Biomedical subjects
Publications and source records attributed to W H Stone.
We describe a new antiserum that has the unique ability to distinguish homozygous from heterozygous genotypes in the G blood group system of rhesus monkeys. With this new typing serum (reagent), all 10 possible genotypes in this system can be distinguished and the utility of blood typing for genetic studies has been greatly increased.
Nonhuman primates are excellent animal models for human diseases because of their close relationship to humans. Indeed, comparisons of the chromosomes and DNA homologies between primates and humans testify to the commonality of the genetic material between these phylogenetically related species. Not surprisingly, this close relationship at the genotypic level extends to the phenotypic level. Thus, the patho-physiological responses of humans and nonhuman primates to internal and external insults are remarkably similar. Two types of human diseases for which nonhuman primates are paramount animal models are discussed. One type includes diseases with defined, single agent etiologies and to which all members of the species are genetically susceptible. Examples of these are leprosy, AIDS, hepatitis and Parkinson's disease. A second type represents diseases that have a substantial genetic component, but are multifactorial and are greatly influenced by the environment. Examples of these are diabetes, lymphoma, atherosclerosis, alcoholic cirrhosis and anxiety disorders. Nonhuman primates are also ideally suited to the role of animal models in the new area of human gene therapy. In the future, biomedical research will focus increasingly on genetic manipulations such as the transfer of genes from one individual to another to correct genetic diseases, particularly those diseases caused by single recessive gene defects. Before gene transfers are attempted in humans, they should be done in nonhuman primates. In a real sense, nonhuman primates, as animal models, represent the "step to man."
We have developed a microagglutination test for typing rhesus monkey erythrocytes that is sensitive, accurate and easy to perform. The technique requires only microliter quantities of antiserum and cells, and agglutination is easily detected using an inverted microscope. An advantage of this technique is that the typing plates can be stored at -70 degrees C without loss of activity. The results of typing over 400 rhesus blood samples with this technique were 95% concordant with results using the standard microtitre agglutination technique. Preliminary results indicate that this test is also adaptable to typing human blood.
A colony of fully pedigreed Monodelphis domestica has been used to investigate the maternal-fetal relationship in this unique marsupial species. To determine how immunity is transferred from mothers to young in M. domestica, we hyperimmunized females with sheep red blood cells (SRBC) before and during gestation. Offspring from these females were collected at various times after birth, and saline extracts of the neonates were assayed for hemolysins against SRBC. Antibodies were present in extracts of newborn that had been allowed to suckle their mothers; none were detected in extracts of infants that were not allowed to suckle. Antibodies were present in the milk of immunized mothers, but were not detected in the milk of nonimmunized mothers. The titer of antibodies in the extracts of newborns generally increased proportionately to the time that the newborn had been allowed to suckle. We conclude that the transfer of passive immunity from mothers of M. domestica to their offspring occurs primarily via the milk.
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Hemolytic disease of the newborn does not develop in rhesus monkeys because placentally-transferred maternal antibodies do not induce immune clearance of the newborn's erythrocytes. In an in vitro RBC adherence assay, rhesus peripheral blood monocytes did not bind newborn's RBC which had been coated in utero or in vitro with maternal antibodies. Nevertheless, rhesus phagocytes possess receptors that are specific for the Fc portion of IgC and for the C3b. Using purified human IgG subclasses as inhibitors of RBC adherence, rhesus Fc receptors preferentially bind IgG1 and IgG3. Thus, it may be that maternal antibodies are non-opsonic because they belong to IgG subclasses that do not bind effectively to rhesus Fc receptors. Also, RBC adherence appears to be controlled by the level of antibody coating which in turn is determined by avidity of the antibodies and by the number of RBC membrane determinants. The failure of maternal antibodies to opsonize the newborn's RBC and thus cause hemolytic disease is very likely due to the low avidity of antibodies and to the weak expression of blood group determinants on the membranes of these RBC.
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Hemolytic disease of the newborn has not been observed in rhesus monkeys even though the newborn's erythrocytes may be coated with maternal antibodies. Using a 51chromium-erythrocyte survival assay, we found that maternal antibodies do not mediate immune elimination of newborn's red blood cells. However, certain allogeneic or xenogeneic antibodies mediate clearance via sequestration by the reticuloendothelial system, or by intravascular hemolysis, or by a combination of these. The class of antibody plays a major role in elimination since red cells coated with IgG but not with IgM were rapidly cleared. In addition, the quantity of antibody controls the rate and extent of clearance. A comparison of rhesus alloantisera suggests that coating of multiple antigenic sites is necessary for clearance. Passive immunization with selected high-titered anti-erythrocyte alloantisera can induce some hematologic signs of erythrocyte destruction in newborn monkeys.
Sera from about 1000 cows were tested for cytotoxicity against a panel of up to 100 lymphocyte samples. Cytotoxic antibodies presumably resulting from transplacental immunization of the cow by her calf were found in about 45% of these sera. The antibody titers of sera from parous cows rarely exceed 4(2), some persisted for over one year, but decreased notably at calving. Thirty-five immune sera were also produced by alloimmunization with lymphocytes. They usually reached peak titers of up to 4(4) at 2 or 3 weeks after the initial immunization. Subsequent immunizations produced sera with very high titers but they were much more polyspecific. High-titered antibodies were also produced by skin graft recipients. Useful cytotoxic antibodies were found in 19 of 111 colostrum whey samples. Studies on 13 dam-calf pairs showed that the newborn calf may acquire cytotoxic antibodies from its mother's colostrum, but the only cytotoxic antibodies detectable in this calf's serum are those not directed against its own lymphocyte antigens. It is concluded that efficient lymphocyte typing requires antibodies from a variety of sources.
