Lens antibodies and eye development. Jan hangman and Harry Maisel

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1 Lens antibodies and eye development Jan hangman and Harry Maisel When 32 hour chick embryos were treated with different lens antibodies, cellular degeneration and gross abnormalities, such as anencephalus, rachischisis, and anophthalmia, were observed only in embryos treated with antisera prepared against total lens and alpha crystallin, but not in those treated with beta or gamma crystallin antiserum. Since the restdts of treatment with total lens and alpha crystallin antibodies were identical in nature, it is concluded that the antibody effect is due exclusively to the interaction of alpha crystallin antibodies and their corresponding cellular anitgens. The cellular degeneration and gross abnormalities were restricted to the brain and eye, that is, those organs in which the presence of lens antigens was previously demonstrated by means of immunologic and fluorescent antibody techniques. None of the other tissues and organs in the abnormal embryos showed any effect of antibody treatment, thus indicating a high specificity of the alpha crystallin antigen-antibody reaction. Abnormalities found in the 24 hour series were identical to those obtained in the 32 hour series, indicating that in both cases the alpha crystallin antibodies had interfered with the same developmental processes. Treatment of 42 hour embryos was ineffective, suggesting that at this stage of development the antigen-antibody interaction either has become impossible or is no longer able to interfere with morphologic development. Since in many of the grossly abnormal embryos no indication of cellular degeneration was found, it seems likely that an antigen-antibody interaction does not necessarily lead to cellular degeneration and necrosis, but may also interfere with cell life in a less damaging manner, such as inhibition of growth and rechanneling of normal differentiation capacities. When rabbits were immunized with chick lens extracts before and during pregnancy, lens antibodies were demonstrated in the maternal circulation throughout pregnancy, in the yolk-sac fluid at the tenth day of development, and in the fetal circidation at the twenty-eighth day of development, by means of immunologic methods. None of the treated rabbit fetuses, however, showed any abnormalities. I t has recently been suggested that lens antibodies circulating in the maternal bloodstream may interfere with lens development in the mammalian embryo and cause cataracts based on an antigen-antibody interaction. 14 Even more surprising is the observation of Barber, Willis, and From the Department of Anatomy, McGill University, Montreal, Quebec. This work was supported by a Fight for Sight Research Fellowship from the National Council to Combat Blindness, Inc., New York, to one of us (H. M.), and by grants from the National Research Council and the National Cancer Institute of Canada. 396 Afeman, 1 who immunized mice against brain extract and found congenital lens abnormalities as a result of this treatment. Careful analysis of these and other experiments with tissue antibodies 3 ' G shows that in some experiments the actual presence of circulating antibodies has not been demonstrated, whereas in others degeneration was found in tissues and organs which do not contain antigenic components corresponding to the circulating antibodies at any stage of development. It is thus evident that the results obtained in these experiments are difficult to explain in a logical manner. Since a careful analysis of an antigenantibody interaction in mammalian em-

