and it forms a conventional epithelial interdigitation into the endometrium (Samuel et al 1975).
Syndesmochorialplacentation is found in ruminants. Binucleate trophoblast cells migrate from the fetal trophectodermal epithelium and fuse with endometrial cells to form trinucleate cells.
The fusion is formation of large of with result of this migration and . areas syncytium numerous nuclei (Wooding 1982).
invasive The endotheliochorial placentation of carnivores is the result of even more trophoblast. The chorionic epithelium comes in contact with endothelium of maternal capillaries.
32 In the haemochorial placenta all three layers of maternal tissues are absent so that the
trophoblast cell are bathed in maternal blood. This is in the human and other primates.
This variation in placental structure and degree of invasivenessof trophoblast causesthe
differences found between species and will influence the exposure of the trophoblast to the
maternal immune system.
1.3.2 Placentation in man.
Implantation and development of the placenta occur in a stepwise manner. After fertilisation
the zygote divides and forms a mass of 8-16 blastomerscalled. the morula. In most mammals
after the morula enters the uterus, fluid from the uterine cavity enters into the intercellular
space between outer and inner cells of the morula. As a result a single cavity on one side of
the central cell will be made. The increase in the amount of fluid will create the cavity and causethe to become flattened. At this stage of development,the conceptus is known as a -Aputer cells blastocyst and separatedinto two cell types (Johnson& Everitt 1988):
a. Trophoblast cells as the outer layer which actually makes the wall of the blastocyst and
gives rise to the placenta.
b. Embryoblast or inner cell masswhich give rise to the embryo.
In human pregnancy, 4-5 days after fertilisation, the zona pellucida disappears and on
This approximately day 5-6 the blastocyst attaches to the endometrial epithelium. attachment the embryo itself the is called implantation and it happens byybreaching the epithelium and embedding in invasion into the endometrial stroma. Proliferation of trophoblastic cells commences and
stroma begins (Moore 1988).
33 1.3.2.1 Cytotrophoblast and Syncytiotrophoblast.
The invading trophoblasts which initially surround the entire conceptus, differentiate to give rise to a number of different trophoblast cell populations. Theseform two layers: a) An inner layer, the cytotrophoblast which consists of mononucleatedcells that either fuse to form the syncytia or invade the uterus as extravillous (intermediate) cytotrophoblast (Yeh and Kurman 1989).
B) An outer layer, syncytiotrophoblast which is formed from the underlying cytotrophoblast.
A syncytium is a collection of cells having no boundaries between the individual cells.
1.3.2.2 Chorion and Villi.
Formation of the chorion occurs after proliferation of the trophoblastic layer and formation of
an additional layer. Locally proliferating cytotrophoblast cells differentiate into the
syncytiotrophoblast. A separate population differentiates to form the extravillous
cytotrophoblast column (Fisher et al 1989 ). These cellular viii surrounded by syncytium are
called primary chorionic villi. The secondaryvilli are formed from a mesenchymalcore which
is surrounded by an inner layer of cytotrophoblast and outer layer of syncytiotrophoblast. they become (Ross Finally when fetal blood vessels appear in the cores tertiary villi et al
1989).
leads Cytotrophoblast cells in the villi continue to divide and this to outgrowth of the
to causes them to grow laterally syncytiotrophoblast. Attachment of these cells endometrium the stem. Thus a thin layer forms around and meet with similar extensionhof neighbouring villous the This is interrupted at the the trophoblast and is called outer cytotrophoblast shell. shell
intervillous Willi that branch from the sides of maternal vessels which connect with spaces. ( Sadler 1990). stem villi are called terminal villi
34 1.3.3 Placentation in horses.
The placenta of the Equidae is a distinctly diffuse and of the non-deciduate type. The outer layer of the chorion (trophoblast) is in direct contact with uterine epithelium, thus, its category
is epitheliochorial (Amoroso 1952). Implantation does not occur until a comparatively late
stage of development (end of seven weeks). The equine conceptus at day 21 is covered
externally by a single layer of trophoblast cells which constitutes the chorion. During the next
two weeks vascularisedmesoderm develops between endodermand the chorion. The allantois
appears at day 21, enlarges rapidly and fuses with the chorion. This fusion gives rise to
allantochorion which has established a stable microvillous attachment with the endometrial
epithelium at day 42-45. Primary and secondaryfolding of trophoblast and adjacent uterine
epithelial layers leads to formation of the microcotyledons (Samuel et al 1974,1975,1976).
On the outer surface of allantochorion, thousands of microcotyledons exist. When they
mature the diameter of each is 1 to 2 mm, so they are visible and actually they make the red
velvety appearanceof the outer surface of the dischargedplacenta (Ginther 1979). It should is be pointed out that the epithelium of each macrovillus or branch of cotyledon coated with
is in later The microvilli. The nonvillous area at the cervix quite prominent pregnancy. two horn is larger horns of the placental sac differ in size and the pregnant always than the non-
invariably pregnant and the tip of the horn is oedematous.
forms Ad isected horse conceptus at day 35 shows two of trophoblast: the non-invasive discrete, invasive allantochorion which forms the definitive placenta and the annulate, girdle.
is similar to that found on human The microvesicular border of non-invasive trophoblast cells
syncitiotrophoblast plasma membrane(Samuel et al 1974). The chorionic girdle cells attach
35 to the endometrium and girdle cells invade and phagocytose the epithelium of the endometrium. The girdle cells then migrate into the endometrial stroma and form endometrial (Allen cups et al 1973). These represent a unique form of trophoblast development in
Equidae. The invasive trophoblast, unlike the non invasive forms of equine trophoblast expresses conventional major histocompatibility compex (MHC) Class I antigens (Donaldson et al 1990). Pregnant mares develop circulating cytotoxic antibodies to these fetal MHC antigens (Allen 1979, Antzack et al 1984).
1.4 Expression of antigens by trophoblast.
Investigation of the microvillous part of the human full term placenta revealedmany proteins with various biological functions. The following section briefly explains some of these components.
1.4.1 Presence of an 80kDa antigen on microvillous plasma membrane.
Jalali et al (1989,1995a) showed that there is an 80kDa trophoblast antigen on the maternal surface of the microvillous border. They designatedthis protein R80K. The antigen is highly polymorphic and in all term placentae studied is saturated with maternal antibody. The antibody can be eluted by acid conditions. The eluted antibody binds back to isologous acid
from treated vesicles and 16.7% acid treated preparations other placentae. Trypsin treatment
fragment of acidified microvesicles released a soluble 5OkDa which reacted with the eluted be antibody. It has been speculatedthat immunity to this antigen of paternal origin may associatedwith successfulpregnancy.
36 1.4.2 MHC Class I and ILL
Although HLA proteins have been extensively investigated, and Class I genes are present on all other cells, the trophoblast cells are unique exceptions, and suppressionof expressionto varying degreeshave been found in different kinds of trophoblast cells.
The human major histocompatibility complex WC) genes are located on the short arm of
chromosome 6 (Ploegh et al 1981). MHC Class I antigens are encoded by HLA-A, B and C
gene loci. They are non-covalently associatedwith 02 microglobulin which is encodedby a
gene on chromosome 15. These are the classicalMHC ClassI genes. In addition to the HLA-
A, B and C genes loci on chromosome 6, cloning and characterisation of human Class I
sequenceshave revealed other genesand pseudogenes(Shulda et al 1991). Thesegenes have
been termed non-classical Class I genes and include HLA-E, F,G, H, I and others (Koller et al
1989, Chimini et al 1990 and Schmidt & Orr 1991). HLA-G, previously designated HLA 6.0
be (Geraghty et al 1987) shows some differential characteristics, e.g. it appears to
monomorphic.
The MHC Class II antigens are encodedby HLA-DR, DP and DQ genes. Expressionvaries
be detectable the is induced considerably on other cells, and may only readily when expression have been found to Class by cytokines. Cytotrophoblast and syncytiotrophoblast not express
II antigens even at low levels.
(MAb's) have that there Immunohistochemical studies using monoclonal antibodies shown are
Class I Class II on normal villous syncytiotrophoblast(Faulk & no classical HLA or antigens
37 Temple 1976, Goodfellow et al 1976, Bulmer & Johnson 1985). This data now have been confirmed by in situ hybridisation using specific molecular probes (Hunt et al 1988).
Sunderland et al (1981) showed that extravillous cytotrophoblast cells in first trimester placentae were HLA Class I positive. Subsequently Hsi et al (1984) showed that
cytotrophoblast cells in the chorion membrane were also HLA Class I positive. In these
experiments, however, the expressed antigen showed unusual characteristics, e.g. bound only
to W6/32 MAb (a mouse MAb to the shared determinant of HLA-A, B and C) (Hsi et al
1984) and its molecular weight of about 40,000, is lower than that of the classicalpolymorphic
Class I molecules (Ellis et al 1986).
Kawata et al (1984), isolated placental cell suspensionsby trypsin digestion and analysedthem
fluorescence with MAbs against HLA-A, B and C and human trophoblast (anti-Trop-2) on a
B C activated cell sorter. They investigated the transcriptional control of HLA-A, and
hybridisation found antigens. They used cDNA probes and Northern blot and that normal
B C The level human cytotrophoblast have extremely small amounts of HLA-A, and mRNA.
found in lymphoid that of 02 microglobulin mRNA was 10% of that cells, showing regulation
for lack HLA Class I the at the level of transcription is also responsible the of antigens on
villous trophoblast.
lacked Class I HLA mRNA, but chorionic Hunt et al (1988) showed that syncytiotrophoblast
of Class I HLA antigens or a non- cytotrophoblast cells may express truncated versions
C like Using different MAbs against HLA-A and B, they classical HLA-A, B and antigens.
trophoblast, chorionic cytotrophoblast cells were considered that unlike placental villous
NIAb W6/32 but failed to bind MAb 61D2 (anti-Class I HLA). They speculated positive with
38 that this differential expression may be necessaryfor the development of an immunological relationship between the mother and the fetus. Placental syncytiotrophoblast forms an uninterrupted single cell layer, is continuously washed in maternal blood and lacks HLA Class
I II Soluble and antigens. HLA-G might directly suppress the maternal cytotoxic responseby binding to the receptors of cytotoxic cells and blocking the recognition of non MHC targets on
trophoblast.
Two choriocarcinoma cell lines derived from trophoblast, Jar and BeWo, have been widely
studied to evaluate the expression of MHC Class I on trophoblast cells. It has been shown
that BeWo cell lines have non-classicalHLA Class I antigen expressionsimilar to that on the
chorionic cytotrophoblast cell membrane(Trowsdale et al 1980, Ellis et at 1986). Ellis et at
(1990) isolated and sequencedan HLA Class I cDNA clone derived from the BeWo cell line
and cDNA from normal extravillous trophoblast. They showed evidence that nucleotide
sequences from the cDNA library from BeWo cells had a high degree of homology with the
published sequence of HLA-G. Using PCR the same sequenceswere demonstrated in isolated
cells from term chorionic membrane.
Kovats et al (1990) looked for BLAG a chain in HLA-A, B and C null transferent cells
(BLAG +, LcL. 221), in first and third trimester cytotrophoblast cells as well as
JEG. Their indicated that the choriocarcinoma cell lines BeWo, Jar and results amount of isolated from highly HLA G a-chain in third trimester trophoblast term placenta was
This implies is decreasedin comparison to first trimester cytotrophoblast. that there a possible
linkage between developmentalstage of the placentaand expressionof BLAG.
39 1.4.3 Trophoblast Lymphocyte Cross Reactive Antigens (TLX).
Allotypic trophoblast cross reactive (TLX) antigens are membraneantigens expressedon trophoblast lymphocyte and surface membranes,described first by Faulk et al in 1978. They
human used rabbit antisera against term syncytiotrophoblastand detectedtwo major groups of
antigens termed TAI and TA2. TA2 was found to be present on trophoblast, peripheralblood lymphocytes (PBL) and platelets. In spite of sporadic claims about the identification of these
molecules, the nature of the antigensand their exact characterisationis not clear.
Present data have revealed a heterogeneousgroup of antigens with a variety of molecular
weights. Kim (1989), isolated TLX proteins from solubilised lymphocytes using an
antisyncytiotrophoblast membraneSepharose immunoaffinity column. He applied the isolated
lymphocyte membrane proteins to 17.5% SDS-PAGE under reducing conditions. Western
blot studies using anti trophoblast membrane revealed a major membrane protein band at
35kDa. Stern et al (1986) using mouse monoclonal antibody H316, which was raised against
purified syncytiotrophoblast membrane,identified two proteins from lymphocyte membranes
55kDa 65kDa. Purcell (1990) that reactedwith the antibody with molecular weights of and et al
investigated whether the TLX antigens were the same as HULy-m5 (CD46), a 56-66kDa
protein composed of two non-disulphidebonded chains, expressedat the surface of almost all
human cells, or the membraneco-factor protein (MCP), a molecule of the complementsystem
CO. They which functions as a cleavagecofactor of C3b and used the monoclonal antibodies MCP), RA-m5 (rabbit H316 (a mouse MAb against TLX), RA-MCP (rabbit antiserum to
(a MAb detected HULy-m5) for blocking antiserum to HULy-m5) and E4.3 mouse which
MAb H316 RA-MCP blocked the binding FITC studies. Their results indicated that and of
labelled E4.3 to the surface of PBL. In their sequential immunoprecipitation studies they
40 showed that MAb E4.3 removed all molecules detected by MCP and TLX specific antibodies, demonstrating that epitopes detected by these antisera resided on the same molecule. They concluded that the CD46 glycoproteins are the same as MCP and TLX glycoproteins. MCP decay and accelerating factor (DAF) modulate C3 activation by the complement pathway (Holers et al 1985). It has been shown that DAF is expressedon trophoblast (Holmes et al
1990). Hsi et al (1991) postulated that MCP and DAF provide protection of the trophoblast
from complement attack when antibodiesare bound to the cell surface.
O'Sullivan et al (1982) claimed that the TAI group antigens are present in the blood of
pregnant women using an immunoradiometric assay with radiolabelled antibodies to
trophoblast membraneantigens. The presenceof this group of antigensincreases as gestation
continues, peaks at the time of delivery and disappearsafter the first postpartum week.
1.4.4 Placental Alkaline Phosphatase (FLAP).
Placental alkaline phosphatase is a tissue specific enzyme. A gene cluster located at
chromosome 2q 34-37 encodes for PLAP and other tissue specific alkaline phosphatases
(ALPs) (Griffin et al 1987, Milan and Manes 1988, Martin et al 1989). This isoenzymeshows
far have been one of the highest degrees of polymorphism among human enzymeswhich so
PLAP is heat studied (Slaughter et al 1981, Millan et al 1982). a 70kDa protein resistant to is denaturation and is usually present as a homodimer (Costa et al 1993). PLAP present on
from (Fishman the microvilli of the syncytiotrophoblast the 12 weeks of pregnancyuntil term
is into during et al 1976). SynthesisedPLAP released the maternal circulation pregnancy.
Brook & Barron (1988) showed an increase of maternal plasma PLAP in women who
delivered an infant of low birthweight (<2.5kg). Ind et al (1994) showed subsequently - in Down's Makiya & that PLAP levels were significantly reduced cases of syndrome.
41 Stigbrand (1992) suggested PLAP as the Fc-receptor of trophoblast responsible for the transport human of IgG from mother to the fetus. Below it will be seenthat there are other Fc human receptors on trophoblast which would also be contendersfor the transport of IgG.
1.4.5 Fc Receptor, ligands for IgG in the Human Placenta.
Three different classes of human leukocyte Fc y-receptors (hFcyR) have been identified on cells of all the major hematopoietic lineage with the exception of erythroid lineage cells
(Anderson & Looney 1986, Simmons & Seed 1988, Allen & Seed 1989, Micklem et at 1990
and Ravetch & Kinet 1991). These are designated hFc'RI (CD64), Fc'yRII (CD32) and
hFcyIII (CD16). Interaction of these hFc'Rs with immunoglobulin causes some biological
responsesto be triggered. These are phagocytosis, antibody dependentcellular cytotoxicity
(ADCC), endocytosis and the releaseof inflammatory mediators.
FcyRI is expressed on monocytes and macrophagesand can be induced on basophils and
neutrophils (Ravetch & Kinet 1991, Hartnel et al 1992). This receptor binds monomeric IgG
with high affinity and its molecular weight is 72kDa. Three highly homologous genes,
hFcyRIA, IB and IC have been recognised(Ernest et al 1992 ).
