W. A. Hemmings

The Relative Transmission of the Fractions of Papain Hydrolyzed Homologous

Molecules of rabbit γ-globulin are split into three nearly equal pieces by papain. Trace labelled with 131I these three fractions are transmitted by the yolk-sac to the circulation of the 24-day rabbit foetus in significantly different quantities. Fraction III is transmitted nearly as readily as whole γ-globulin, whereas fractions II and I are transmitted only 1/5 and 1/10 as readily, respectively, as fraction III. It is suggested that fraction III, which contains most of the antigenic groups of the original molecules, also has the configuration recognized by the cells as homologous γ-globulin.

\gamma

It has long been known that maternal circulating antibodies pass into the foetal blood in rabbits during the latter half of pregnancy. The allanto-chorionic placenta has been assumed to be the site of this transference, the number of tissues separating the two blood streams being reduced to a minimum in rabbits at these stages. It was shown in a recent paper that, at a stage before the establishment of the embryonic circulation, maternal circulating antibodies pass the bilaminar omphalopleur into the yolk-sac cavity. It is shown in this paper that in 24-day embryos antibodies pass from the maternal circulation by way of the uterine lumen and the yolk-sac splanchnopleur into the foetal vitelline circulation, and do not pass by way of the allanto-chorionic placenta. The method employed involved injection of immune rabbit serum either into the uterine lumen or the maternal blood and interruption of the foetal vitelline circulation of some of the embryos by ligaturing the yolk-sac stalk.

-globulin from the Uterine Cavity to the Foetal Circulation in the Rabbit

Active immunity to Brucella abortus was induced in adult female rabbits. They were mated a week after the last injection of antigen and were killed and the yolk-sac contents of the embryos tested for agglutinins 8½ days after copulation. Specific agglutinins were found to be present in the yolk-sac contents in all cases. The titre varied significantly from embryo to embryo in the same litter, and was in some as high as that in the maternal serum at the time of killing. Passive immunity to Br. abortus was imparted to female rabbits 7 to 9 days pregnant by intravenous injection of immune serum of high titre. The rabbits were killed and the yolk-sac fluid of the embryos tested for agglutinins 10 to 17 hr. after injection. Specific agglutinins were present in most of the embryos from five of the six rabbits injected before 8 days post-coitum. All the embryos in the sixth rabbit were regressing. Specific agglutinins were not found in any of the embryos from two rabbits injected after 9 days post-coitum, by which time the yolk-sac fluid has ceased to increase in volume. Positive results were obtained both when rabbit and bovine immune sera were used. Active immunity to Br. abortus was induced in pregnant rabbits by injections beginning after the 15th day post-coitum. The serum of the newborn young, removed from their immune mothers before they had suckled, was tested and specific agglutinins were found to be present with a titre corresponding to that of the maternal serum. It was concluded that agglutinins, whether actively or passively acquired, pass freely from the maternal circulation into the yolk-sacs of 7- and 8-day rabbit embryos. This constitutes a delicate test of the passage of protein without alteration through the yolk-sac wall. The yolk-sac wall does not appear to be selective, since it is at least as permeable to foreign proteins as it is to those of maternal origin. Agglutinins pass from the maternal circulation into the embryo after the disappearance of the bilaminar wall of the yolk-sac also, either by way of the yolk-sac splanchnopleur or the allantochorionic placenta or both. The bearing of these results on current theories of placental permeability are discussed.

The Route of Antibodies passing from the Maternal to the Foetal Circulation in Rabbits.

Antibodies pass from the uterine lumen of pregnant rabbits into the foetal body fluids. It has been shown that antibodies formed in rabbits enter the foetal circulation by this route much more readily than those formed in horses or cows. It is the intention of this paper to investigate further this selective admission to the foetal circulation, to determine if there is a differential selection of heterologous antibodies according to the species in which they were produced and if this selectivity changes with foetal age. Antitoxins were employed throughout and were prepared in rabbit, man, guinea-pig, dog, horse and cow, against diphtheria or tetanus toxin or Clostridium welchii α-toxin. Mixtures of sera from two or three of these species, each serum containing a different antitoxin, were administered so far as possible. The antitoxin concentrations observed in the experimental samples were corrected to unit concentration in the serum administered, the term concentration quotient (c.Q.) being used for this quantity. Measured in this way the entry of antitoxin to the foetal serum varied with the species in which the antitoxin was produced; rabbit, man, guinea-pig, dog, horse and cow forming a descending series in that order. The entry of antitoxin into the amniotic fluid, or the foetal stomach contents derived from it, does not vary with the species in which the antitoxin was produced. This is so also for the maternal serum. Thus the species in which an antitoxin is produced affects the concentration at which it will appear in the foetal serum but not that at which it will appear in the other foetal body fluids or in the maternal serum. The entry of antitoxin prepared in rabbits to the foetal circulation increases with foetal age from 20 to 26 days after copulation and decreases thereafter.

The Passage of Antibodies from the Maternal Circulation into the Embryo in Rabbits

The yolk-sac splanchnopleur of 24-day rabbit embryos freely admits to the foetal circulation, antibodies prepared in the rabbit whereas antibodies prepared in cattle or horses are almost, but not entirely, excluded. This selectivity appears to be independent of the molecular dimensions of the antibody particles, for both rabbit agglutinins and haemolysins are admitted equally freely, whereas both equine antitoxins and bovine agglutinins are equally retarded. Selection appears to depend on the species-specific origin of the antibody molecules themselves, being independent of the other species-specific characters of the rabbit, bovine and equine sera employed. The selectivity of the yolk-sac splanchnopleur as between rabbit and bovine agglutinins is relatively slight at 20 days post-coitum but develops progressively thereafter and is marked at 24 days post-coitum. Individual embryos, even in the same litter, vary significantly in their capacity to admit ungulate antibodies. The theoretical significance of the selectivity of the foetal splanchnopleur is discussed in relation to passive anaphylactic sensitization. The technique of direct injection of immune sera into the lumen of the pregnant uterus was employed. The antisera used throughout were the natural sera separated from the blood of immunized animals not having been subjected to any procedures for their refinement, purification or concentration.