2 Volume 1 Number 3 Lens antibodies and eye development 39 bryos is exceedingly difficult, it was decided first to test this interaction in the chick embryo. Hence, 24, 32, and 42 hour chick embryos were treated with antisera prepared against total chick lens extract and with antibodies prepared against the isolated lens proteins, alpha, beta, and gamma crystallin. In the second experiment, rabbits were immunized with chick lens extract before and during pregnancy and the effect of this treatment on the embryos was analyzed. Materials and methods White Leghorn eggs incubated at 38 C. for 24, 32, or 42 hours were cleaned with 0 per cent alcohol. After a small hole was punctured in both ends of the egg, an opening was sawed in the shell and the membrane lifted to expose the embryo. Whenever the embryo remained attached to the shell membrane or was not located directly under the opening, the egg was discarded. Subsequently, 0.1 ml. of antiserum was deposited over the embryo. The openings in the top and at both ends were then closed with cellophane tape, sealed with paraffin, and the eggs incubated for an additional 50 to 0 hour period. After this period, the embryos were removed from the eggs and examined under the dissecting microscope for the presence of abnormalities. Deformed embryos were fixed in Bouin's fluid, sectioned, and stained with hematoxylin and eosin for histologic examination. Preparation of antibodies. A 25 per cent adult lens extract suspended in Freund's adjuvant in a ratio of 3:2 was injected subcutaneously into a number of rabbits in the amount of 1 ml. at each of 5 widely separated sites. This procedure was repeated 4 to 10 times at weekly intervals. The serum of the rabbits was obtained 14 days after the last injection. Alpha, beta, and gamma crystallin were isolated by running adult lens extract containing 15 mg. protein per milliliter in a Spinco Model CP apparatus for continuous flow electrophoresis. The separation was made at 4 C. in Veronal buffer at ph 8.6, ionic strength 0.02, and current 60 Ma. The fractions were collected, dialyzed, concentrated to a protein value of 35 mg. per cent, and tested for homogeneity in the ultracentrifuge and by the agar-diffusion method. The isolated fractions were suspended in Freund's adjuvant and injected into rabbits as oudined above. The antisera obtained are referred to as alpha, beta, and gamma crystallin antisera. In some experiments the chick embryos were treated with complete antiserum, whereas in others only the gamma globulin fraction was used. This fraction was prepared by mixing the antiserum with an equal amount of saturated ammonium sulfate. After leaving the mixture overnight in the cold, the precipitate obtained was suspended in distilled water and dialyzed against 0.9 per cent saline, until the dialysate was free of ammonium sulfate. The gamma globulin solution was then adjusted to a protein content of 20 mg. per milliliter. In the second experiment, young adult rabbits were injected intravenously with 10 per cent chick lens extract 3 to 4 weeks before the onset of pregnancy. When the amount of circulating lens antibodies was sufficientiy high (titer 1:10,000), the rabbits were fertilized. A number of these animals were operated on at the tenth day of pregnancy, the uterus exposed and quickly frozen with carbon dioxide. In this manner, it was possible to peel off the uterine wall and tissues surrounding the yolk sac and to collect the yolk-sac fluid as a spherule of ice. Subsequently, the concentration and number of lens antibodies in the yolk-sac fluid were determined by means of Boyd's- precipitin technique and OuchterlonyV 5-1G double diffusion method. In another series of rabbits, the fetuses were removed at the twenty-eighth day of pregnancy; fetal blood and amnionic fluid were collected and examined for the presence of antibodies. Simultaneously, the eyes of the fetuses were removed, fixed in Bouin-Hollande, sectioned, stained, and examined for the presence of eye abnormalities. Results The influence of lens antibodies on chick embryos. Although, sham operation, treatment with Earle's solution, or normal serum did not affect the development of the 32 hour chick embryos, treatment with lens antibodies either resulted in death of the embryo or caused degeneration of the brain, retina, iris, and lens. In addition, it was observed that a number of embryos showed gross abnormalities such as anencephalus, rachiscliisis, cyclopia, and vaiious degrees of anophthalmia without any sign of degeneration. In the series of chicks treated with undiluted antiserum, the cytotoxic effect was usually of such a serious nature that the embryos died shortly after the beginning of the treatment. In those treated with diluted antisera, it was noticed that the degeneration was located more on the left

3 398 hangman and Maisel Investigative Ophthalmology June 1962 Table I. Effect of total lens antiserum on 32 hour chick embryos Treatment Evibryos treated Embryos surviving after 0 hr. treatment No. % Surviving embryos with abnormalities No. % Sham operation Earle's solution Normal rabbit serum (undiluted) Lens antiserum (undiluted) Lens antiserum (diluted 1:1) Lens antiserum (diluted 1:2) Table II. Effect of antisera prepared against isolated alpha, beta, and gamma crystallin on 32 hour chick embryos Treatment Normal rabbit serum (undiluted) Alpha crystallin antiserum Beta crystallin antiserum Camma crystallin antiserum Embryos treated Embryos surviving after 0 hr. treatment No % Surviving embryos with abnormalities No. % Table III. Effect of antisera prepared against isolated alpha, beta, and gamma crystallin on 24 hour chick embryos Treatment Embryos treated Embryos surviving after 0 hr. treatment No. Surviving embryos with abnormalities No. Sham operation Earle's solution Normal rabbit serum Alpha crystallin antiserum Beta crystallin antiserum Gamma crystallin antiserum Table IV. Effect of antisera prepared against isolated alpha, beta, and gamma crystallin on 42 hour chick embryos Treatment Embryos treated Embryos surviving after 50 hr. treatment No. Surviving embryos xoith abnormalities No. Sham operation Earle's solution Normal rabbit serum Alpha crystallin antiserum Beta crystallin antiserum Gamma crystallin antiserum