FcyRII (CD32) is expressed on B-cells, monocytes, macrophages, basophils, eosinophils,
hFc'yR. Three hFcyRIIA, IIB endothelial cells, platelet and it is the most abundant genes, and
hFc'yRII (Qiu 1990). IIC which are located on 1q 23-24 encode et al
large Fc'yRIII is expressed on some T-cells, neutrophils, monocytes, macrophages and granular
hFc-yRIIIA hFcyRIIIB Iq lymphocytes/NK cells (Braakman et al 1992). Two genes, and on as (Qiu 1990). 23-24 have been identified ncodingthis receptor et al
42 The fetus is protected against prevalent pathogens by maternally derived passive immunity. IgG antibodies are the only class of immunoglobulin which cross the placenta. McNabb et al (1976) IgG showed that 1 and IgG3 bound to receptors on placental membranes,IgG4 bound to a lesser degree IgG2 bound (1987) and the least of all. Einhorn et al by analysisof fetal blood demonstrated that IgG, especiallysubclasses 1 and 3 are transferredintact acrossthe
placenta.
The Fc-receptor on human placentawas first demonstratedon Hofbauer cells by Moskalewski
et al in 1975. Matre & Johnson in 1977 reported the presenceof different FcR on human
placenta. In 1978 Matre and Haugen demonstrated Fc-receptors on trophoblast cells.
Recently the presence of all three subtypes of Fc'yR in human placenta has been studied.
Down et al (1989) isolated human placenta syncytiotrophoblast proteins by detergent. They
demonstrated three IgG binding proteins (M. W. 68000,52000-56000,40000) by their ability
to bind to human IgG in immune complexes with sheep or goat anti human IgG. They
designatedthem placental IgG binding proteins PIBP68, PIBP56 and PIBP40.
Stewart et al (1989) showed that hFcRII-C was expressedin placental syncytiotrophoblast.
Kristoffersen et al (1990) using different monoclonal antibodies against Fc yRI, FcyRII and
FcyRM studied the heterogeneity of FcyRs in human placenta. Their studies together with
in studies of Kameda et al (1991) and Sedmak et al (1991) revealed FcyRI Hofbauer cells,
FcyRII in Hofbauer and endothelial cells of term placenta and Fc'RIII in trophoblast cells. In
(FcyRM) the study of Kristoffersen et al (1990) monoclonal antibody 3G8 also stained
(1994) trophoblast and stromal cells but did not stain the endothelium. Bright et al also using
FcyRs that Fc'RI fibroblast monoclonal antibodies against three showed was expressedon
FcyRII found cells of first trimester and term chorionic villi. was on capillary endothelial cells
43 of first trimester and term placenta and FcyRIH was expressed on syncytiotrophoblast of immature and term villi, but no Fc'yRwas detected on cytotrophoblast cells.
In is doubt conclusion, there no that the human placenta possessesFc'y receptors, IgG
do antibodies of all subclasses passfrom the mother to fetus and FcyR ll (CD 16) is expressed on trophoblast cells. One group of investigators accumulated data that the placental Fcy- receptor and placental alkaline phosphataseappear to be the same molecule (Makiya &
Stigbrand 1992), but CD 16 is found in phagocytic cells while PLAP is restricted to trophoblast. As my work is primarily with the horse placentait is worth emphasisingthat IgG transport does not occur across the horse placenta and Fc y receptors are thus not required
(Jeffcot 1975).
1.5 Alloantibodies in the Sera of Pregnant Women.
The immunological relationship of pregnant mothers to their fetus has been studied by many investigators. The fetus has antigensinherited from both the father and the mother, thus it is a semiallograft to the mother. Despite this it is not rejected. The placenta constitutes an anatomical and functional barrier between the maternal and fetal circulation. Maternal responsesto fetal and placental alloantigensare known to occur frequently during pregnancy.
MHC-antigens, some trophoblast cell surface componentsand some molecules present on B-
initiate immune in cells and activated T-cells have been shown to an response the mother
(Winchester et al 1975, Davies 1985a & b, Reed et al 1991, Arkwright & Redmann 1991 and
is defined. Jalali et al 1989 & 1995a). The nature of most of these antigens still not well
immunity (CMI) Mothers may produce both humoral and cell mediated to placental and
in first Their to erythrocyte paternally derived antigens or subsequentpregnancies. response
44 leukocyte and antigens are well known. CMI to placental antigens also has been studied by some investigators (Sargent et al 1982).
Presence of plasma factors such as antibodies which suppressmaternal reactivity against the fetus could be one of the explanations for the survival of the allograft (Pence et al 1975,
Rocklin et al 1976). Many researchersover the last three decadeshave worked to find out the
nature and characteristics of such factors. They have used migration inhibitory factor assay,
microcytotoxicity, inhibition of mixed lymphocyte reaction (MLR), fluorescent antibody
analysisand enzymelinked immunosorbentassay (ELISA) to detectthese factors in the sera
of pregnant women. The following sectionsexplain some of theseinvestigations.
1.5.1 HLA Antibodies.
Van Rood et a] (1958) showed that some women produced leukocyte antibodies during
pregnancy. Payne (1962) and Doughty & Gelsthorpe (1971) demonstratedpresence of these
antipaternal lymphocytotoxic alloantibodies (APCA) in the sera of multiparous women who
had successful pregnancies. Reed et al (1991) investigated the time course of maternal
allosensitisation to fetal HLA antigens. They found that the mother produced antibodies
against some but not all of the HLA antigens as early as the eighth week of pregnancy. IgG (predominantly Gilman-Sachs et al (1989) showed that most of these antibodies are
T B IgGI), some are IgM and both of them are againstpaternal and cells.
in is The absentin sera The role of antipaternal antibodies pregnancy unclear. antibodies are
(Mowbray et al 1983, Johnson et al of most women with recurrent spontaneousabortion fetus. Successfulpregnancies 1984). These antibodies are not harmful to the trophoblast or
they be enhanced by maternal allogenic may require some of them and may markedly
45 (Coulam recognition et al 1991). Coulam (1992) showed that the presence of APCA is fertility associated with rather than infertility. The fertile women had a higher frequency of lymphocytotoxic antibodies to paternal cells than primary and secondary recurrent spontaneousaborters and infertile women.
The incidence of antipaternal antibodies varies in different studies from 6%-50% (Jensenet al
1964, Terasaki et al 1970, Tongio et al 1972 and Stastny 1972). Regan et al (1991) found
that the incidence of APCA in 256 normal pregnancieswas 32% compared with 10% for 50
pregnancies which aborted. It has been speculatedthat the detection of APCA occurs only
when the antibodies are not fixed and absorbed onto fetal tissues expressing the relative
antigens (Ahrons 1971) or by placenta (Wegman et al 1979). Gilman-Sachs et al (1989)
investigated the presence of antipaternal antibodies in women with recurrent spontaneous
abortion who had been immunised. They detected the antibodies by two colour flow
cytometric analysis and standard microcytotoxicity. Two thirds of couples assayedby flow
cytometry had non-cytotoxic antibodies, whereas only one third of couples were positive by
classical microcytotoxicity. Other investigators reported that HLA-antibodies (Ab1) induced
the development of anti-idiotypic antibodies (Ab2) followed by disappearanceof Ab I
(Agrawai et al 1994).
1.5.2 Mixed Lymphocyte Culture (MLC) Blocking Antibodies.
between The MLC assay has been used as an in vitro model for studying allogenic reactions
individuals. In reproductive immunology it examines the proliferative response of maternal factor(s) in lymphocytes to paternal lymphocytes. The presenceof blocking the sera of some
pregnant women could inhibit the reaction.
46 Bissenden et al (1980) using MLR between mother and father found that sera from pregnant women contained immunosuppressive factor(s) which could be detected by 29 weeks gestation and were more effective by 36 weeks of pregnancy. The presence of serum blocking factors that prevents recognition of the antigens may provide an
explanation for the apparent lack of maternal reactivity againsttrophoblast.
Coulam (1992) showed that the prevalence of MLC inhibitors to husband's lymphocytes in
sera of women also increasedwith the duration of pregnancy. In her studies 4% of infertile
20% 44% fertile had women , of women with recurrent spontaneousabortion and of women a
MLC inhibitor. It should be mentioned that there is some correlation between antipaternal
cytotoxic antibody production and MLR blocking activity (Mowbray et al 1983).
The activity of blocking factor is suppressedin sera of women with RSA. Unander & Olding
(1983) investigated the immunologic responsivenessof women with recurrent spontaneous
Cimino abortion (RSA). 75% of them displayed no blocking activity in one way MLC. et al
(1991) studied the activity of the blocking factor in women with at least two consecutive
45.5% lack abortions. They also used one way MLC and showed that of these women however, is detected in the adequate blocking activity. The blocking activity, not always sera (Rocklin 1982). Bjerk (1994) has that of women who have had normal pregnancies claimed do the local immune in MLR assaysof peripheral blood lymphocytes not seemto reflect state
These data that more the uterus, either in the pregnant or non pregnant state. suggest
the of blocking factor antibodies sensitive and specific assaysare needed to assess presence disorders. and to identify immunologiccauses of reproductive
47 1.5.3 Anti-Idiotypic Antibodies.
Antigenic epitopes that occur in the variable region of the Fab portion of an antibody molecule are called `idiotopes". The `idiotype" of a particular immunoglobulin is the sum of the individual epitopes (Janeway & Travers 1994). If an antibody is used as an immunogen, it is possible to induce the production of anti-idiotypic antibodies. These antibodies structurally resemble the original epitopes. In fact, individuals make immune responsesto their own idiotypes. It has been suggestedthat the resultant idiotypic network regulates the immune response(Jerne1974).
Suciu-Foca et al (1983a & b) showed that sera from parous women contained anti-idiotypic
antibodies. These antibodies reacted with maternal T lymphocytes that had been primed in
vitro with HLA-Class II antigens either from the partner or an unrelated donor. Agrawai et al
(1994) re-tested 7-9 months after delivery sera from women who were initially positive for
anti HLA antibodies in pregnancy. Ten out of thirty one women were found to have lost the
antibodies. They tested these sera for the presenceof anti-idiotypic antibodiesby inhibition of
complement dependentcytotoxicity (CDC) with the positive sera. All the sera contained CDC
blocking antibody by this inhibition technique. They assigned the anti-idiotypic antibody
CDC blockingfactor. which was directedagainst the anti HLA antibodyto the
idiotypic in habitual They Behar et al (1991) investigated the role of the network abortion.
found 64% of sera from pre-immunised patients did not contain anti-lymphocyte antibodies. HLA Class I These sera, however, inhibited the activity of antibodiesto the antigensexpressed for binding HLA by the spouse. This suggested that they are anti-idiotypes the site of
from by immunisation with paternal antibodies. Sera women who were treated successfully
48 cells contained both cytotoxic IgG anti-HLA antibodies and inhibitory IgM anti-idiotypic antibodies.
Torry (1991) et al proposed that anti-idiotypes might regulate( the production of antipaternal in antibodies and normal pregnancy immunoregulatory effects of both antibodies may be
necessary. Sugi et al (1991) suggestedthat treatment of women with recurrent spontaneous
abortion by immunisation using paternal lymphocytesis actually due to the production of anti-
idiotypic antibodies.
1.5.4 Anti-trophoblast Antibodies.
Different subsets of trophoblast epithelium are exposed to the maternal immune system and
constitute major elements of allogenic recognition in pregnant women. The
syncytiotrophoblast of the chorionic villi which forms the major fetal interface in the placenta
is bathed in maternal blood. This layer does not expressMHC-molecules. Some investigators
have assessedthe presenceof antigens other than HLA-antigens which may elicit an immune
response. Davies (1985a) developed an enzyme linked immunosorbent assay (ELISA) for
detection of antibodies to syncytiotrophoblast plasma membrane. He used sheepanti human
bacterial that IgG or IgM antiserum -conjugatedto a enzyme, urease, and showed antibodies
were present in sera of some pregnant women.
Davies (1985b), Davies & Brown (1985a & b) and Billington & Davies (1987) attempted to
identify the characteristicsof these antibodiesand the epitopesto which the antibodiesare IgG4 less directed. The antibodies were predominantly IgG (IgGI, IgG2 and subclasses)and
IgG in 77% IgM in 48% pýparous sera. were IgM. They detected the and of pregnancy but, IgM had declined The IgG response could be detected throughout the pregnancy to
49 background levels by the tenth week of the pregnancy. Formation of antibodieswere in predominantly a first pregnancy, and were found to a lesser degree early in a second Women in pregnancy. their third and subsequentpregnancies had only weakly detectable The antibodies. reasonfor this gradual declineremained unclear. They speculatedthat the
allogen eic recognition which occurs in a first pregnancy generates some form of immunosupression. Exposure to a pregnancy involves the activation of suppressormemory
cells. The epitope(s)and their characteristicsare still unclear.
MacLeod et al (1989) prepared TLX and TBX (trophoblast B-cell cross reactive) antisera.
They used the standard long lymphocytotoxicity assay and showed that antibodies to TBX
antigens were cytotoxic to B but not to T lymphocytes and they were not directed against
HLA-DR. Blocking studies showed that activities of TLX antisera against peripheral blood
lymphocytes (PBL) and TBX antisera againstB-cells were not decreasedwhen Class I and II
antigens were blocked by non-cytotoxic MAbs to human Class I and II antigens. This
suggeststhat antibody content in TLX and TBX antiserais not directed towards the classical
HLA Class I or Class II antigens.
50 1.6 The Aim of My Study.
1. The major aim has been to investigate the inheritance of the R80K allotypes in sibships, to see if there were unequal transmission of the paternally derived antigen into the next generation.
There is claimed to be an immunological defect of maternal antibody to trophoblast in
aborting women, and the common pattern is to abort all the pregnancies. Since the
phenomenononly occurs in about 1 in 500 couples,it is likely that a woman would only
fail to make antibody to one of the two paternal allotypes. As she aborts all pregnancies
this implies that only one antigen is passedto all the offspring. The aim was then to
test this hypothesis in a study of the inheritance of the paternal allotypes of R80K, a
highly polymorphic trophoblast antigen, which in all normal pregnancies is completely
covered by maternal antibody.
In the time available,it was not possibleto get sufficient samplesof sibling placentaein
humans. I was lucky enough to be given the opportunity to work, in collaboration, on
thoroughbred horses. It was possible in two breeding seasonsto collect half sibling
placentae from the offspring of three stallions, each descended from the famous
stallion, Nearco.
it If there were unequal transmission of the two paternal allotypes, would:
a) Support the hypothesis above. b) Suggest that R80K is indeed the important trophoblast protein to which a maternal immune response must be made to protect normal pregnancies.
the R80K to 2. The study of the reaction of human maternal antibody and antigen, see in the correlation between presence of the antibody normal pregnancies, and protection against abortion. in 3. To study whether the development of the antibody women undergoing immunotherapy correlated with success of subsequent pregnancy.
51 2. MATERIALS AND METHODS
2.1 Preparation of microvesicles.
Microvesicles were prepared by a modification of the method of Smith, Brush and Luckett
(1974).
2.1.1 Equine placentae.
Forty placentae were collected between 1 and 12 hours after delivery and the placentae were collected by Professor W. R. Allen or Miss Susanna Mathias of the Thorouhbred Breeders'
Association Equine Fertility Unit as follows:
10 placentae from foals sired by Never So Bold
10 placentaefrom foals sired by Soviet Star
4 placentaefrom foals sired by Primo Domini
5 placentaefrom foals sired by Nomination
Also 11 placentaefrom random horses,each fathered by a different stallion.
free to free large blood The placentae were washed in calcium and magnesium salt solution
25 25cm to clots. To crack off the microvesicles, a x square piece of placenta was stapled a
board. This times similar sized 2mm thick polyvinyl was rinsed several with complement UK) fixation test (CFT) solution (Oxoid, Unipath LTD, Basingstoke, and then placed on a
buffer (PBS)(Oxoid, Unipath LTD, rocking chamber containing 800m1of cold phosphate saline for hour 4°C. Basingstoke, UK). This was shaken at about 90 reciprocations per min one at
by Susanna Mathias in Newmarket. To remove the large cellular The above steps were done
52 debris from fluid the the supernatant was centrifuged at 800g, at 4°C for 20 min in an MSE 4L
(MSE, London, UK) centrifuge. The microvesicle containing supernatant was carefully removed for and centrifuged 60 min at 4°C and 18000g using a refrigerated ultracentrifuge (ICE International centrifuge model B20). The supernatantwas discarded and the pelleted microvesicles in were resuspended lOml of chilled PBS and centrifuged at 500g for 20 min in a bench (MSE, London, UK) centrifuge to remove aggregated material. The supernatant was
into 2m1 made aliquots and stored at -70°C (Cliffco deep freeze) until required. All preparations were labelled.
2.1.2 Human placentae.
Full term human placentae were collected within 30 min after delivery from normal
uncomplicated pregnanciesin the labour ward at St. Mary's Hospital, London, UK.
The placentae were rinsed 3-4 times with calcium free cold CFT solution to completely
remove any adhering clots. The membranes were removed and the placenta was transferred to
a beaker containing 400 - 500m1 of cold PBS. The solution was agitated using a mechanical
agitator (Vibromixer, Chemap AG, Bonn, Germany) for 30 min at 4°C. Agitation in a
magnesiumand calcium free solution causedthe microvesiclesof the brush border to crack off.