We describe a technique to produce high-titered bovine lymphocytotoxic antisera using skin implants. The main advantage of this technique is that the skin does not need to be processed prior to implantation and no surgical skill is required. In addition, the skin can be stored for up to 2 weeks and can be shipped to other laboratories without special handling and without loss of immunogenicity.
Detection of linkage between genetic loci in cattle has been hampered by the lack of large full -sib families. A unique source of full-sib families is now available from embryo transplantation. Lymphocytes from six full-sib families, ranging in size from three to seven siblings, were tested for serologically defined BoLA antigens (BoLA-A). In addition, mixed lymphocyte reactivity (MLR) was tested between all paired combinations of cells within each family to distinguish BoLA-D specificities. Serologically identical siblings within each family were reciprocally nonreactive in MLR, and vice versa; thus, no recombinants were detected between the BoLA-A and the BoLA-D loci. Classical genetic linkage analysis revealed that these loci are significantly closer than 11.9 centimorgans.
Bovine histocompatibility antigens cross-react highly with HLA antigens in comparison to their homologues in other nonprimate species. A panel of 34 anti-HLA monoclonal antibodies was tested for binding activity with lymphocytes from five breeds of cattle. Eleven antibodies demonstrated extensive cross-reactivity. Lymphocytes from several species of ungulate and subungulate were then tested with the same panel of antibodies. The buffalo and hyrax also displayed some cross-reactivity, though not as extensive as seen with the cattle lymphocytes. Other ungulates showed essentially no cross-reactivity. The significance of these results is discussed in terms of the structure and evolution of histocompatibility antigens.
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Blood samples were collected from more than 100 animals in each of 2 Spanish cattle breeds (Retinto and De Lidia), 2 Portuguese breeds (Alentejana and Mertolenga), and American Longhorn cattle. All samples for the 4 Iberian breeds were tested for 20 polymorphic systems; American Longhorn were tested for 19 of the 20. For each breed an average inbreeding coefficient was estimated by a comparison of the observed and expected heterozygosity at 7 or 8 codominant systems tested. All breeds had positive values but only 3 breeds had estimates of inbreeding that were statistically significantly different from 0: De Lidia with f = 0.17, Retinto with f = 0.08 and Mertolenga with f = 0.05. The De Lidia breed especially may be suffering from inbreeding depression since this high value is greater than expected if all of the animals were progeny of half-sib matings. Genetic distances were calculated from the gene frequency data on these 5 breeds plus 9 other European breeds. Analyses of these distances show a closely related group of the 4 Iberian breeds and American Longhorn, confirming the close relationships among the Iberian breeds and the Iberian, probably Portuguese, origin of American Longhorn cattle.
There is good evidence that susceptibility to Plasmodium vivax infection and to P. knowlesi erythrocyte invasion is influenced by certain human Duffy (Fy) blood group antigens. Since P. knowlesi readily infects rhesus monkeys (Macaca mulatta), it was not surprising to find an Fy-like antigen on rhesus erythrocytes. Using human Fy antisera in elution and absorption experiments, we found that all 40 rhesus monkeys tested displayed the Fy(a-b+) phenotype. Furthermore, the rhesus Fyb antigen was inactivated by chymotrypsin but not by trypsin, suggesting that it is homologous to the human Fyb antigen. Preliminary serological analyses and enzyme hydrolysis experiments suggest that none of the 13 blood group systems that we have described in rhesus are analogous to the human Fy system. Thus, it appears that there is no Duffy-like polymorphism in rhesus monkeys.
An algorithm is presented for clustering antisera by computer. It has two novel features: the leading serum to which all other sera in the cluster are compared is chosen as the most centrally located serum in the cluster; the similarity between two sera is defined from the 2 X 2 table of serum reactions as s = 2a/(2a + b + c). This similarity index is a better measure of the similarity between two sera than conventional measures of similarity such as the correlation coefficient. Finally, the identification of cluster and serum subsets provides a more complete analysis of cross-reactivity and multispecificity, and suggests which absorptions might yield monospecific typing sera. A computer program which performs this serum cluster analysis is available upon request.
Eleven lymphocyte antigens have been defined in cattle using 81 lymphocytotoxic sera. These sera (typing reagents) were selected from over 1,000 normal sera, 35 alloimmune sera, and 111 samples of colostrum whey. Absorptions revealed that nine of the 11 antigens detected by these reagents are serologically independent. Segregation of the genes controlling these antigens was observed among 470 calves from mating of 56 heterozygous sires and 360 dams, indicating that each of the 11 antigens is controlled by one of 11 alleles at a single autosomal co-dominant locus BoLA-A (Bovine Lymphocyte Antigen, locus A). To determine the relation between BoLA antigens and histocompatibility, skin allografts were exchanged among four unrelated adult cows. Incompatibilities for the SD antigens, but not MLR or blood group antigens, were predictive of early rejection of allografts. Furthermore, graft recipients produced antibodies against the SD antigens of incompatible donors. These results indicate that the BoLA-A locus is part of the major histocompatibility complex (MHC) of cattle.