4 Volume 1 Number 3 Lens antibodies and eye development 399 than on the right side of the heada fact attributed to the 90 anticlockwise rotation of the embryo shortly after the beginning of the treatment. Since lens antiserum used in the first experiment contained antibodies against all lens proteins, in the following series the embryos were treated with antisera prepared against isolated alpha, beta, and gamma crystallin. The results (Table II) clearly indicate that alpha crystallin antiserum is responsible for the production of abnormalities, whereas beta and gamma crystallin antibodies do not interfere with embryonic development. The abnormalities found in this series of experiments were similar to those produced by total lens antiserum. Table III shows the effect of antisera prepared against isolated alpha, beta, and gamma crystallin on 24 hour embryos. Despite the difference in age from that of the embryos of the previous series, it is evident that, again, the alpha crystallin antibodies are responsible for the high rate of death and the resulting abnormalities in the survivors. Since the abnormalities produced in the 24 hour series were similar to those in the 32 hour series, it is suggested that the antibodies in both cases interfere with the same developmental processes. The treatment of 42 hour embryos (Table IV) was ineffective. Despite the presence of alpha and beta crystallin molecules in various organs of the embryo during the treatment period, no anomalies were seen. Histologic observations. Degeneration and necrosis in the abnormal embryos appeared to be restricted to the tissue of the eye and brain. Although in the most seriously affected cases the left side of the head showed complete necrosis (Fig. 1), frequently the degeneration was found to be localized in the nervous retina only (Figs. 2 and 3). It was surprising to note that in these cases the central part of the nervous retina was degenerated, whereas the peripheral parts were completely unaffected. Fig. 1. Section through cephalic end of 100 hour chick embryo treated with undiluted lens antiserum at 32 hours of incubation. All tissues of the left eye are degenerated, whereas the right eye is normal. Note extensive necrosis of brain tissue. (x80.) With regard to the lens, it was observed that in some specimens this organ was completely degenerated (Fig. 4), and in others the cytotoxic effect was restricted only to the anterior or posterior pole. In general, however, the lens appeared to be less frequently affected than the nervous layer of the retina. In addition to these cytotoxic manifestations of the antibody effect, it was surprising to notice that many grossly abnormal embryos showed no sign of cellular degeneration or necrosis. In cases of anophthalmia, it was observed that the optic vesicle either had completely failed to develop or had formed but failed to contact the surface ectoderm. A further example of this noncytotoxic effect was seen in specimens in which the optic vesicle had completely developed, had subsequently induced a lens, but had then failed to invaginate (Fig. 5). Upon further examination of these cases, it was found that the prospective nervous layer of the

5 400 hangman and Maisel Investigative Ophthalmology June 1962 Fig. 2. Schematic representation of degeneration of nervous retina of 90 hour embryo treated with alpha crystallin antisemm at the 24 hour stage. Degeneration is restricted to central part of nervous retina and has occurred shortly after lens induction. Fig. 3. Schematic representation of degeneration in nervous retina of 96 hour embryo, treated with alpha crystallin antiserum at the 32 hour stage. Degeneration is restricted to central part of nervous retina and has occurred after complete invagination of optic vesicle. retina did not show any mitotic activity, but had then formed pigment granules (Fig. 6). The influence of lens antibodies on rabbit embryos. Table V represents the data found by testing yolk-sac fluid and maternal serum for the presence of lens antibodies on the tenth day of pregnancy. Although the titer of lens antibodies circulating in the rabbit blood stream varied from 1:5,000 to 1:12,800, the yolk-sac fluid showed a titer varying from 1:3,200 to 1:12,800a fact which indicates that the lens antibodies had penetrated into the yolk-sac fluid in a concentration almost equal to that in the maternal serum. By testing yolk-sac fluid by the agar-diffusion method, 6 to 8 precipitin bands became visible, indicating that substantial amounts of 6 to 8 different lens antibodies were present in the yolk sac. Similarly, maternal serum showed the presence of 6 to 9 lens antibodies. When, on the twenty-eighth day of pregnancy, the titer of the lens antibodies was determined, it was found to vary from 1:6,400 to 1:12,800. In the fetal blood, however, the concentration of lens antibodies was low (1:640) or nonexistent. No lens antibodies were detected in the amnionic fluid. Histologic examination of the fetal eyes did not reveal any lens abnormalities. It may thus be concluded that despite the presence of chick lens antibodies in the maternal blood stream throughout pregnancy, in the yolk-sac fluid at the tenth day of embryonic development, and in the fetal blood stream at the end of pregnancy the antibodies did not interfere with lens development in the embryos. Discussion The abnormalities observed in the chick embryos are restricted to the tissues of the eye and brain, that is, those organs and tissues in which the presence of lens antigens has been demonstrated previously by