Any large particles of tissue were removed by a household sieve. To remove red blood cells
(RBC) and cellular debris the fluid was centrifuged at 800g for 15 min at 4°C. The pellet was
discarded and the supernatantcentrifuged at 18000g for 60 min using an ultracentrifuge. The
(MV) from in lOmis pellet containing the microvesicles the plasma membranewas suspended bench 500g. 2ml cold PBS. Aggregated material was removed using a centrifuge at aliquots of
the supernatantsolution were labelled and stored at -70°C until required.
53 2.2 Elution of IgG antibody bound to microvesicles.
The IgG antibody bound to human and horses vesicles was prepared by acid elution at pH 3. Samples were centrifuged (ICE, International Refrigerated Centrifuge Model B20) at 18 000g
4°C for 1 hour. at The pellets from each human placenta. or those from the horses placentae were weighed exactly and resuspendedin 4m1of 0.1M citrate buffer pH 3 (Appendix A). The incubated mixture was at room temperature (RT) for 60 min. The acid treated vesicles (Va) were removed by centrifugation, 18000g, 20 min and at 4°C, washed three times in 5m1 of PBS and resuspendedin 4m1of PBS.
The supernatant containing acid eluted antibody (Ae) was dialysed (Tube size 1-8/ 32",
Medicell International LTD, London, UK) againsttwo litres (1) of PBS at 4°C overnight. Acid eluted antibody was separated into aliquots and stored at -70°C until used. The concentration of Ae antibody from human placentaewas detectedby radial immunodiffusion (Section 2.4) and that from horse placentaewas detected using solid phase radioimmunoassay(Section 2.5) and adjusted to contain the same concentration of IgG (74 gg/ml) in each case.
54 2.3 Isolation of IgG from Human Sera.
Sera dialysed were overnight at 4°C against 0.02M phosphate buffer pH 6.2 (Appendix A). Samples were centrifuged at 800g for 20 min in an Eppendorf microcentrifuge 5415 (Eppendorf
AG, Hamburg, Germany). Diethylaminoethyl (DEAE-Whatman 52) (Pharmacia Fine
Chemicals, Uppsala, Sweden) anion exchangerwas preparedin 0.02M phosphatebuffer, pH 6.2 in beaker a and allowed to settle. Excess buffer was discardedand 5ml aliquots were put into bottom round tubes (Sherwood, Medical, Ballymoney, Northern Ireland). I ml of dialysed serum was added to each tube. This was incubated at RT for 1 hour with stirring. Tubes were centrifuged at 800g (MSE, London, UK) for 20 min at 4°C. The supernatant taken off contained the IgG fraction. The other proteins stuck to the DEAE. The concentration of IgG was detected by radial immunoassay. SDS-PAGE (Sodium dodecyl sulphate polyacryl amide gel Electrophoresis) and FPLC (Fast protein liquid chromatography)were used to monitor the
degree of purification.
2.4 Single radial immunodiffusion (SRID).
Single Radial Immunodiffusion was done according to the method of Mancini et al (1965). 1.5g
of agarose (Sigma, Poole, Dorset, UK) was dissolved in 150m1of gel buffer (Appendix A) to
make a 1% agarose. The suspensionwas placed in a boiling water bath and stirred occasionally until the agarosewas completely dissolved. 30ml of 1% agarosewas cooled down to 60°C and
0.6m1 of rabbit anti-human IgG (Immunopathology Department, St. Mary's Hospital Medical
School) was added to make a 2% preparation of agarose anti IgG mixture. The mixture was
bubbles. mixed as thoroughly as possible and attention was taken to avoid It was poured onto a 100 100mm 2mm This previously prepared double plate sandwich measuring x to give a gel. frame was allowed to solidify (20 min) and then the top plate and the rubber were removed. 2mm bore The Circular wells were punched out in the gel 1cm apart using a pipette. small
by by Each filled 5 cylinders of gel cut out the pipette were removed suction. well was with µ1
55 of sample. Three wells filed known were with a amount of human IgG. The plates were kept in a horizontal strictly position in a moist box incubated and at 37°C for 24 hours. The size of the precipitated ring shape was measured carefully and the concentration of IgG was calculated by the following formula:
D2-d2
XS
s2-d2
in which S is concentration IgG, d is diameter of standard of wells (2mm), D= diameter of
precipitated ring sampleand s is diameter of standard.
2.5 Measurement of equine IgG Concentration.
The IgG concentration of equine acid eluted antibody was detected by competition solid phase
radioimmunoassay in 2 steps, as follows:
2.5.1 Standard Curve.
25µg of equine IgG (Sigma, Poole, Dorset, UK) was coated onto LP3 PVC tubes (Luckham,
Sussex, UK) in 2m1 of sodium carbonate-bicarbonatebuffer 0.05M pH 9.3 (Appendix A) overnight at 4°C. Tubes were rinsed once with 2ml of PBS. Subsequentlytubes were blocked in 2% (w/v) Marvel (Cadbury Ltd, Stafford, UK) in 0.05% (w/v) Tween 20 (BDI, Poole,
Dorset, UK) in PBS (TPB S) for 1 hour and washedthree times using TPB S. Various amounts of radiolabelled protein G were addedto tubes in duplicate and in 2ml of 0.1% Marvel in 0.05%
TPBS. Tubes were incubated for 45 min at RT and then washed 5 times with 0.05% TPBS.
The radiolabelled protein G bound to equine IgG was counted in a gamma counter (Prias 2,
United Technologies Packard, Panghourne,UK) and this was plotted against original counts per min (cpm) of labelled protein G which was added to tubes. In order to establisha standard
(O-10µg) in curve, dilutions of a known concentrationof equineIgG made 2m1volume with the
56 same Marvel in PBS/Tween buffer were used. 15000 cpm of radiolabelled protein G was added to each tube. Tubes were incubated for 45 minutes at RT and then washed 5 times with 0.05%
TPBS. The radioactivity was counted and cpm of bound labelled protein G was plotted against known concentrations of equine immunoglobulin.
2.5.2 Concentration of Samples.
25 gg of equine IgG was used to precoat tubes and the tubes were blocked as in section 2.5.
22µl of samples and 15000 cpm of radiolabelled protein G was added simultaneouslyin 0.1%
marvel in 0.05% TPBS. Tubes were incubated for 45 min at RT and then washed 5 times with
0.05% TPBS. The radioactivity (cpm) was counted in the gammacounter and then by referring
to the standard curve the concentration of equine IgG antibodies in the acid elution was
calculated.
2.6 Lymphocyte preparation.
(1982). Human lymphocytes were separatedfrom peripheral blood by the method of Lamm
(Analar, BDI-, 7.3, 10ml of blood was taken by venepuncture into 1ml 3.8% sodium citrate pH
diluted in in distilled water and autoclavedbefore use). The blood was then an equal volume of 1.077 PBS. This diluted blood was layered over an equal volume of Ficol-Hypaque solution
in bottom (Sherwood Medical, (Lymphoprep special, Nycomed, Birmingham, UK) round tubes 700g for 15 Ballymoney, Northern Ireland) and centrifuged (MSE bench centrifuge) at min.
the lymphocytes, was The thin layer of cells, a band above the Lymphoprep, containing
in Hank's balancedsalt solution (BSS) (Flow Labs, removed into a clean tube and washedtwice pipetted off and the pellet was Ayr, Scotland). -After the final washing the supernatantwas
BSS give 1x 106cells/ml. resuspendedin Hanks to
57 2.7 Preparation of B-lymphocyte suspension.
B-lymphocyte suspension in was prepared two steps as follows: -
2.7.1 Preparation of 1% Sheep Red Blood Cells (SRBC). B-lymphocytes were prepared by using SRBC (Tissue Culture Services,Berkshire, UK) treated 2- with Aminoethylisothiouronum bromide (AET) as follows:
Ig of crystalline AET dissolved in 20m1 distilled was of water and the pH adjusted to 9.0 with IN NaOH. The volume was increasedto 25m1with distilled water. 10m] SRBC of was washed three times in 50m1 of PBS and 3m1 of packed SRBC was resuspendedin l0ml of AET solution and incubatedat 37°C for 15 min with stirring. The AET SRBC treated were washed four times in 50m1 of PBS. 20m1 of fetal calf serum (Flow
Laboratories, UK) and 30m1 of Hank's BSS was added to 0.5m1 of packed washed cells to make 1% SRBC.
2.7.2 Separation of T and B-lymphocyte.
Lymphocytes were isolated as describedin section 2.6.3ml of 1% AET treated SRBC (section
2.7.1) was mixed with 3ml of lymphocytes resuspendedin Hank's BSS and the mixture was centrifuged (MSE, Bench Centrifuge) at 200g for 5 min at RT and again gently nixed. The mixture was layered on top of the 5m1of Ficol-Hypaqueand centrifugedat 700g for 15 min.
The enrichedB-cells at the interphasewere harvestedand washedtwice in Hank's BSS. The final pellet was resuspendedin Hank's BSS to give a suspensioncontaining 5 or 1x 106cells/ml as required.
2.8 Preparation of cells by dextran sedimentation.
iml lOml of horse or human blood was added to 0.5m1of 0.2M EDTA (pH 7.6) and mixed with
dextran 400 000 500 000 (BDH Biochemicals LTD, Poole, Dorset, UK) in of 6% (w/v) - mw
58 0.9% (w/v) NaCl. When the erythrocytes settled (about 30 - 40 min, depending on individual blood), the cloudy leucocyterich supernatantwas removedand washedthree times in EDTA- saline (pH 7.6) (Appendix A). The final pellet was resuspendedin saline to give 5x 106 cells/ml.
2.9 Monocyte preparation.
Monocytes were separated by the method of Böyum (1983) with modifications by the manufacturer (Nycomed, Birmingham, UK). 5- 6ml leucocyte rich plasma, from dextran prepared cells (Section 2.8) was layered over 3m1 NycoprepTM 1.068 (Nycomed, Oslo,
Norway). This was centrifuged at 600g (MSE, Bench Centrifuge) for 15 min and then the clear plasma was removed down to about 4 mm above the interface. The remainder of the plasma was collected and all of the Nycoprepm 1.068 solution down to just above the erythrocyte pellet. The cell suspensionwas diluted with 0.9% NaCl containing 0.13% (W/V) EDTA and
1% fetal calf serum (FCS) to give 6- 7m1.This was centrifuged (MSE, Bench Centrifuge) at
600g for 7 min and repeated twice. The cells were resuspendedin 5 ml of the same solution and the dilution adjusted to give 5x 106cells/ml.
2.10 Staining of peripheral blood cells. into Cytospins were prepared by pipetting 100µl of 1x 106 cells/ml a cytofunnel which in holder Shandon sandwiched a filter guard between itself and a glass slide a metal slide of a The Cytospin 2 rotor. The cells were spun for 7 min at 22g. slides were carefully removed and
for 10 An Shandon the cells fixed in absolute methanol mies. automated staining machine, MAY- Varistain 12, with 8 pots and slide carriers was used to stain the slides using the 600 50% May-Grunwald GRUNWALD-GIEMSA (M. G.G. ) stain(Dacie &Lewis1995). mis of
Dorset, UK) 600 of 10% Giemsa stain (BDH) were freshly prepared stain (BDH, Poole, and ml LTD, Surrey, UK). Reagents in buffered water pH6.8 (Merica-Diagnostics were added to the
59 pots of the following staining machine the manufacturers instructions. Slides were loaded into a carrier the and carrier progressedthrough the automated staining cycle. After the cycle was completed the carriers were removed and tilted on the bench in front of a hot air dryer. The
slides were carefully removed and a cover slip mounted using Styrolite.
2.11 Collection of sera.
5ml blood by of was taken venepuncture into sterile tubes from normal pregnant women,
women with recurrent spontaneous abortion (RSA) or normal male individuals. Blood was
allowed to clot. The tubes were centrifuged (MSE, Bench Centrifuge) at 500g for 5 min. The
LP3 serum was pipetted off and transferred to two clean tubes. Tubes were stored at -70°C
until used.
2.12 Radioiodination of Proteins.
Proteins, staphylococcusAureus protein A and Streptococcusprotein G (Sigma, Poole, Dorset,
UK) were radiolabelled using the modified chloramineT method of Greenwoodet al (1963) and
the manufacturer's guide to radioiodination (Amersham 1993). 1 mCi of Nat I (Amersham,
Buckinghamshire, UK) was added to 5µg of the protein to be labelled. 5µg (l0gl) of 0.5mg/ml
of chloramine T (BDI4, Poole, Dorset, UK) in 0.5M phosphatebuffer pH 7.5 (appendix A) The solution was added and the mixture was incubated for 45 secondsat RT. reaction was (BDH, Poole, Dorset, UK) in stopped using 20µg (20µ1 of 1mg/ml) of sodium metabisulphate PBS to lmi. Separation free 0.5m1phosphate buffer pH 7.5. The volume was adjustedwith of by dialysing (dialysis tubes Size 1-8/32", iodine and the labelled fraction was carried out either 4°C by Medicell International LTD, UK) against 51 of PBS at with several changes or gel
Using (Prias 2, United filtration chromatography (Section 2.18.1). a gamma counter (usually 51l) the Technologies Packard, Pangbourne, UK) a small volume was counted and
Aliquots at until needed. total radioactivity was calculated. were stored -70°C
60 2.13 Reaction of acidified vesicles with acid eluted IgG antibody. 125I-labelled protein A G or protein was used to measure binding of the IgG to human and equine acidified vesiclesrespectively.
2.13.1 Equine vesicles.
50gl of acid eluted IgG (Ae) antibody was added to 40µl of acidified vesicle (Va) suspensionin the 50µ1 0.05% presence of of TPBS using 1.5ml microcentrifuge polypropylene tubes (Treff
Lab, Schweiz, Switzerland). These incubated were for 60 min at RT. The surface of vesicles blocked 10 was using tl of 0.1% solution of Marvel in PBS to cover Fc- receptors and other binding non-specific sites. In the next step the vesicles were washed twice in lml of 0.05%
TPBS then in 200µl Tween PBS (0.05%). 1251 and resuspended of 18000 cpm of - labelled
protein G was added in solution of 0.05% Tween, 0.15M NaCl and 0.02M phosphate buffer pH
8. Tubes were incubated at RT for 30 min. The vesicles were washed twice in 1.5ml of 0.05%
Tween, 0.15 M NaC1 and 0.02 M phosphatebuffer pH 8. The pellet was resuspendedin lml
PBS and transferred to LP3 tubes. Radioactivity (cpm) was counted by using a gamma counter.
2.13.2 Human placental microvesicles.
401Llof acidified vesicles (Va) were mixed with 50 µl of acid eluted antibody in the presenceof
2Oµ1of 0.05% Tween 20 in PBS in microcentrifuge tubes. The surface of the vesicles was blocked by adding 10 d of 0.1% of Marvel in PBS. The mixtures were incubatedfor 1 hour at
A RT and then washed twice using lml of 0.05% TPBS. 18000 cpm of radiolabelledprotein
incubated RT for 30 three times was added to each tube. Tubes were at min and washed using (cpm) 0.05% TPBS. The pellets were transferred to LP3 tubes and radioactivity was counted
in the binding to IgG bound to by using a gamma counter which cpm measures protein vesicles.
61 2.14 Methods for detection of the RSOK antigen on the surface of peripheral cells.
A variety of techniquesfor detection of surfaceantigens have been devisedincluding: a microdroplet lymphocyte cytotoxicity described by Terasaki et al (1964) and modified by
Mittal et al 1968), immunofluorescenceand radioimmunoassay(Zeltzer & Seeger 1977, Brown et al 1977). In order to detect antibodies to cell surface antigens, i have used a 12'I- labelled protein A or protein G.
Protein A, a component of the cell wall of StaphylococcusAureus cowan I binds with high affinity to the FC-portion of human IgG1, IgG2 and IgG4 but not IgG3 (KronvaU 1973 and
Akerström et al 1985). IgG3 is only 8% of total human IgG (Roitt 1994), so 125I-Labelled protein A assaywas used to detect' human IgG bound to cell surfaces,acidified vesiclesor in solid phaseradioimmunoassay.
Protein G, from Group G streptococcus, reacts with a larger number of IgG isotopes and has a Akerström higher affinity to immunoglobulins comparedto protein A (Björck & Kronvall 1984, Guss et al 1985). It binds strongly to equine IgG (Kronvall et al 1970, Sjöquist et al 1972). et A for al (1986) also showed that protein G has a much higher affinity than protein polyclonal
IgG from horses. Thus 1251labelled protein G was used to detect equine IgG bound to cells,
acidified vesicles (Va) and in the solid phaseradioimmunoassay.
blood 2.14.1 Binding of acid eluted IgG antibody to peripheral cells.