Selection of antitoxins by the foetal membranes of rabbits.

The sera of non-pregnant adult rabbits which had been hyperimmunized to Brucella abortus antigen, the sera of pregnant female rabbits which had not been immunized, and the sera, exocoelomic fluids, amniotic fluids and stomach contents of 25-day-old foetal rabbits were examined electrophoretically and ultracentrifugally. The serum of pregnant rabbits differed both in total protein concentration and in the proportions of the components from that of non-pregnant hyperimmunized rabbits. The foetal sera contained components corresponding to albumin, α-, β- and γ-globulin, but the proportions of these components, as well as the total protein concentrations, differed widely from those of the sera of both pregnant and non-pregnant adults. Foetal exocoelomic fluid, amniotic fluid and stomach contents contain similar electrophoretic components and resemble each other closely in the proportions of the components, though differing in the total concentrations. The components resemble those of sera in mobility, but the proportions of the components differ widely from those of the sera, both foetal and adult. Various experimental procedures to which rabbits were subjected resulted, after 24 h, in a significant increase in the total protein concentrations, without any corresponding change in the proportions of the components, of the foetal sera. No corresponding changes were detected in the maternal sera. The effect on the sera could be explained by withdrawal of water alone from the foetal circulation. Using antibodies as markers, it was shown that both β- and γ-globulin enter the foetal circulation from immune rabbit serum injected into the uterine lumen. The importance of the contribution of maternal serum proteins to the foetus is discussed in the light of the immunological and electrophoretic results.

The Selective Admission of Antibodies to the Foetus by the Yolk-Sac Splanchnopleur in Rabbits

Natural and pepsin-digested rabbit antitoxins were compared with respect to their transmission to the foetal circulation in rabbits. The experiments were so designed that the foetuses were exposed simultaneously to the two antitoxins in rabbit serum injected into the uterine cavity. The natural antitoxin was transmitted readily, but the pepsin-digested antitoxin was not transmitted at the lowest concentrations tested, so that concentration quotients for pepsin-digested antitoxin ranged from < 1/6 to < 1/30 of those for natural antitoxin. It is concluded that the pepsinized moiety of an homologous antitoxin molecule is transmitted less readily than the whole.

Electrophoretic studies of serum proteins of foetal rabbits

Bovine γ-globulin, trace-labelled with 131I, reaches the circulation in substantial amounts although bacterial agglutinins from bovine antisera appear to be completely excluded. Thus when the bovine globulin is both isotopically labelled and immune, the labelled protein which reaches the circulation is still precipitable by antisera to bovine globulin but is without antibody activity. When the labelled bovine γ-globulin that has reached the circulation from the gut of the young rat is fed a second time to another young rat it enters the circulation in relatively larger amounts. Such increase in the relative amounts absorbed is not produced by dilution of the original bovine globulin with young rat serum nor by intravenous screening of this globulin in young rats and must be attributed to the previous passage through the gut wall It is thus confirmatory evidence that such passage effects a change in the γ-globulin fed.

The relative transmission of natural and pepsin-refined homologous antitoxin from the uterine cavity to the foetal circulation in the rabbit.

Antibodies can be detected in the amniotic fluid and in stomach contents of rabbit foetuses exposed to immune serum following injection into the uterine lumen or into the maternal circulation. It is shown that the positive reactions obtained with foetal stomach contents were due to the presence of specific antibodies derived from the immune sera to which the foetuses were exposed. By means of injections of immune rabbit and bovine sera into the embryonic membranes of 24-day rabbit foetuses, it was demonstrated that the antibodies in the stomach contents are derived from the amniotic fluid and not from the foetal circulation. There is a pronounced tendency for antibodies to be concentrated in the stomach, concentrations of 32 and 64 times the titre of the amniotic fluid being observed. Although antibodies of rabbit origin readily pass through the yolk-sac splanchnopleur into the foetal circulation, they do not do so through the stomach wall in detectable amounts.

Changes in

Antitoxins were employed to measure the relative rates of entry of antibodies into the foetal circulation, and into the amniotic fluid, from the uterine lumen in rabbits on the 24th day of gestation. Antitoxic sera prepared in rabbits, cattle and horses against diphtheria toxin, tetanus toxin and Clostridium welchii α-toxin were used, since tests for antitoxins are both sensitive and quantitative. These sera, each immune to one antigen, were mixed and the mixture was injected into the uterine lumen. Analysis of the relative concentrations attained in the foetal sera and amniotic fluids showed that whereas rabbit antitoxins entered the foetal blood at a rate at least fifty times greater than bovine or equine antitoxins, all three antitoxins entered the amniotic fluid at almost identical rates. The concentrations attained in the substance of the membranes traversed provide evidence suggesting the operation of at least two distinct mechanisms. The entry of rabbit antibodies into the foetal circulation may depend on an active process of absorption and secretion by the cells. The entry of both homologous and heterologous antibodies into the amniotic fluid and of heterologous antibodies into the foetal blood, on the other hand, may depend on a process of ‘seepage’ between the cells.