6 Volume 1 Number 3 Lens antibodies and eye development 401 means of fluorescent antibody and immunologic techniques. 5 ' 10 > ]2-13 Indeed, histologic examination of the other tissues and organs in abnormal embryos revealed that none showed any sign of degeneration or gross morphologic change. Moreover, since abnormalities were found predominantly in the alpha crystallin antiserum group, it appears that the antibody effect is principally due to the interaction of alpha crystallin antibodies and their corresponding cellular antigens and not to the action of gamma and beta crystallin antibodies. This is surprising because substantial amounts of beta crystallin are present in the cells of the invaginating lens placode during the period of treatment with beta crystallin antiserum. 12 That gamma crystallin antibodies were ineffective was more or less anticipated since the presence of these molecules cannot be demonstrated in the embryo until the tenth day of development. The question therefore arises of why alpha crystallin molecules are susceptible to antibody action, whereas those of beta crystallin are not. With regard to this problem a number of possibilities has to be considered: First, do the antibodies against alpha and beta crystallin have equal opportunity to penetrate into the direct environment of the cells containing their corresponding antigens? In 24 and 32 hour embryos, alpha crystallin antibodies can easily reach the cells of the optic vesicle and brain by way of the optic stalk and the wide-open neural groove. Similarly, beta crystallin antibodies will not have any difficulty in reaching the cells of the invaginating lens placode containing their corresponding antigens since these are located on the surface of the embryo. Hence, there appears to be nothing to prevent either antibody from penetrating into the immediate vicinity of the antigen-containing cells. Another factor to be considered is the concentration of the antiserum. Since dilution of antiserum considerably reduces the antibody effect (Table I), it may be that the titer of beta crystallin antibodies is too low to produce any morphologic effect. When, however, the concentration f. Fig. 4. Section through lens of 96 hour embryo treated with undiluted lens antiserum. This represents one of the few cases in which the lens was seriously affected by lens antibody treatment. Fig. 5. Cross section through eye of 90 hour embryo treated with alpha crystallin antiserum at die 32 hour stage. Note epidermis, lens vesicle, optic vesicle which has failed to invaginate, connective tissue, and brain.

7 402 hangman and Maisel Investigative Ophthalmology June 1962 Table V. Titer and number of lens antibodies in maternal serum and yolk-sac fluid at tenth day of pregnancy Rabbits Titer of lens antibodies in maternal serum Precipitin bands in agar plate Titer of lens antibodies in yolk-sac fluid Precipitin bands in agar plate 2 3 1:5,000 1:6, :5,000 a. 1:6,400 b. 1:3,200 c. 1:6,400 d. 1:6, :10,000 1:5, :12,800 9 a. 1:6,400 b. 1:12,800 c. 1:6,400 d. 1:12,800 of alpha and beta crystallin antiserum was compared by means of Boyd's precipitin test, 2 it appeared that the titer of beta crystallin antiserum was almost twice as high as that of alpha antiserum. Consequently, this possibility was abandoned. As a final explanation for the different activities of the two antisera, the location of the antigenic components in the cell was considered. It was thought that alpha crystallin might be located at the surface of the cell, enabling it to react with its environment, whereas, on the other hand, beta crystallin might be located in the interior of the cell, unable to react with its surroundings. Supporting this assumption is the fact that beta crystallin molecules are the important components of the lens fibers, that is, structures which are found in the interior of the cell. Moreover, since the effective components of the antisera are gamma globulins, macromolecules with a high molecular weight and unable to penetrate into the inside of the cell, it seems reasonable to suggest that the antigen-antibody interaction occurs on the surface of the cell. Consequently, it is likely that alpha crystallin is located at the surface of the cell and that the alpha crystallin antigen-antibody reaction is a surface reaction. It is apparent, however, that this reaction is restricted in time to certain stages of development. As soon as the embiyo has passed the 42 hour stage (13 to 15 somites), the antibodies apparently become ineffective (Table IV). This may be due either to the inability of the antibodies to reach the antigen-containing cells or to a change in the cell membrane preventing the antigens from reacting with the antibodies assumed to be at the surface. At the moment, the second possibility seems to us the most likely one. When in previous experiments optic vesicles covered with ectoderm obtained from to 20 somite chick embryos (30 to 50 hours) were explanted in a medium containing lens antiserum, it was found that the antibodies were able to interfere with development only in explants from embryos younger than 16 somites (45 hours), but not thereafter. 10 Hence, despite the presence of antibodies in the medium, explants from embryos older than 16 somites proceeded normally with development. This may indeed be considered as an indication of a Table VI. Titer of lens antibodies in maternal serum and fetal blood at twenty-eighth day of pregnancy Rabbits Titer of lens antibodies in maternal serum 1:12,800 1:6,400 1:12,800 Titer of lens antibodies in fetal blood 1:320 1:640