G bound to 1"I-labelled protein A or protein was used to measure antibodies cell surface '25I-labelled incubated firstly with antibody and then with antigen. In these assays,cells were bind IgG boundto cell surfaceantigens. proteins which to antibodies 0.1% TPBS for 1 hour at RT. Tubes were rinsed LP3 polystyrene tubes were preblocked using 2.8) 0.05% TPBS. Ix 106 horse or human dextran prepared cells (Section were once using
62 mixed 20 diluted with µl of 1/100 normal male equine or human serum respectively. Subsequently tubes were mixed with 50µl of the acid eluted IgG antibody in the preblocked tubes in the 50µl 0.05% Tween. presence of In the case of equine acid eluted IgG antibody of the (Ae), Ae from hree Never So each groups, Bold, Soviet Star and Primo Domini, were used 50µ1 ). The incubated of Ae mixture was at RT for 45 min and then washed once with 2m1of 0.05% TPBS. 18000 125I-labelled cpm of protein A or protein G was added to each tube for human or equine samples respectively. Tubes were incubated at RT for 30 min and then cells were washed three times using 0.05% TPBS. Radioactivity (cpm) was counted using a gamma counter.
2.14.2 Detection of antibody using FACS.
Cells were prepared using dextran as describedin section 2.8 and sera as in section 2.11.1 X
105 cells were mixed with 20 µl of 1/100 diluted normal male serum and subsequently with 10µl of serum or 50 µl of Ae in LP4 tubes (Becton Dickinson, Oxford, UK) and the tubes were incubated for 45 min at RT. Cells were washed once using 2ml of EDTA-saline (Appendix A).
The pellets were resuspended in 200gl of EDTA-saline and 2.5µl of fluorescein-labelled rabbit anti-human IgG (DAKO, Glostrup, Denmark) was added to the cells and incubated at 4°C in a dark place for 15 min. Cells were washed once in EDTA-saline and resuspendedin 2ml of
EDTA-Saline. If read later, 2ml of 1% paraformaldehydein 0.85% NaCl pH 7.6 was used and
FACSLITE laser (Becton Dickinson, the cell suspension kept at 4C. A FACS 4 analyser with a
Oxford, UK) was used to detect the binding of IgG to cells.
2.14.3 Complement dependent cytotoxic antibody test.
test the long NIH The complement dependentcytotoxic antibody was carried out using method
(1968). of Mittal et al
63 A Terasaki (Flow microtitre plate Labs, Ayr, Scotland) was oiled (liquid paraffin) and either 1µl a of serum 5µ1 or of acid eluted antibody was placed in each well beneath the oil using Hamilton a syringe with dispenser (Philip Harris, London, `repeating UK). Two wells of each plate contained normal male serum as a negative control and two other wells contained a positive control (kindly donated by Tissue Typing the Service, Transplant Services,Bristol, UK). 1µI of CCA, lymphocyte either a suspensionor 1µl of one of the homozygous cell line 017bßSTRV and LHM (UK Transplant Services, Bristol) or K562 human erythroleukemia cells (Gift from Ashley King, Cambridge, UK) was added to the antibody. The cell lines had been cultured in
RPMI 1640 medium supplemented with 0.2 m/1 L-glutamine, 0.002g/ml sodium hydrogen
carbonate and 10% FCS at 37°C, 5% CO2 and subculturedtwice weekly by my colleagueSue
Surridge and tested that during culture they did not change. The cells before use were
centrifuged at 300g for 10 min (MSE, Bench Centrifuge) and resuspendedin Hank's mediumat
1.5 x 106cells/ml.
Plates were incubated at RT for 30 min. Sµ1 of adult rabbit complement (Buxton Rabbit Co
LTD, East Grinstead, Surrey, UK) was then added to each well and plates were left one hour at
RT. To stain the cells 1µl of 5% eosin (BDH 'Limited, Poole, Dorset, UK) was added to each
well after incubation. The plateswere left for 5 min at RT to equilibrate,the cells were fixed UK) The using 1µl of 36% formaldehyde (BDH, Poole, Dorset, titrated to pH 7.4. plates were
(Wild, Switzerland). The dead dye read using an inverted microscope cellstook, up the eosin darker live because their membrane became, permeable and as a result became than cells.
the kill 20% background. The actual Positive samples were scored when was ' . above
for When or sera were tested percentage kill was recorded each sample. acid eluted antibody incubation times were increased to twice that described. Pure B- against B-lymphocytes all 2.7. lymphocytes were prepared as in section
64 2.15 Detection R80K of anti antibody in sera of pregnant women and women with recurrent spontaneous abortion.
Cells from 55 husbands were prepared using dextran as describedin section 2.8. and sera from
13 pregnant 42 had women and women who recurrent pregnancy loss were prepared as described in section 2.11.
An 125I-labelled A protein assay was used to measured antibodies bound to the cell surface LP3 antigens. polystyrene tubes were blocked in 2% Marvel in PBS for 1 hour at RT. Tubes
were rinsed once with PBS. 1X 106 of dextran prepared cells from the husbandwere mixed
with 4Oµ1of wife's serum. 40µl of normal male serum was used as a control. All tests were
done in duplicate. The samevolume of serum was added to preblocked tubes with no cells as
another control. Tubes were incubated 60 min at RT. The cells were washedonce with 2m1of
PBS. Cells were resuspendedin 20Oµ1of PBS. 18000 cpm of' I-labelled protein A was added
to each tube and the mixture was incubated at RT for 45 min. The cells were washed three
times with 0.05% Tween in PBS. Radioactivity (cpm) was counted using a gamma counter and
IgG bound to cells was calculated as follows:
cpm of (cells + partners serum) - cpm of (serum with no cells)
cpm of (cells + normal male serum) - cpm of (normal male serumwith no cells)
2.16 Trypsin digestion of microvesicles.
from digested Human or equine antigens were prepared microvesicles with trypsin after
Acidified mixed 4m1 lmg/ml antibody had been eluted with acid. microvesicles were with of
Dorset, UK) for 15 in 0.15M Tris HCl 8.1 (Appendix A) trypsin type IX (Sigma, Poole, min pH
20µl of 10'2M Phenylmethanesulphonyl fluoride at 37°C. The reaction was stopped using
65 (PMSF)(Sigma, Poole, Dorset,UK). Sampleswere centrifuged at 18 000g for 20 min and 4°C.
The 1 immediately supernatantwas taken off and stored as ml aliquots at -70°C. SDS-PAGE analysis (Section 2.17) and solid phase radioimmunoassaywere used to examinethe antigenic fragment.
2.17 SDS - PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). SDS-PAGE was done according to Laemmli's (1970) method. The plates were cleanedand the apparatus (Bio-Rad, Watford, Herts, UK) was assembled. A 10% separationgel (Appendix B) was made up and degassedin a vacuum flask for 20 min. 30 i1 of temed (Bio-Rad, Herts, UK),
0.5m1 of 10% SDS (Appendix B) and 0.25m1 of 10% ammonium persulphate (AMPS) (Bio-Rad
Laboratories, Herts, UK) was added. The mixture was gently shakenso that no bubbleswere produced and it was transferred into slabs and allowed to polymerise (45 min). This was
overlayeredwith 0.5m1(10mm) of distilled water.
The stacking gel (Appendix B) was prepared and degassedfor 20 min and 0. im! of 10% SDS, the liquid from 0.02ml of temed and 0.1 ml of AMPS was added. The the top off ,s`eparation gel was inserted. The gently removed and the stacking gel was poured and a comb was gel was again When inserting allowed to polymerise (20 - 30 min) and the comb was then removed. and bubbles the because removing the comb attention was made to not trap under the arms of comb,
Excess liquid out gently. oxygen causes the inhibition of polymerisation. was sucked
B) heated for 4 in boiling buffer. The Samples were placed in sample buffer (Appendix and min
Hamilton (Philip Harris Scientific, Park Royal, London). samples were loaded using a syringe (BDH, Poole, Dorset, UK) 200,97,77,55 Standards of the following molecular weights and
the molecular of 42 kDa were run in one well of each gel to enablethe measurementof weight
buffer (Appendix B) and bubbles at the bottom of the the samples. Running was overlayered The lid the tank it was run at 60mA using a Bio- tank were trapped gently. was placed on and
66 Rad power unit. When the dye front reached to the bottom of the gel, the powerpack was switched off and the apparatuswas disassembled. The gel plates were removed and the plates gently The from bottom parted. corner the of the gel that was closer to the number one well the was cut and thergels were stained.
2.17.1 Staining and Destaining of Gels.
The by Coomassie gel was stained blue. The gel was put into 0.1% w/v Coomassieblue in for methanol - acetic acid 30 min and then destainedin 40% methanol, 10% acetic acid and
40% H2O for at least 2 hours with severalchanges.
2.18 Purification of proteins.
Proteins were purified either by gel filtration chromatographyor affinity chromatography.
2.18.1 Gel Filtration Chromatography.
A gel filtration column of sepharose 6 prep grade connected to a fast purification liquid
chromatography (FPLC) system (Pharmacia, Uppsala, Sweden) was used. Samples were
concentrated (pressure dialysis system) to give 1ml fractions. These were centrifuged at an 10000g for 10 min, using Eppendorf Centrifuge 5415, to remove any particulate matter and then passedthrough a 0.2µm filter (Sartorius InstrumentsLtd, Epsom, Surrey,UK).
the 3 bed The column was equilibrated according tmanufacturers manual with volumes of in EDTA-saline at 30m /hour flow rate and RT. The sample was loaded a 500µl volume and
buffer flow The in i eluted with the same and same rate. eluted samples were collected nil fractions. Those which formed peaks were collected and subjected either to SDS-PAGE or
The re-equilibratedwith 2 solid phaseradioimmunoassay to check the purification. column was bed volumes of EDTA-saline.
67 2.18.2 Purification of acid eluted IgG antibody from human placentae by affinity
chromatography.
A 2m1 plastic disposable syringe barrel was filled with 2m1 of Aff"gel (Protein A) (Bio-Rad Laboratories, Watford, Herts, UK) and then equilibrated with PBS. -Acid eluted IgG prepared from human microvesicles was centrifuged at 10000g for 10 min (Eppendorf centrifuge 5415)
and then run through the column using a very fine tube. Contaminatingmaterials were removed
by washing the column with EDTA-saline (Appendix A) solution. Bound IgG was eluted from
the column using 0.1m citrate buffer pH 3. The optical density of the eluted IgG was estimated
by taking a1 ml fraction. This was read on a spectrophotometer(Carl-Zeiss, PMQ 11, Hamburg,
Germany) at 280 nm. The concentration of protein present in each samplewas plotted. The
samplesfrom the peak were pooled and dialysed against PBS at 4°C overnight to remove the
acid. Sampleswere given a number appropriate to their origin and stored at -70°C until used.
IgG affinity purified protein A sampleswere analysedby SDS-PAGE according to Laemmli
(1970).
2.19 Statistical methods.
Spreadsheet used investigate Standard computer programmes i. e. Epistat and programmes were to
the statistical differences in group means. Student's t- test was used to test the samplemean in PBC binding differences. Fisher's exact test was used to test for associations antibody and
to the of expressionof the outcome of pregnancy. A binominal test was used calculate probability
paternal antigen on all the half siblings of one stallion.
68 3.1 Results
3.1.1 Acid elution of antibody from human microvesicles.
IgG which is bound to an epitope on the trophoblast can be dissociatedin the presenceof acid.
Microvesicles from 56 human full term placentae were treated with citrate buffer pH3.0 to dissociate the IgG. The concentration of each samplewas measuredby radial immunodiffusion against anti human IgG. All microvesiclesstudied had IgG bound to trophoblast antigen.
The amount of IgG elutable from 56 human microvesiclespreparations was measured. The wet weight of the vesicles was also measured. The mean± SD detectableIgG from the vesicleswas
340 ± 124 .tg /g of wet vesicles.
by SDS-PAGE Acid eluted antibody was purified on a protein A affinity column and examined
light bands (50 25 and Western blotting with anti human IgG antibody. Heavy and chain and
kDa) of IgG can be seenin plate I.
69 Lane No 1234567 kDa zoo--t
97 ---ý 77-olo-
55'--f 42 -ý
Plate I. SDS - PAGE of acid eluted IgG antibody purified by a protein A affinity column.
Heavy and light chain bands (50 and 25 KDa) of IgG can be seenin track 3. Tracks 4 and 5 are two different preparations of acid eluatesand track 2 is a marker.
3.1.2 Acid elution of antibody from horse placental microvesicles.
IgG antibody was dissociatedwith citrate buffer pH3.0 from vesicles of 40 thoroughbred horse full term placentae. The amount of each eluted IgG antibody was measuredby a competitive
is radioimmunoassay(Section 2.5). A standardcurve of the assay shown in figure 3. The wet Using for horse weight of the vesicleswas also measured. this assay 40 thoroughbred placentae
IgG was found bound to all vesiclesstudied.
70 The amount of each antibody preparation was variable ranging from 200 to 515 µg/g of
The vesicles. mean elutable IgG from the equinevesicles was 346 ± 76 µg/gram wet weight of vesicles.
10,000
I' 1000
100
00 20 40 60 U 100
pgWAd
Figure 3: Standard curve of equine IgG over the range of 0- 10 pg IgG/ml. Results are
(pg/ml). plotted as cpm of radiolabelled protein G binding againstamount of IgG
3.1.3 Reaction of human eluted antibody with acid treated microvesicles.
bound IgG (V) Five vesicles preparations were studied. The was measuredon these or acidified Ae (Va + Ae). IgG vesicles (Va) and on acidified vesicles exposedto their own was presenton (Table III). It be that the the V before its removal by acidification, but not afterwards can seen
from its own placenta. acid eluted IgG binds back to the neutralisedvesicles
71 Table M. Elution and rebinding of IgG from microvesicles.
izsI -labelled protein A binding (cpm) V Va Va + isologous Ae
2907 601 3075
1874 595 2129
2395 627 2308
3149 685 3028
2537 611 2480
Five different microvesicle preparations were acidified, and after neutralisation the eluate(Ae) reapplied. The amount of IgG bound was measuredby the binding of radiolabelled Protein A.
Results shown are the means of duplicate binding to the vesicles (V), acidified vesiclesVa and test, acidified vesicles with antibody rebound (Va + isologous Ae). Using Student's tit = 9.3 p=7 Ae. The difference x 10 for V versus Va, t= 10.32 p=4x 10'4for Va versus Va +
between V and Va + Ae was not statistically significant (p = 0.7).
3.1.4 Determination of optimal conditions of the detection of binding of cells and eluted
antibody. bind father's To find how much cellular antigen is neededto the eluted antibody, one cells were
from his placenta. Different numbers of cells were used to bind the acid eluted antibody child's (Figure 4). Maximal binding of eluate occurred with 1x added to find the optimal conditions
106 cells.
72 1000
800
.0 600
116 400 ew 0
a 200
0
104 105 106 107 numberof PBC -0- PBC + AeIgG -s- PBC +normal male IgG
Figure 4: Dose responsecurve of binding of Ae to the father's PBC. One preparation of Ae was reacted with isologous dextran prepared cells. Normal male IgG, the sameamount of IgG a present in the Ae, was also reacted asontrols. The amount of IgG bound was measuredby the subsequentbinding of 125I-labelledprotein A. Results are plotted as cpm of protein A binding against different numbers of ceUs. Maximum binding of eluate occurred with 106cells, and this was usedin subsequentassays.
Radiolabelled protein A binds to eluted antibody bound to either dextran cells or Lymphoprep
binding to dextran prepared cells. Measurement of bound IgG showed that cell preparations (Figure 5). was much greater than to Lymphoprep prepared cells
73 Dextran 1200 preparedcells cells
1000
800
cpm 600
400
200
0
Ael Ae2 Ae3 Ae4
Figure 5: Comparison of 125I-labelled protein A binding to bound Ae of dextran or
Lymphoprep prepared cells. Four different Ae were reacted with their own isologous dextran or Lymphoprep prepared cells. Binding of radiolabeUedprotein A was used to measurethe bound IgG. Results are the means of duplicate counts of labelled protein A to the cells after
binds lymphocytes background subtraction. These indicate that the antibody to and probably
dextran also to some other cells present in the preparation.
3.1.5 Eluted human antibody reacts with the father's cells rather than a third party father's The binding of eluted antibody from placenta was studied on the cells and unrelated
binding demonstrable to the paternal antigen (Table M. cells to see if there was specific '23I-labelled A IgG Data Bound IgG was measuredby binding protein to the on cells. show that
74 Ae antibody binds to the PBL preparation from the father and there is only a small background binding to the non-specific PBL.
Table '"I IV: Binding of labelled protein A to the eluted IgG which bound to PBL.
Protein A bound (cpm)
Eluate Paternal PBL Unrelated PBL cm cm
Ael 690 76
Ae2 414 66
Ae3 732 128
Ae4 586 136
Four different paternal PBL were reacted with their isologous Ae and on unrelated PBL. The
125I-labelled A. Values amount of IgG bound was measuredby binding of protein are meansof for difference duplicate counts minus backgrounds. Student's t-test, t=7.8 p=4x 10'3 the between isologous and unrelated lymphocytes.
75 3.1.6 Microscopy of dextran and Lymphoprep prepared human peripheral blood cells.
There are T and B cells and a few monocytes in the Lymphoprep preparation (plate II). A preparation of dextran cells can be seenin plate M. These contain monocytes, lymphocytes(T
&B cells) and other leukocytes.
Plate IL Lymphoprep prepared cells. PBL were cytocentrifuged and the preparations were
G.G. ) Lymphocytes stained using the May-Grunwald-Giemsa (M. technique. are present with a
few monocytes which are not seenin this photomicrograph.
76 Cý ýýý
I ýý Ap 0 dO# "C
Ai .ý
i
Plate III. Dextran prepared cells. Cells were cytocentrifuged and preparations were stained
using the M. G. G. technique. All types of leukocytes can be seen,but the majority of cells are
polymorphonuclear.
3.1.7 Reaction of human antibody with cultured lymphoblastoid B lymphocyte cell lines
homozygous for LILA.
In order to see if there was an association of the polymorphic antigen, R80K, with HLA
antigens, the microvesicle preparations were tested for cytotoxic activity against four different
EBV transformed lymphoblastoid B-cell lines. All cells were homozygous for HLA Al, B8 ed four lines (Table V). When and Dr3. Most eluates fail; AZ to react with any of the cell an eluate Ae2 line 4 Ae3 reacted with one cell, it did not react with all, e.g. reacts with cell whereas the reacts only with cell line 1. This showed that the specificitiesof eluted antibodies are
independent of HLA specificities (Table V). This work was a collaborative study with Prof Ben
Bradley and Dr Jenny Underwood and Reza Jalali.
77 Table V: Reaction human of acid eluateswith four different EBV transformed lymphoblastoid lines cell all homozygous for HLA Al, B8 and Dr3.
Acid Cell 1 Cell 2 Cell 3 Cell 4 eluted (% kill) (% kill) (% kill) (% kill) antibody 1 80 80 50 50 10 10 20 20 2 20 20 10 10 10 10 70 70 3 40 40 20 15 15 20 30 25 4 20 20 20 20 10 15 15 20 5 20 20 40 40 50 60 20 10 6 20 20 100 70 70 70 30 25 7 20 20 10 20 10 15 20 15 8 20 20 10 10 30 30 70 70 9 20 20 10 10 60 70 15 40 10 60 50 10 10 30 30 15 20 11 20 20 10 10 20 20 20 20 12 20 20 10 15 10. 10 30 25 13 20 20 10 15 40 60 10 20 14 20 20 50 70 10 10 15 25 15 20 20 30 20 10 10 20 30 16 20 20 40 70 20 15 80 70 17 20 20 10 10 10 20 30 30 18 20 20 70 60 20 15 15 20 19 20 20 10 10 15 15 80 75 20 20 20 40 50 50 40 15 20 21 20 20 15 20 10 10 30 30 22 20 20 50 65 ND ND 30 25 23 20 20 20 10 ND ND 80 80
- control 20 20 10 10 10 15 20 20 + control 100 100 100 100 100 100 100 100
Complement dependentcytotoxicity is shown as the meanpercent killing in duplicate wells.
Numbersin bold indicatekilling significantlyabove negative controls. ND = not done
78 3.1.8 Reaction of equine eluted antibody with acid treated vesicles.
The horse reactionsof eluates with Av were tested similarly to those on human samples. Four different preparations of equine Va were reacted with their isologous Ae. These samples,four preparations of equine V and four preparations of Va without antibody were reacted with "'I- labelled protein G and the binding measured(Table VI). As demonstratedfor human placentae
(Section 3.1.3), unacidified vesicles did not bind eluted antibody but after acidification, like the
human experiments, equine Ae boundback to neutralisedVa from its own placenta.
Table VI: Elution and rebinding of equine Ae to microvesicles.
125I-labelled protein G binding (cpm)
V Va Va + homologousAe
318 101 289
277 118 284
342 166 326
317 142 316
357 132 358
333 147 384
284 116 247
261 107 263
to Four preparations of equine microvesicles were acidified and after neutralisation exposed
by binding "51-labelledprotein G. Duplicate their own Ae. Bound IgG was measured of results
By Student's V Va, t= 19.9 p= 10'6 Va + Ae versus Vat = 14.1, p= are shown. t-test versus .
79 2x 10'6. The difference between V and Va + Ae is not significant like the human results (Table r[l)"
3.1.9 Reaction of acid eluted antibody with different preparation of peripheral blood cells.
Evidence for monocytes being responsible for some of the binding of eluted antibody to peripheral blood cells, was obtained. Three different types of father's peripheral blood cells were exposed to eluted antibody from their babiesplacenta or from an unrelatedplacenta.
Bound IgG was measured using '25I-labelled protein A. Dextran prepared cells show the highest value for specific binding. A roughly equal binding of IgG occurs with Nycoprep prepared monocytes and the Lymphoperep prepared cells (Figure 6). Thus the highest binding observed with dextran prepared cells may be related to the extra monocytesin this preparation.
1000 900 $0o 700 CPU 600
500
400
300
200
100
I Dextrin lymphoprep Nycoprep
dextran, Lymphoprep Figure 6" Cells from peripheral blood were separated using and from the Nycoprep" Concentrationswere adjustedso that eachcontained cells samevolume of
80 blood. Each preparation was exposed to specific or non-specific eluted antibody and bound
antibody was measured using radiolabelled protein A. Pure monocytes and Lymphoprep
preparations bound to IgG, and dextran prepared cells bound about twice as much IgG as either other of the,4wo cell preperations. It will be seen that the best differentiation between specific and
non-specific binding occurred with the dextran prepared cells. Confirmation of this result is
found in figure 7 where FACS analysis showed that only a fraction of Lymphoprep prepared
cells reacted with the Ae, but virtually all the cells prepared by Nycoprep reacted with the
antibody (Figure 7).
Control Control T+B lymphocytes
Specific Specific T+B lymphocytes
Cri numbers
Fluorescence
Nycoprep cells stained specific or Figure 7: FACS analysis of Lymphoprep and prepared with
control eluates.
81 Preparations of paternal antigen on Lymphoprep or Nycoprep prepared cells were exposed to Ae from FITC-labelled the placenta. antihumanIgG was used to identify bound antibody. Only some the-lymphocytes in Lymphoprep of the prepared cells showedbinding of the positive but were antibody, virtually all the monocytes prepared by Nycoprep ; stained.
3.1.10 Acid eluted IgG antibody is speciesspecific.
The between human cross reaction and horse antibodies and antigenswas studied. Antibodies from 10 eluted equine placentaewere tested against five human.lymphocyte preparationsusing long NIH the microcytotoxicity test (Table VII), looking for cross reactions between human therebyindicating that and equine acid eluates from the microvesicles. No exampleswere found j the antibody is probably speciesspecific.
Table VII: Percent kill human of a panel of male peripheral blood lymphocyte (hLyl - hLy5) by acid eluates of 10 horse placentae(Ael - Ae 10).
Human lymphocyte
Equine Ae hLy 1 hLy2 hLy3 hLy4 hLy5
Ael 10 10 10 15 15
Ae2 10 15 10 15 10
Ae3 10 15 15 10 10
Ae4 10 10 10 15 15
Ae5 10 10 15 15 10 Ae6 15 15 10 10 15
Ae7 15 10 10 10 10
Ae8 10 10 15 10 10
Ae9 10 10 10 15 10 Ae10 15 15 15 10 15
Negative control 15 20 15 20 15
Positive control 100 100 95 100 100
82 The long NIH microcytotoxic test (Mittal et al 1968) was used with rabbit serum as complement source. Values are meansof duplicate percent kills.
3.2.1 Reaction of the 50kDa fragment with acid eluted antibodies.
Eluted bind antibodies to unpurified trypsin treated acidified vesicles (Te). Preparations of different Te (Section 2.16) were reacted with their isologous Ae or unrelated Ae in a solid phase assay. The amount of IgG bound was measuredby radiolabelled protein A (Table VII).
Presented data indicate that trypsin treated Va binds Ae antibody.
1251-labelled Table VIII: protein A binding to Ae bound to different preparations of human Te.
Radiolabelled Protein A binding to human
Te + Isologous Ae Te + unrelated Ae 380 155 560 207 369 274 218 165 448 73 316 227 599 145 565 210 334 335 323 295
isologous Five different preparations of trypsinised Va were coated on tubes and or unrelated
bound by 12I-labelledprotein A. Results Ae was ; added. The IgG which was measured shown
binding duplicated tubes from which counts bound to Te (without are counts of protein to have been The differencebetween these samplesis significant addition of any eluate) subtracted.
sum p=0.004 using wilc0xon rank
83 3.2.2 Trypsin digestion of horse vesicles.
As Jalali et al (1995a) have shown with human vesicles, after acidification an antigenic fragment the protein is released by trypsinisation of 50 kDa which binds the eluted antibody. To seeif the horse had an analogous protein, equine acid treated vesicles were digested with trypsin. In a solid phase assay trypsin treated acidified vesicles (Te) were exposed to the isologous or unrelated acid
eluates. Bound antibody was measured using 'uI-labelled protein G (Table IX). Trypsin
treated Va bound to isologous Ae, but not with unrelated antibody.
Table -IX: Binding of '2sI-labelled protein G to equine antibody bound to trypsin treated Va
coatedtubes.
cpm of 12,51-labelledprotein G binding to
e ni ne
Te + isologous Ae Te + unrelated Ae
364 36
257 7
420 29
290 46
184 44
227 189
299 62
312 26
Va to isologous Ae or unrelated Ae. Four preparations of equine trypsin treated were exposed G. Results counts of protein Bound antibody was measuredby radiolabelled protein shown are
84 binding in duplicate samplesminus the counts for protein binding to Te only. Student's t-test
(t=6.2and p=4x 104).
By SDS PAGE Western blotting - and using the eluted antibody the trypsin fragmentappeared at 60-70 kDa, about compared with the 50kDa human fragment (Jalali et al 1995a).
3.3.1 Cross reactions of human trophoblast by binding assays.
Preparations human Ae of were exposedto isologous or unrelated Va. Bound antibodieswere by 'uI-labelled measured protein A (Table X). Each Ae preparationbound back to the Va from it which was eluted, and cross reactions of other Va preparationswere found. The neutralised the Ae bound withWVa preparation from some other placentae (eight of forty eight = 16.7%). This indicated considerable alloantigenic polymorphism.
Table X shows reactions of Va from 10 human placentaewith Ae from the human placentae.
Val Vat Va3 Va4 Vas Va6 Va, Vas Vag Va, o
Ael 2.16 1.41 2.06 1.09 0.88 0.71 0.79 0.87 0.90 0.74
Ae2 1.61 2.45 1.17 1.94 0.91 2.27 1.88 0.75 0.56 2.40
Ae3 1.38 1.04 1.82 0.74 1.60 0.65 0.46 0.78 0.54 0.5
Ae4 0.90 0.47 0.57 1.67 0.89 0.96 0.64 1.18 1.27 1.01
Ae 1.03 1.09 1.14 1.07 2.40 0.86 0.95 2.43 0.72 0.79
Ae6 1.52 1.06 1.00 1.09 1.48 3.02 1.02 1.08 1.64 1.06
Ae7 0.38 0.70 0.56 1.05 0.90 0.55 1.24 0.33 0.79 0.29
85 Aeg 1.06 2.09 1.26 1.05 1.16 1.56 2.32 3.46 1.57 1.43
Ae9 0.65 0.55 0.50 0.29 0.70 0.62 0.38 0.92 3.42 0.73
Aelo 0.42 0.56 0.69 0.69 0.43 0.79 0.56 0.64 0.99 2.88
10 Ae preparations of were exposed to the isologous Va and Va from the other placentae. Antibody bound was measuredby binding of radiolabelledprotein A. The values shown are the duplicate means of estimations which have been assessedstatistically in two steps. The cross
reaction values for each placenta were averagedand the individual values divided by this mean
are shown in the table. Emboldened numbers indicate the reaction with self.
Statistical analysis of these reactions is difficult where the amount of vesicle antigens cannot be
standardised. The approach used here compensate for variation between microvesicle
preparations and the cross reaction' values in the study. In figure 8 is shown the distribution of
values for cross reactions, and in the seconddot plot the values for reaction with self. It will be high is seenthat the results of cross reaction binding show a skew towards values, that possibly
deviation low, a true cross reactions. The standard of the presumednegative values were then is higher the true calculated by calculating the mean, which than mean of negative samples,and
from it. This for deviation from subtracting all samplesbelow the mean produceda set of values was This is then the deviation the mean, and the root mean square of thessalculated. standard of
the lower part of the distribution.
limit has then to be taken the To show the results an upper of negative cross reactions as mean
86 + 2SD, and is indicated in the figure (dot line). Eight forty-eight of (16.7%) sampleswere above the upper limit of 1.84
4.0
3.5 -.I 0 0
3.0 -.I o 0
2.5 1 2 00 'U »o 0 i 0) 2.0 C ...... "...... 0 C 0 1.5 " 0" M 0 1.0 ýMMM
0.5 0NM
0 cross reaction reaction with self
Figure 8: Distribution of values for cross reaction and reaction with self for Va from human
placentae reacted with eluted antibodies.
O= reaction with self "= cross reaction.
The line indicates the calculated upper limit of negative reactions as explainedabove.
87 3.3.2 Cross reaction of human eluted antibodies with Lymphoprep and dextran prepared cells by a binding assay.
Different preparations of eluted antibodies were exposed to a panel of unrelated lymphocytes. Bound antibodies were measured by binding of radiolabelled protein A. Table XI shows the raw data and table XI a shows these data with the background subtracted.
Table XI: Raw data of reaction of human Ae to a panel of Lymphoprep prepared cells.
PBLI PBL2 PBL3 PBL4 PBLS
Ael 433 281 324 312 454 369 246 395 337 513 Ae2 442 242 316 429 393 382 163 557 416 414 Ae3 640 206 304 394 354 798 236 337 321 420 Ae4 312 304 344 374 620 360 248 372 359 411 Ae5 320 203 395 416 392 331 246 314 338 343 Ae6 407 154 315 415 361 462 262 321 446 418 Ae7 375 464 434 261 349 453 339 312 326 396 Aes 343 279 505 373 425 372 221 479 249 478 Ac9 484 212 491 452 291 339 259 413 327 467 Aeio 495 226 314 445 423 451 259 322 373 368 Aell 329 294 643 649 381 384 271 742 796 353 218 344 356 398 no Ae (Bkg) 284 397 184 299 297 351
from Eleven Ae preparations were reacted with five lymphocytespreparations unrelated normal 125I-labelled A. Results human donors. Bound antibodieswere measuredusing protein shown
in duplicate for are CPM eachreaction.
88 Table XIa: Data of table XI with the background subtracted.
FPBL3 PBL PBL2 PBL4 1 PBL 5 Ae 1 60 62 38 -2 109 Ae 2 71 1 115 96 29 Ae 3 20 31 12 Ae 4 -4 75 36 40 136 Ae 5 23 33 50 -15 -7 Ae 6 94 7 -3 104 15 Ae 7 73 200 51 -33 -2 Ae 8 17 49 170 -15 77 Ae 9 71 34 130 63 4 Ae 10 128 41 -3 82 21 AeEe1 16 81
There is no specific sampleto consider as a positive control. The values of mean and SD have been calculated. The values greater than the mean+2 'SD' which was equal to 247 have been considered as positive samples. By means of this calculation three of fifty-five (5.4%) samples were above this value and consideredpositive.
Binding of eluted antibody to unrelated cells prepared by dextran sedimentationis shown in
( Table XII. Seven out of eighty one positive reactions were found 8.6%), more than with
Lymphoprep prepared cells, but not statistically significantly so.
89 Table XII: Reaction 13 of preparations of human Ae to dextran cells prepared from unrelated normal donors.
PBC1 PBC2 PBC3 PBC4 PBC5 PBC6 PBC7
Ae 1 189 115 116 118 35 - - Ae 2 220 434 17 29 117 - - Ae 3 99 47 142 116 - 10 - Ae 4 64 150 345 - 97 - Ae 5 202 516 191 156 - 98 - Ae 6 199 44 17 101 147 94 70 Ae 7 7 -62 204 -49 189 84 260 Ae 8 149 32 270 '7 91 383 123
Ae 9 230 34 290 190 159 129 35
Ae 10 266 148 326 1 122 372 4
Ae 11 271 296 312 173 250
A e 12 149 499 fº> 410 242 <> 269 ...... Ae 13 169 526 412 465 Ü 296 375
dextran from donors. 13 preparations of human eluates were exposed to cells normal Amount
1251 A. Results of antibody bound was measured by protein shown are counts of the protein bound to duplicate samples after subtracting the counts of protein binding to cells without
SD' 744 7/ 81(8.6%) addition of any eluate. The mean +2` = and samples were above this
in value, shown shaded the table.
90 3.4.1 Transmission of paternal antigens in a human family.
Ae from placentae of two grandchildren was used to type the grandparentsand other family members ( Figure 9). Both placentae had the same antigen/antibody,and detected this antigen in all the family tested except the grandmother. Transmissionof this one paternal R80K allele is shown in Figure 9.
all, placenta2 I' placenta 1
NIS Positive
00 Not tested for 10 Negative * sourcesof eluted antibody testing
humanfamily. Figure 9: Transmission of paternal antigen in a human family were reacted with eluted Dextran prepared cells from different members of a by binding from the two The bound antibodies were measured of antibodies placentae.
91 radiolabelled protein A. Antibodies were eluted from the placentae 1 and 2. Cells were prepared by dextran sedimentationfrom family membersincluding the paternal grandfather and grandmother. Antibodies eluted from both placentaewere tested againstthe cells of the family. The bound '25I-1abeUed antibodies were assessedusing protein A. Identical results were obtained with the two eluates, showing that both reacted with the cells of the paternal but grandfather, not tothoseof the paternal grandmother. Counts of protein A binding to each preparation is also shown in Table M.
Table XM: Protein A binding to eluted antibodies, from the two placentae.of figure 9. Cells
were reacted with eluates from the placentae. The bound antibody was measuredwith protein
k
Dextran prepared cells from Protein A bindin cm to Ael Ae2
Paternal grandfather 1098 855
aternal grandmother -95 -30
Brother of paternal grandfather 1033 751
Daughter of paternal great-uncle 851 581 Husband 1 1027 -
Husband 2 - 843
less the bound Values shown are mean counts of protein A binding to cells, counts to cells
significant positive binding. without antibody. Values above 152 cpm were statistically
92 3.4.2 Typing Nearco family of members using acid eluted antibodies of individual placentae.
Horses of family Nearco for of were typed R80K antigen, using antibody eluted from placental microvesicles. Tables XIV XV and show results of typing placental microvesicles, and table XVI that of typing peripheral blood cells.
Table XIV reactions of acid eluted antibodiesand acid treated microvesiclesfrom 21 equine full term placentae sired by 3 stallions.
Footnote
Nearco, a descendant of the famous stallion St Simon was bred in Italy by Federico Tesio in 1935. He won all fourteen races for which he was entered, and subsequentlybought by Martin Benson for Beech House Stud in Newmarket, where he spent the next nineteen years.
He was an astounding horse at stud, being the leading sire in 1947 and 1948 and for more than a decadewas never out of the top ten. He sired a number of very famous stallions, including Dante, Nasrullah & Nearctic. When he died in 1957 some eighty of his offspring were standing at stud throughout the world.
To have been able to use his descendants,with their known pedigrees,has been both an honour and very advantageousto my study.
Reference: Lambton A& Offen J. Thoroughbred Style, Racing Dynasties - the horses, the owners, the studs. Published Stanley Paul & Co,London 1987 pp 42-43.
93 '! D I ý ý " .r .QQr ý+ r rQ r r- r ------o ßi--g-0 - r- rN rrrrrr ýiW1JýDEi Ni vbi N) 0 N N Nrrrrp AO0 0 OOV i NO ýD 00 Oý r Vi "ý. C t4 O pwmr N~ `r f. + -" 0- mm pO r+ r- N e" m ti NOOW WO OOb', ÖV Ö :w be i^ww «" r' 3 . "+wJW A + 00 %ä0" Omaolb . ombMß Q OOr ýl 1J0rr 00000 O rdrrý. J 00 ýD i. 00 ýG +rrN, """N""" " V 0p r N+Ö iý w ýD ÖO Oý ýO N Öý 11 JV VN ` ý.y Ob Iý 00 1 r . . ýi ý"+ Q 1. . + rQCO Orr ä.... wo- Qp O Irr ýJtýtýý... ý" 1ý+ om 1.+ Nr N 1.+ NNNo r t ob 0o W ÄNÄN:... ioOýfýOýON JZ& -c o a V v: rr QrrO Fr OO 14 is äpOOrrO rr M r+ wr Mr m rr N OÄ%OÄýOMVA bi e 00 1 4-4 0 --Oýa00o.... ýt bboo 4--P9--->p A Oý u Oý OW o ý.. yý W N CO 0o W N ö .iÖ ch rNrNOD V 0 0VJJV ll Oarp. , ' Nr wr - rpr i. +oýC tJ b-rooýi. ºN: " ýwaýo«. aýNiý ö d OO to ÄOwZ. ýl r"" NN Vý IJ CA 00 bb w ýO j .ýW c> Ot ,% to ; O e ý oJ V N r-p pý%000 rN00 00-000- NpN«rr0CD r ßl0 'b Ä ý -o r46CYN N 1C r001Jjaý v' NrW p+r. ar + p QNOP O VýNýÖýIÖÖVý Nr -+ r ýr rr O ýi OOr+brQ JÄ ý IJ0ý C 1 1 ý W0 Vºr%0p, N . a+ 0 s .1 C D pßj ON ch OrpO wr p+ r. r opON wtwNtA N; M+ rOOO Ni Q !J h+ mrrr N Cwpw ; rr r+ r pwv{ ý VJawrwl. lN fOoboeoe NatAJW ft -
or0. orrNo ö Öý ýJýÄ ý-+ to Oý rbp"Ä tN m «.. ' P r. o1. !ý ýQ ý" ýA t/1 J 10 O 00 N ý/1 "r ro 00 r"- mObOOýO yr Qý W 0C«r -J O !Je Or O _ ý- r- ti "-p rr! d mN !. i r OOOrOrQrr p W o JÖ YD iA *, V1 Ni J -1 J 00 6A t* 00 %0 - ýJ rrp Ni bi r ;DN ü.. C3N rQ pr rrrorO O -p ÖDO%W 00 «"+IJýptýW e. a00 ýºtý1ANrraD CýoWNtAtnfANÄýGÖ "'rO y m IA b p Wei b m Ä dNui C0 000º ýýwiwäONä ý " c CD O w ^'r CD CD CD W rýJOýJrýr W,, rein mm rrW 000 'ý(n wV iDbe W bONN«-" Ä im ww4b"b X A%AZI JtA WÖ 0-eÄ; 0 jýiÄJ tu> bpjo"u1 0ý1bOpirr O .J rrrýi-OQ OrprOrOrpýr v WO %A %A i4. NC fN .iN b im N ;p rWp W ) v ßj -i N1 OÖ bAAÖ %0 tt «-rCD N00«-"rCD ß+r00 Nr"! - ej Jean Qß+000! -+prON dobi wb', eAbi to.) CD ;0W- b% Ni Ji , ch Nab AL cýCD -% Ch - b>, "W
wo
N ." r? W ýO! 14 oo .O Op-+CD QCD rCD . a000rý+0 - i 0 WAOP Wr+ .lOA Ä N JpI Äb e f JJ i + w LA 0 bi .rÄA O -0b.. OONOO. + W. pr r0Or0CD CD ppp«-pr Orr Ä ;DÖ4Ji Ö {W NN " ÄÖ 0o i Z^ Ö be i. .A .to w ir O pr-I400-0«-+O NNON OOOO; rOO o- Cp000000 - 00 ÖO 00 00 %A J VA !JAO N W; D U A Wwo w *, "3 0% O Vl JV %O Or 2 s" NOOrOOO O WNNM r" ^' Q me rp' O m" QQr rr rr :" -4. Ö0%0 1J0 b% 1 i 0N aa0'4 0. PJ : bº'. NÄ; mi4 o . , r .
bPp U+00CD . +OOr+p JrCD O l. i0A00«'bi r'N rbrfflA i lb: A9AAb :> -400 Ni Aw +00 -A 1 00 b, rýJNr 01Jýk- NlpJ- a, 0 . . . pprrCD 00ý+Ob C + O:. Oý+ r; r0000 .. rpApý"r«rý; `C Go bý 00 %A ww Z) ;D be i') ý+ 41 yA p> %A IJ OD be j vÄ ÖJ3 00 0-0 O - %0 J Nr0 b w NOo0 Zw C p. ObOOOO ý".O «. 0 O-"tJ %r WÄ:..gor ý-+ %oý,, tJ "0 : : b. be i. J p. -o ;D ber NÄw4.. P) " Q ýb %A O r "+ rÄ .b . _Q r'i pr r' OQQpWON OWOrr rm r p r+ rrrrO Id :iÄ ;D ;0 .a 0O p0 ;CÖr t> %0 0o O, A t OÖ OC v t* bi r - rA J N ý+ bOOm. rOO --' Oar O rM '' &rOrrrOOrr+Q « pe rr VýppA1A . 00b r(> WW WrýDýDNÄ Abc> rO "'ýrr rä W yý br t- por! -p O_OO'rprý+O_ p000 . -- - Wwä x%00 WýJVýýC 0 i% A- %CpfJ. A v; 0: NA ýNÄOý. -r . 86 rW-" rp A Nl"iOr OÖrý'OCDmr*rý. i J OO; ýNOrý"' rOrý'Orr Ö %0 b Ök Öti%0 00 . wÖÄÄdbih o La ÖoÖý'ý"' fP O N (DN O ^ *. ,., W O(JO"rmg 0000CD ýr"ýr ripp COpOOOr fJ tA Nd NA ýýOwl, %D W0 do J 00 V. Nr L* 00 A CA 00 00 O G ä OÖr AOA x U ö' e %< 1 50 . c ýa Q ä 29 preparations of equine Ae were exposed to the isologous Va or unrelated Va preparations. 21 Of these preparations were derived from the full term placentae of horses fathered by 3 stallions i. e. Primo Domini (P.D. ), Soviet Star (S.S. ), Never So Bold (N. S.B. ) and 8
from preparations the placentaeof horses fathered by random stallions. Bound antibodieswere measured using radiolabelled protein G. Results shown are the ratio of net protein binding of reacted G by its protein the sample minus background to the mean net value for the vesicles with the
be for other eluted antibodies. A high ratio can seen the reactionsinvolving their own vesiclesand elutedar
The eluted antibodies of any one sibship also reacted with the vesicle: preparation from all individuals of that samesibship.
To assessthe significance of the findings shown in table XIV, the mean and SD of the binding
of the family antibodies with the vesicle was calculated, and also the mean and SD of the
reactions with antibody from all unrelated placentae.The confidenceinterval for the difference
between the meanswas calculated as twice the root meansquare of the two standarddeviations.
Values outside that level were considered as positive binding of the antigen to the eluted
antibody (Table XV).
Table XV. Statistical analysisof the family studiesshown in table XIV.
PLACENTA ANTIGEN
1 2 3 PD1 0.7 0.9 PD2 0.7 0.7 PD3 0.6 0.7 PD4 1.0 SS1 1.2 1.0 0.8 SS2 <<`' 1.5 0.9 SS3 F»< 0.4 0.7 SS4 0.4 1.1 SSS 1.1 0.9 SS6 1.1 0.9 SS7 0.9 NSB1 NSB2 1.3 0.8 NSB3 1.4 0.9 <>:. NSB4 0.8 0.8 >>>>'><>; NSB5 0.1 0.6 NSB6 1.0 0.5 NSB7 0.9 1.1 :< NSBS 0.0 0.8 NSB9 0.8 1.2 NSB 10 1.2 0.9 95 Eluted antibodies in three groups detecting antigens 1,2 and 3 were reacted with acidified microvesicles of placentae from foals of Primo Domini(PD), Soviet Star(SS) and Never So Bold (NSB). All placentaefrom one sibship reacted significantly with antibodiesof one antigen specificity, but two NSB9 random cross reactions are seen , with antigen 2 and SS2 with antigen 2.
In found each sibship, all the half sibs were to react only with antibodiesof a single antigen type, labelled however, as antigen 1,2 or 3. There are, occasionalreactions with odd vesiclesfrom the other sibships, showing possibly a reaction with the maternalantigen present on the vesicles (Table XIV). It should be rememberedthat all the sibs have different mothers, but all are the foals of one stallion.
3.4.3 Cross reaction within the family shown by eluted antibody binding to horse PBC.
When the placentae were not available and blood sampleswere, I typed some horsesusing the eluates from the families. Dextran prepared peripheral blood mononuclear cells from foals fathered by three stallions, Nureyev (N), Primo Domini (PD) and Never So Bold (NSB) were assessed. These preparations were exposed to eluted antibodies from the placenta of horses
Soviet Star(SS) (a foal Nureyev) Never which :had been sired by either of Primo Domini, of or
So Bold. Bound IgG was measuredby ' I-labelled protein G (Table XVI). It can be seenthat were G bound IgG siblings who joffspring of a radiolabelled protein to the on the cells of related
in but to the of anotherstallion. common stallion each group not progeny
96 Table XVI: Binding of equine eluted antibodies to PBC of 18 foals of three half sibships. Dextran cells were prepared from four. foals of Dominion, 13 foals of Nureyev and one foal of
Never So Bold. Cell preparations were exposed to the eluates of placentaefrom the PD, SS and NSB families. Bound antibodies were measuredby radio-labelled protein G. All samples
done in duplicate. were ,
1 2 3 4 5 6 7 8 9 Ae Ae Ae Bkg PD SS NSB Horse Father from from from + + + PD SS NSB mean mean mean Yearling 1 Dominion 765.,; 193 487 204 2.0 0.0 1.0 Yearling 2 Dominion 1077 406 1128 369 0.1 1.5 Yearling 3 Dominion 1207 295 734 305 2,.Q:.:.. 0.0 0.1 Exotic Forest Dominion 1232 394 308 381 3.2 0.0 0.0 True Power N. S.B. 511 281 954 402 0.6 0.0 Only Star Nureyev 263 1403. 306 326 0.0 31 0.0
Wolf Hand Nureyev 816 :::::::::2346 l 889 1159 0.0 2 0.0 496 1254 237 355 0.5 2.9 0.0 Devils Needle Nureyev .. Petronella Nureyev 356 466 335 357 0.0 3.8 0.0 Marienuna Nureyev 237 752 346 306 0.0 3.2 0.3 Agama Nureyev 328 1622: 304 409 0.0 35 0.0 Anodyne Nureyev 248 3422 257 309 0.5 2.5 0.0 Pastoral Nureyev 261 1377 454 392 0.0 3.2 0.2 Pechenga Nureyev 370 3197 368 350 0.0 3.0 0.0 1.5 Russian Countess Nureyev 437 913 863 405 0.1 1.4 0.0 5.0 Sonic Lady Nureyev 795 1627 : 482 805 0.0 Vitikala Nureyev 407 986 501 274 0.4 2.0 0.6 Nuravia Nureyev 748 1062': 418 314 1.0 1.7 0.2
G binding for Values shown in Column 3,4,5 and 6 are total counts of protein each sample. binding the to the Values in Columns 7,8 and 9 are the ratios of protein of sample meanvalue of backgrounds. Only a the samplesof the samefoal, after subtractingthe one antigen was
97 detected in each group i. e. one antigen in all the Nureyev sibs, one in Dominion sibs and a third in True Power from NSB.
Overall the results in table XVI are in line with those found in the family memberstyped on their placentaeseen in Tables XIV and XV.
3.4.4 Distribution of equine allotypes in the family of Nearco.
The results of the typing of the sibshipseither by vesiclesor peripheral blood mononuclearcells
havebeen mapped to the extendedfamily tree of Nearco(Figure 10).
98 Figure 10 Nearco Family Tree
99 Three different antigens, designated1,2 and 3 have been detected. In each sibship all the half siblings have only one of the three paternalantigens e. g. all progeny of Nureyev have the antigen
1 and the progeny of Soviet Star have the sameantigen. This showsthat the antigen inherited is from the paternal grandfather and there is transmissionof only one of the two paternal allotypes of R80K antigen to all progeny. Interestingly, Swan Ann, mated to Dominion, produced a foal with antigen 2, Primo Domini. This antigen is also transmitted to all of his offspring. When
to Never So Bold foal,True 3 is mated , she produced a power, showing antigen which the alloantigen of all other NSB progeny. The only exception is Polar Falcon who does not inherit the paternal antigen i. e. antigen 1. The expressedantigen is antigen 2, presumablythe antigen of Marie d'Argonne, the paternal grandmother of Queen's Visit and Warning Light. This implies that a switch must have occurred elsewherein the family. Even in this case,the same antigen i. e. antigen 2, is passedon to his two offspring and again only one of the two haplotypes is found in any sibs
100 3.5.1 Detection of antibodies in sera of women with normal pregnancies by binding to
paternal PBC.
Preparations of paternal dextran prepared cells were exposed to the sera of their pregnant 125I-labelled spouses. Antibody bound was measured using protein A. Anti-paternal cytotoxic
antibody (APCA) was also measured(Table XVII). As can be seen, sera from all women with
normal pregnancies contained anti-PBC binding antibody in variable amounts, but APCA was
positive in only 2/ 13. All 13 women had successfulpregnancies.
Table XVII: The values of anti-trophoblast antibody and APCA in sera of women with normal
pregnancies.
Samples PBC Ab APCA Days into No of previous binding pregnancy successful ratio pregnancies
0 1 2.8 - 120 2 2.7 - 120 0 0 3 1.0 - 180 180 1 4 2.0 - 1 5 2.9 - 240 240 1 6 3.0 - 120 1 7 2.9 - 8 1.7 + 180 1 180 1 9 2.9 - 180 1 10 6.0 - 90 2 11 1.2 - 12 4.8 + 195 2 180 0 13 1.1 -
Sera from 13 pregnant women were assessedfor the presenceof anti-trophoblast antibody using
dextran (Section 2.15). Bound antibody their husbands prepared cells was measured using A. radiolabelledprotein
101 The values shown for PBC binding antibody are:
Counts bound to paternal cells with pregnancyserum - Bkg Counts bound to paternal cells with normal male serum - Bkg
These ratios varied from 1.0 to 6.0, mean ± SD = 2.7 ± 1.4. Positive values for APCA were ted when killing was more than 20% above the background.
3.5.2 Detection of antibody in the serum of women with repeated abortion.
PBC binding anti-trophoblast antibody was assayedin sera of women who had more than two
pregnancy losses. A preparation of the partner's dextran prepared cells was exposed to the
women's sera. None of these women had had a successfulpregnancy. In all samplesthe ratio
was low, ranging from 0.5 to 1.4 (Table XVIH). A ratio of 1 indicates no binding above the
control. Anti-paternal antibody (APCA) was positive in only 1 of 26 samples.
102 Table Xvm: The values of anti-trophoblast antibody and APCA in the sera of women with repeated abortion.
Sample PBC binding APCA No. of previous antibod abortion 1 0.5 - 3 2 0.9 - 3 3 0.8 - 3 4 1.0 - 3 5 0.7 - 3 6 0.8 - 3 7 1.3 - 3 8 0.8 - 3 9 0.8 - 3 10 1.0 - 3 11 0.8 + 3 12 1.1 - 3 13 0.5 - 3 14 0.9 - 3 15 0.9 - 3 16 0.8 - 3 17 1.0 - 4 18 0.7 - 4 19 0.8 - 4 20 1.1 - 4 21 1.4 - 4 22 0.9 - 5 23 0.5 - 5 24 0.8 - 5 25 0.8 - 5 26 1.0 - 6
for Sera from 26 women with repeatedabortion were tested anti-trophoblastantibody using by '2I-labelled their husbands' dextran prepared cells. Antibody bound was measured protein
A,. Values shown for anti-trophoblast antibody and APCA were calculated as explained for was SD for 0 86 Table XVII and sections2.15 and 2.14.3. The mean± anti-trophoblast antibody. f
0.2.
103 3.5.3 Detection of antibodies to husbands' PBC in sera of immunised women.
Sera of women with recurrent spontaneous abortion before and after immunisation with husbands cells were tested for both APCA and PBC binding antibody. It can be seen (Table
XIX) that all PBC binding antibody levels increased after immunisation. APCA was only detected in 4/16 samplesafter immunisation.
Table XIX shows the value of anti-trophoblast antibody and APCA in sera of some women before and after immunisation.
Sample PBC binding antibody APCA Pregnancy number outcome before after before after immunisation immunisation immunisation immunisation
1 1.0 1.2 - + A 2 1.6 1.7 - + A 3 1.1 1.5 - - L 4 0.7 1.2 - - L 5 1.0 1.9 - - L 6 0.9 1.1 - - A 7 1.0 1.1 - - L 8 0.7 1.7 - - L 9 0.6 1.8 - + L 10 0.5 0.7 - - A 11 1.1 1.3 - - A 12 1.1 2.2 - - L 13 1.3 2.3 - - L 14 0.7 1.1 - - A 15 1.0 2.1 - - L 16 1.0 1.4 - + L
for PBC binding Sera of 16 women with repeated abortion were tested paternal antibody and
before immunisation 0.9 ± 0.4 ApcA. The mean PBC -binding ratios and after were
It be that 1/16 had a ratio > 1.5 before treatment, and 1.57 ± 0.5 respectively. can seen and
104 8/16 after immunisation. There was no correlation between PBC binding antibody ratios and
APCA after immunisation, although APCA seroconversionoccurred in 4/ 16.
Table XX shows the combined values for tables XVII, XVIII and M. It can be seenthat the mean PBC binding antibody ratios increased following immunisation. Using the value of 1.5 as the limit of the antibody negative range, the correlation with successjust fails to reach significance, but the numbers are small ( Fisher's exact p=0.056 ). When the increment after immunisation is used, with an upper limit for the negativesof 0.4, there is a greater significance value for the correlation with successfuloutcome ( Fisher's exact p=0.03 5 ). Neverthelessthe numbers of results available is too small for more accurateanalysis.
S. Mean± BC Fraction Success
Clinical category antibody APCA +ve (%) binding ratio
Women before immunisation 0.9 f 0.4 0/17 0%
with after L 1.7 f 0.4 2/10 63%
RSA immunisation A 1.18 f 0.3 2/6 27%
2/13 100% Normal pregnant women 2.7 f 1.4
1/26 Untreated recurrent abortion 0.86 f 0.2 -
105 4. Discussion
Women with recurrent pregnancy failure usually lose all of their pregnancies,or all but the first. Recurrent spontaneousabortion is a relatively rare phenomenon,partner dependent, and occurs in about two couples per thousand. The loss of all pregnanciesin these couples implies that if an immunological phenomenonis responsiblefor prevention of abortion, then a similar process must be involved in abortion and all the offspring should have the same antigen. As stated previously there is an 80kDa protein molecule (R80K) which is a component of microvesicles prepared from the human syncytiotrophoblastplasma membrane and is highly polymorphic (Jalali et al 1989,1995a). I have shown that the antibody to this protein is present in sera of normal pregnant women and on all term placentae. This antibody, however, is absentin sera of women with recurrent first trimester early pregnancy loss and immunotherapy is highly efficient at reversing this. This may protect the conceptus immune from maternakattack and prevent subsequentabortion. Furthermore, it has been shown that a mouse MAb to R80K can prevent resorption in three mouse models (Jalali et al, AJRI in press). This suggests that the anti-trophoblast antibody may be an essential part of a successful pregnancy.
As the antigen is highly polymorphic, it would be expectedthat most males would have two frequency. allotypes, and would transmit either of these to the offspring with equal in Therefore, if the lack of antibody to one paternally derived R80K allotype the trophoblast
half were the cause,women would be expectedto abort their pregnancies,as only very rarely derived haplotypes. Abortion half would a woman fail to respond to both of the paternally of
is, less than loss the of a woman's pregnancies however, much common of all, or all after discrepancy first pregnancy. This investigation was set up to study this by studying the inheritance of this paternally derived protein, which could be explained if there were uneven
106 distribution of the paternal haplotypes in the offspring. If only one paternal allotype was expressed in all trophoblast in any progeny, and the mother did not make antibody to it, abortion would be an all-or-none phenomenon rather than occurring, in half of the pregnanciesof affected couples.
The study of the distribution of the R80K antigen in horsefamilies
The study of the inheritance of the R80K alloantigenwas carried out in horsesbecause it was possible, in about seven weeks of one season to get placentae from a large number of halfsiblings. That a stallion may father up to about thirty foals in one breeding season,each of which then has a common father, but a different mother (half sibling) made possible the study. If I had tried to do this in humans,it would have taken ten to twenty years to collect equivalent placentaewithin a sibship.
Optimisation of the methodsfor typing family
In order to type horsesaccurately, a number of conditions neededto be optimised for the use of eluted antibody to detect the R80K antigen on cells or microvesicles
a) Cell typing: By cell typing, it was possible to type individuals without accessto their placentae. This is a great advantagefor family studies in severalgenerations when placentae
are not available. In order to find out the best cell preparation for typing, three different
It found methods of cell preparation were compared. was that all three preparations will by dextran bind the antibody, however, cells prepared sedimentationgive a higher degree of binding than the other preparations. The dextran prepared cells provided more R80K
(1989,1995a) that the R80K is the antigen. Jalali et al showed antigen , expressed on
lymphocytes. This finding that in to B surface of human B suggests addition cells, the antigen blood FACS might also be expressedon other peripheral cells such as monocytes. analysis
107 confirmed theseresults and it can be seenin Figures6 and 7 that eluted antibodiesbind to monocytes in dextran prepared cells as well as B lymphocytes. Therefore dextran prepared cells were used to study the inheritanceof R80K allotypes.
In because some experiments of lack of availability of fresh cells, it was necessaryto use formaldehyde fixed cells. Experiments showed that formaldehyde fixation does not affect binding antigen capacity. Possible lysis of the fixed cells by tween 20 was also studied and it was observed that using a low concentration of detergent(0.05%) did not have any effect.
b) Measurement of antibody binding: A variety of assayshave been employed for measuring binding of antibodies to the cell surface antigens, and I have used a conventional radioimmunoassy with a number of major modifications. The assay relies on the use of radiolabelled protein A or protein G.
The value of Staphylococcusprotein A and Streptococcusprotein G as successfullaboratory reagents have been well documented. It has been shown that protein A binds with high affinity to the Fc portion of human IgGI, 2 and 4 subclasses(Kronwell 1973; Akerström et al 1985). In general IgGI, 2 and 4 account for 92% of human IgG. Equine IgG only weakly binds to protein A, but a cell surface protein of group G streptococci, protein G, binds more
Protein strongly to horse IgG (Gus et al 1986). G also binds to mouse monoclonal IgG
(Björk and Kronwell 1984). These characteristicsmake these reagents very useful. In all
human IgG, I have 125I-labelled A in experiments for detection of used protein and the IgG MAb, G has been experiments to detect equine or mouse radiolabelled protein used.
This technique provides exquisite sensitivity, compared with other methods. The biohazard
however, argument for not it. This be by of this assay, makes an using can overcome formulated for following the rules which have been protection from the hazardsof radiation.
108 c) Oxidation damageduring radiolabelling: During the labelling processusing chloramineT, oxidative alteration of protein conformation is well known, and can result in loss of binding efficiency. Detailed attention was paid to the radioiodination of proteins using the well established T chloramine method of Greenwood et al (1963) and the manufacturer's guide (Amersham 1993). Precautions were taken to minimise the exposure of the proteins to
In oxidising agents. order to know the yield and specific activity of binding, some designed to experimentstimatt the . exact amount of chloramine T needed for optimal labelling were carried out. It was found that a ratio of 1:1 for the amount of protein A or G to be labelled to chloramine T neededwas optimal for the detection of antibody bound to vesicles. With these conditions the efficiency of labelling was up to 50%.
d) Preliminary experiments were carried out to establishthe optimal concentration of acid eluates and acidified vesicles in typing studies. It was found that the 50 µl of 74 µg/ml of acid eluted antibody and 40 µl of acid vesicles give the optimal ratio for maximum binding.
It was not possible to standardisethe amount of microvesiclesin each assay,so that a sample with a low yield of vesicles would give lower binding results. Thus a vesicle preparation tested against many antibodies of equal concentration showedless variation than comparing binding of one antibody to several different vesicles of different concentrations. This becamethe basis of the statistical evaluation of the family typing data.
it found e) Blocking buffer: In all experiments was that the presence of Tween 20 was binding necessaryin all buffers for better and prevention of non-specific binding. This may inhibit non-specific binding of some sticky proteins present. Various blocking reagents, including normal male serum, normal horse serum, purified human or equine IgG, normal
bovine haemoglobin Marvel A rabbit serum, serum albumin, and were used. solution of o i% Marvel in PBS was found to give the highest ratio of specific to nonspecific binding .
109 when in combined with washing a solution of 0.05% Tween 20,0.15 N NaCl and 0.02 M phosphate buffer at pH8. Using theseoptimal conditions reliable detection of the paternal allotypes in horse placentaeor cells was possible.
Transmission of the R80K paternal allotypes into the next generation The results described in this thesis show that each eluted antibody reacts significantly with the acidified vesicles prepared from the samefamily. When placenta were not available and cells were used for typing, it had been possible to get much better assays with lower nonspecific binding. These results also showed that all the halfsiblings carried the same paternally derived allotype. This implies that the same paternal antigen was inherited in all sibs of a family.
If there were a normal distribution of the two allotypes, then reactions of eluted antibody from one placenta with acid treated vesicles from another placentae with a common sire random would be expected to expression of the two allotype (50%). Only 50% of the half siblings share a similar antigen which was a random expression of the two allotypes.
There is, however, clear non-random expressionof the two haplotypes and only one of the paternal haplotype appearsto be expressedon all the half siblings of one stallion rather than the expected 50:50 distribution of the two haplotypes,as can be seenin table XV.
The grandpaternal allotype is usually transmitted
Figure 10 shows that the progeny of Soviet Star, expressedantigen 1 as their cell surface.
This is the antigen which passedfrom Nureyev, the grandfather of this progeny. Similarly,
been detected in the Primo Domini; antigen 2 has all of progeny of again the antigen of their Dominion. Furthermore, Swann Ann Dominion foal grandfather, mated with produced a
110 with 2, Primo Domini antigen and all his progeny also had antigen 2. Mated with Never So Bold (antigen 3) she produced a foal with antigen 3 which is found in all the Never So Bold These findings progeny. suggestthat usually the grandpaternalallotype is transmitted.
It be should mentioned that the maternal antigen is expressed,but obviously will not be covered with maternal antibody. This is suggestedby the scattered reactions with IgG from antibody unrelated placentae. As these cross reactions are only with a single placenta in a sibship, it appearsto be detecting another allotype other than that binding the isologous maternal antibody. It should be rememberedthat in the sibships all siblings have different mothers, and the maternal antigens are different in each case. This, then suggeststhat there is a maternal component detectableon at least some of the microvesicle preparations.
Other examples of unequal transmission ratio
Transmission ratio distortion (TRD) of the t haplotypes genes in mice provides one of the best studied examples for anomalous transmission of a paternal gene. Male mice, could be heterozygous for at haplotype and a wild type form of the t complex (+/t)xpected to transmit the t chromosome to 50% of their offspring, but they transmit the t haplotype to their progeny with an abnormal frequency, usually more than 50%, depending on the presenceof the Tcr locus and the number of the Tcd loci (section 1.2.1).
My results, however, show that only one paternal haplotype is expressednormally and there is no expressionof the other haplotype. It should be mentioned that a degree of transmission distortion of shared HLA haplotypes has been shown in studies of an inbred society, the
Hutterites, by Ober et al (1992).
111 Possible mechanismswhich might explain the expression of only one of the two paternal R80K haplotypes
The in results presented this thesis indicate that in each sibship only one antigen was detected in all the half sibs. This found by was typing of extensions the -family of Nureyev, using the binding of eluted antibodies to either vesicles or peripheral blood mononuclearcells. In most casesthis was done using eluted antibody and vesiclesbut in 18 casestyping of dextran prepared cells were carried out. Although the exact mechanism for such anomalous inheritance is not clear, there may be a number of possibleexplanations and these include:
1- Selective loss of half of the sperm
Expression of only one of the paternal allotypesin all term placentaeof one sibship implies that there is differential transmission of the paternal R80K alleles. During spermatogenesis the primitive germ cells undergo meiotic division resulting in formation of fully mature sperm which are haploid, i. e. each sperm carriesonly one paternal allele. Therefore, individual embryos contain only one of the two paternal allotypes.
In the case of R80K alleles it is possiblethat the unequal transmissionarises from preferential
death of sperm with the two alleles.
Consider two paternal R80K alleles A and B. If sperm carriyng A alleles survive and those with the B are lost, then only embryos with the A allele would be generated. It is possible that expression of the R80K gene product on the surface of the sperm may allow distinction of those sperm carrying the A from those carrying the B allele. It is unlikely that if both alleles are expressedthat antibody in the man could result in recognition of the two
in his body he immunisedby alleles, as these are self antigenson other cells and would not be
112 his If own allotypes. the two alleles are expressed,maternal antibody to both would be the usual expected result, so that this could not explain differential loss of one allele.
It has been shown that R80K is a target for NK cells. This is particularly interesting as do sperm not express Class-I HLA antigens,and would be thus susceptibleto NK attack, if ed they expresR 80K on their surface. Other cells which expressR80KOn their surface e.g. B cells and monocytes are protected by their MHC molecules. If there were differential expression of the two R80K alleles in sperm,NK attack might affect the final distribution of the two. Such differential expressionmight occur by imprinting of the gene for the A allele
Those sperm carrying the A gene, would not expressthe R80K antigen and would survive.
Those carrying the B allele, not imprinted, would express the R80K protein and could be killed by NK cells. By this mechanismit might be that A gene is subject to imprinting whereas B gene is not. If the B sperm were killed by NK cells in the male genital tract, then the ejaculated live sperm would not have detectableR80K on the surface, and in preliminary studies in this laboratory this was found using FACS analysis of sperm stained using a monoclonal antibody to R80K ( Jalali et al, AJRI in press). This finding would preclude killing of the B allele sperm in the female tract, but it would not be necessaryif they had all been killed in the male tract. There are some CD56+ cells in the semen (Mowbray, unpublished data) and it is well known that NK cells are frequent in the endometrium.
Maternal imprinting of trophoblast genes is common but this postulate would predict the survival usually of the grandmaternalgene, which is not what I found. It would be necessary for the imprinting to be of the grandpaternalgene. Although in trophoblast it is the maternal
be genes which are imprinted, in spermatogenesisany gene could affected of either parental be by R80K is in origin. The hypothesis would testable showing that the gene unpaired the in DNA One however, sperm, but paired the genomic of the man. could argue, that there are hardly any NK cells in normal semen. Neverthelessthis could be explained by the fact that
113 NK kill one cell may many sperm. Second, semen contains a large quantity of
Prostaglandins,particularly E, which can inhibit NK cell activity in vitro (Tarter et al 1986 Vallely and et al 1988). Third, it is known there is a correlation between human NK killing and the absenceof HLA Class I expression, although Pena et al (1989) and Nouri et al (1993) failed to show such a correlation. The effect of HLA classI in the sperm hypothesis, however, is not obligatory, but only permissive.
2- Selective loss of half of the embryos
If the above were not the explanation is there a possibility that differential expressionmight result in NK killing of trophoblast of early embryoswhich carried the expressedB allele and not the imprinted A allele?
Loss of 50% of embryos might be a consequence of early embryo expression of R80K on
only half, which would then be destroyed by the CD56' granulated lymphocytes in the decidua. When the surviving fraction unimprinted their R80K genes they would be
susceptible to attack, if the mother had not made protective antibody to that allotype. The
early embryo would then in general have the grandmaternal gene expressed, and the
grandpaternalgene imprinted.
Recent work with the BA11 monoclonal antibody to the R80K protein of human and murine it inhibits killing human origin, developed in this laboratory, has shown that NK of and by NK murine NK targets, prevents the killing of murine trophoblast murine cells, and (Jalali AJRI in In the prevents abortion in three well studied mouse models et al, press). it has become that there increased course of the mouse experiments clear was an number of This implies that NK killing limit the embryos in the uteri of treated mice. of embryos may A to half not affect number which survive. reduction of sperm numbers would probably but if this happen. If the number of embryos surviving, the effect was on embryos would hypothesised the the presumed death of embryos were the selection process above, embryos
114 would now both haplotypes. show paternal This may make testable the possibilities discussed least above, and would at be a way of locating the selection processto the female rather than a sperm selection in the male.
A hypothesis which might explain the switch from grandpaternal to granulmaternal antigen in Polar Falcon
The haplotype inherited is normally the grandpaternalhaplotype, but occasionallythis alters, and then breeds true. In the extend family of Nearco transmissionof three different antigens tested have been found. All the offspringelate back to Nearco, who would have had only two allotypes. This implies that the trophoblast antigen in the living animals studied cannot always have been derived form the grandpaternal source, although this is what has usually been found. Occasionally another antigen, of perhapsgrandmaternal origin, was found in the half siblings, but whichever was present all the siblings shared the same antigen. Polar
Falcon is a point where a different antigen is found. The antigen expressedis presumedto be of grandmaternal origin. Interestingly, in spite of the occurrence of this switch, both of his offspring passedon this sameantigen. It seemsthat the antigen of Marie d'Argonne, the
Queen's Visit Worning Light paternal grandmother of and was passed to them. Other informative relatives of Marie d'Argonne were not availableto test. As three antigenictypes
115 in were present the current generation of horses studied, this means that a similar switch have must occurred elsewhere in the family in the past, but it is still true that of the two
paternal allotypes, only one is found in any sibship.
If imprinting is the mechanismfor differential recognition of sperm or embryos, there is a
mechanismby which such a switch might occur. There is now evidencethat the `imprintor'
gene upstream from the imprinted gene is responsiblefor the methylation. If in the process
of spermatogenesisthere is a recombinationbetween the two geneloci, the gene on the other
chromosome will be the one imprinted, and this would remain until a later further
recombination. crossoverbetween the two loci PATERNAL CHROMOSOME imprinted gene imprintor gene'
MATERNAL CHROMOSOME unimprinted gene imprintor gene
PATERNAL CHROMOSOME unimprinted gene
n S.
MATERNAL CHROMOSOME imprinted gene
is for From the results in this thesis it is impossibleto decide which mechanism responsible
the phenomenonI have shown.
The individual hypotheses are testable, most easily if the appropriate gene locus and some It be look to if there is sequence is known. would then possible to at sperm see only one this including father's DNA copy of the allele, and the gene, compar N,yith the genomic which The is have (PCR) would have two. only realistic way to polymerasechain reaction primer
116 to PCR from generate products the genewhich can be sequenced.The sperm product would then be have base expectedto only one at eachposition, whereastwo baseswould be present at some sites in the genomic DNA. To test the possibility of sperm loss before ejaculation it is only required that the R80K be expressed on one of the sperm, whereas if the step occurred later half would be expected to show surface R80K. Preliminary testing, as stated, failed to find R80K on ejaculated sperm, but this would require further work to be reasonably certain of the absence,rather than low level expression. By FACS analysis it is always easier to demonstrate an antigen rather than
convincingly show its absence.
If embryo loss were the mechanism of unequal distribution then it would not necessarily be
needed that sperm ever expressed the antigen, it might be that expression of the unimprinted
antigen only occurs in the fertilised egg. Detection of R80K on all or none of sperm would
not therefore be helpful in attempting to distinguish between the hypotheses of loss of half
of the embryos.
Another possibility which might explain the expression of only one paternal haplotype is that
the father is homozygous for the R80K gene. In this case if the products of homozygous
geneswere immunoreactive,the mother would make antibody againstthe R80K antigen and
therefore this would lead to a successfulpregnancy. If the products of the genes,however,
are nonimmunoreactivethere would be a pregnancylose.
be It is possible that the intensity of paternal trophoblast R80K gene products may not in lack sufficient to induce a strong immune reaction in some mothers. Thereby resulting of if is R80K antibody production and consequent abortion. Therefore the mother not
immunised by trophoblast there will be pregnancy failure. Production of maternal antibody
by immunisation with purified paternal lymphocytes results in a high rate of pregnancy hypothesis, however, the two success. This can not explain the expression of only one of
paternal haplotypes.
117 Anti R80K antibody is a protective antibody
In the experimental conditions used in this study it has been possible to demonstratethat immunisation with purified paternal lymphocytes may produce the protective antibodies. A consensusof randomised clinical trials of immunisationwith purified. paternal lymphocytes reported a statistically significant successof treatment in women with recurrent spontaneous abortion compared with a placebo treated group (Consensus1993).
It has been suggested that immunisation will stimulate a maternal allogenic responseto paternal antigens and this responsestimulates formation of antibodies. These antibodiescan inhibit Fc-rosette formation (Faust & Neppert 1987) and mixed lymphocyte reactions (Unander 1985 Sugi 1991). There is however, et al , et al no evidence, that these are actually the protective antibodies (Mowbray 1986), but cytotoxic antibody was found to be a good indicator of the length of protection after immunisation, although the reason is unknown ( Mowbray & Underwood 1990 )
I measuredthe anti-R80K antibody in the sera of normal pregnant women and women with recurrent spontaneous abortion before and after treatment with purified paternal lymphocytes. As seen in tables XVII, XVIII, XIX and XX a successful outcome is very strongly associated with detectable anti-R80K antibody. This make it likely that, unlike other antibodies found in pregnancy,this is the protective antibody.
Jalali et al (1989 & 1995a) showed that the antitrophoblast antibody was bound to syncytiotrophoblast on all term placentae and the target antigen was also present on peripheral B cells. In my study the presenceof the target antigen on monocytes has been be shown. It is possible that cells prepared with dextran for immunotherapy may more efficient for immunisation than lymphocytes prepared using a density gradient, because preparation of dextran sedimentationcells will include more monocytesand B lymphocytes.
Previously Winchester et al (1975) found a group of alloantibodies in pregnancy sera which B lymphocytes react with antigens present on and monocytes. These were not detectableon 118 unstimulated T cells. Their data indicated that these antibodies were distinct from HLA antibodies. This be R80K may well analogousto the alloantibodiesto R80K present on the same cell types.
R80K alloantibody is speciesspecific
Antibody from horse eluted the placentaedid not have any cytotoxicity against any human blood lymphocytes peripheral (Table VII). In the binding assay there was no binding between the equine eluted antibody and human dextran prepared cells nor between human
dextran antibody and equine prepared cells. The R80K of both speciesis similar, but like the MHC latter, antigens, species differences prevent analogy between this antigen in other: species.
Possible role of the R80K protein in pregnancy.
Mouse models of abortion and in vitro studies of human deciduate cells indicate that activated NK cells are capable of damageto trophoblast (Chaouat & Clark 1986; King &
Loke 1990). Evaluation of NK cell activity in women with recurrent spontaneous abortion without any treatment have showed that raised NK cell, levels predict abortion in these women (Aoki et al 1995). An increased level of these cells also has been shown in pregnancieswhich subsequentlyaborted (Nichols et al 1994). It is interesting that in vitro studies carried out (Jalali et al AJRI in press) have shown that eluted antibodies could inhibit the activity of NK cells againstK562, a cell line which is the standardtarget for NK cells. In these studies acid eluted antitrophoblast antibody which had affinity for K562 prevented NK killing in a dose dependent manner, suggesting that in the absenceof the antibody, NK cells may attack the placenta and lead to pregnancy loss. More recently Jalali preventing killing et al (AJRI in press) showed that a MAb generatedagainst R80K was also very effective innf
119 trophoblast three These findings and mouse abortion models. suggest that the successof pregnancy may be associatedwith production of antibody to R80K.
Comparison of the R80K antigen with the MHC molecules
The presented indicate that results only one of two paternal haplotypes is expressedin all term from father. placentae siblings of the same Biochemical analysisof the R80K molecule and MHC antigens provides evidence that these molecules are distinct from each other. SDS-PAGE of the humanR80K shows a single 80kDa band which is different from the HLA
Class I II (Section ). or molecules 1.4.1. Furthermore, the results presented in Table V show human that HLA antigens and R80K are assorted independently on HLA homozygous
human lymphoblastoid cultured cell lines. One should conclude that this polymorphic protein is not an MHC surface antigen, however, its location in the genome is still unknown.
Preliminary studies in horse have failed to show any relationship between the equine leukocyte alloantigens (ELA) and the equine trophoblast alloantigen equivalent to human
R80K (Mathias et al 1995). The MAb to equine MHC Class-1 proteins do not detect R80K.
The anti human R80K monoclonal antibodies react neither with equine trophoblast antigen nor equine MHC.
Conclusion
1. It appearsthat only one of the two paternally derived allotypes of the R80K is genetically transmitted. This finding could explain the lose of all pregnancies in women with recurrent spontaneous abortion. Transmission ratio distortion to the degree found here has not previously been reported and it may well be associatedwith the absenceof sperm containing the gene for the missing allotype.
120 2. The presence of antibody to paternal R80K antigen may be essential for a successful
pregnancy in allogenic matings. This antibody is absent in sera from most women with
recurrent abortion, but is presentin those who have normal term pregnancies.
3. Anti-trophoblast antibody can be detected in sera of women who have undergone
immunotherapyand who subsequentlyhave a successfulpregnancy. Thus immunisationis an
effective treatment for this group of patients.
4. Monitoring of R80K alloantibody may be a very useful way of detecting adequate
protection of the embryo in a pregnancy. It may be utilised to assess the need for
immunotherapy, and to monitor the formation of antibody after immunotherapy when further
treatment may still be required.
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140 Appendices
APPENDIX A
PREPARATION OF REAGENTS USED
AET solution.
200mg 2-Aminoetylisothiouronium Bromide (AET) was dissolvedin 5m1distilled water.
The pH was adjusted to 8.0 using sodium hydroxide.
0.2 M stock phosphate buffer.
A. 0.2 M solution of monobasicsodium phosphate.
B. 0.2 M solution of dibasic sodium phosphate.
290 ml solution A added to 710 ml solution B.
Eosin solution for tissue typing.
2% solution in tap water.
Complement fixation test diluent tablets & Phosphate buffered saline tablets.
These were purchasedfrom Oxoid Ltd, Basingstoke,Hampshire, UK
Each tablet was dissolved in 100 ml distilled water with stirring.
0.1 M Sodium carbonate bicarbonate buffer.
A. 28.62g of Na2CO3.10 H2O dissolved in 1000ml of distilled water.
B. 8.40g of NaHCO3 dissolved in 1000ml of distilled water.
300m1solution A addedto 700 ml solution B.
141 0.1 M Citrate buffer.
A. 0.1 M solution of citric acid (21.01 g in 1000 ml).
B. 0.1 M solution of sodium citrate (29.41 g C6HSO7Na3.2H20 in 1000 ml). 46.5 ml solution A added to 3.5 ml solution B and the volume adjustedto 100m1.
EDTA Saline buffer.
74.48 EDTA dissolved in g was 800ml distilled water and pH adjusted to 7.6 using ION
NaOH and made up to 1000m1with distilled water as 200mM stock buffer. 72 g NaCl was added to 200ml of the stuck buffer and volume was adjustedto 7800m1with distilled water(DW).
Gel buffer for Mancini plate.
A. 85 g NaCl, 3.75 g Sodium Barbitone and 5.75 g Barbihiric acid were dissolvedin 1500 ml distilled water and pH was adjustedto 7.4 using 15 ml ION NaOH. The volume was adjusted to 2000m1using distilled water as stock Kabet.
B. 0.2 M EDTA pH 7.6.
40m1of A and 50 ml of B added to 160m1distilled water.
Appendix B
Sample buffer
Distilled Water 4.Oml
0.5M Tris - Hc1 pH6.8 1.Om1
Glycerol 0.8ml
10% (W/V)SDS 1.6m1
142 Mercaptoethanol 0.4m1
0.05% (W/V) Bromophenol blue 0.4ml
Acrylamide 30%
60 gram acrylamide
1.6 gram NN'- Methylenebisacrylamide
make up to 200m1with D. W.
10% SDS
10 gram SDS in D. W. with gentle stirring and bring to 100m1with D. W.
Ammonium persulfate (AMPS)
0.05 in 5m1D. W. gram .
Stacking gel buffer 0.5 M Tris - HCI, pH 6.8
6 gram Tris base
60m1D. W.
Adjust to pH 6.8 with 1N HC1.Made to 100m1with
D. W.
Separating gel buffer 1.5 M Tris - HCI, pH 8.8
54.45g Tris base (18.15g / 100m1)
150m1D. W.
Adjusted to pH 8.8 with 1N HCI. madeup to 300m1
with D. W.
Electrode buffer pH8.3
Tris base 9g
143 Glycine 43.2g
SDS 3g
Adjusted to 3 litres with D. W.
Stain (working solution)
62.5 (0.125% ml stock stain Coomassie blue R-250), 250 ml methanol (50% methanol) and
50 ml (10% acetic acid) were used and madeup to 500m1with H2O.
TBS
4.84g Tris
58.48g NaCl
made up in 2 litres D. W. and pH adjustedto 7.5.
Separating gel preparation 0.375M Tris, pH 8.8 for 10% gel
Acrylamide stock 16.7m1
Tris buffer 1.5 M, pH8.8 8.4m1
D. W. 15.9m1
Degassedfor 20 min.
10% SDS 417 µ1
TEMED 25 µ1
AMPS 208 µl
Stacking gel preparation 4.0% gel, 0.125Tris, pH 6.8 for 10% gel
Acrylamide stock 2.9m1
Tris buffer 0.5 M, pH 6.8 1.67m1
144 D. W. 12m1
Degassedfor 20 min.
10% SDS 167 µl
TEMED 16.6 µl
ANTS 83.4 µ1.
145 Publications arising from this work
1" Jalali GR, Rezai A, Underwood JL, Mowbray JF, Surridge SH, Allen WR & Mathias S.
An 80-kDa Syncytiotrophoblast Alloantigen Bound to Maternal Alloantibody in Term
Placenta. AJRI, 1995; 33: 213 - 220.
2. Mathias S, Allen WR, Rezai A, Jalali GR, Underwood JL & Mowbray. Maternal
Immunoglobulin Equine Placenta. on the Biol Reprod Mono I, 1995; 11 - IT
3. Rezai A, Underwood JL, Jalali GR, Mathias S, Allen WR & Mowbray JF. Anomalous
Inheritance of a Paternally derived Trophoblast Antigen. AJRI in press.
146