8 Volume 1 Number 3 change in the cell membrane at approximately the 16 somite stage, rendering the alpha crystallin molecules unable to react with the antibodies. Summarizing thus far, it may be concluded that the effects observed in our experiments result from an antigen-antibody interaction restricted to one particular molecule, occur only during certain stages of development, and in all probability take place at the surface of the cell. With regard to the degeneration and necrosis observed, it was surprising that many of the 32 hour embryos showed these effects only on the left side of the head. Observing the embryos during the treatment, it was noticed that the antiserum, immediately after being administered, spread out toward the surrounding yolk and albumin. As the subcephalic pocket with its elevated crescentic amnionic folds forms a cavity under the head end of the embryo, some of the antiserum would collect in it and the head of the embryo would be partly or completely submerged in antiserum. Synchronously with the flexion of the embryo, rotation along its length axis takes place. This rotation occurs in such a manner that the left side of the embryo becomes located deep in the subcephalic pocket, and the right side is turned up toward the surface. This would imply that a few hours after the beginning of the treatment, the right side of the head would turn up and become located above the antiserum level in the subcephalic pocket. It is thus thought that the rotation of the embryo may explain the frequently observed left-sided necrosis of the head. One of the surprising observations in our experiments is the fact that an antigenantibody interaction apparently does not necessarily lead to cellular degeneration and necrosis, but may interfere with cell life in a less damaging manner, such as by inhibition of growth or rechanneling of the normal differentiation pattern. It is thought that the essential feature of this difference might be found in the concentration of the antibody molecules reacting with the cell. Lens antibodies and eye development 403 Fig. 6. Top shows posterior pole of 96 hour lens. Some of the nuclei show moderate pyknosis. In direct contact with posterior pole of lens is prospective nervous layer of the retina, which has failed to invaginate. This layer shows pigment granules. If the antibodies present in the immediate environment of the cell are insufficient in number to occupy all the available reactive sites of the antigen molecules, then the cell may still be able to survive but unable to continue with its normal morphologic development and function. Another possibility to be considered is the fact that the affected antigen molecule is only one in a group of different molecules, all building up the molecular population of the cell. Depending upon its "essentialness" for the metabolic processes of the cell and its importance in the interaction with other molecules, the antibody effect may then vary to a great extent. Indeed, in the eye a highly diversified picture was seen as the result of the antigen-antibody reaction. Although in some embryos the optic vesicle did not develop fully, resulting in anophthalmia, in other cases the optic vesicle induced a lens but failed thereafter to proliferate and invaginate. In many of these specimens, however, the anterior part of the optic vesicle, destined to become the nervous retina, subsequently differentiated

9 404 Lansman and Maisel Investigative Ophthalmology June 1962 in another direction, thereby forming pigment granules (Fig. 6). It is thought that in these cases the affected antigen is unable to play its normal role in the interaction of molecules, thus permitting the cell to channel its capacities in another direction. When we started to inject our rabbits with chick lens extract, it was thought that these lens antigens were identical to those of the rabbit lens. This was based on the observation of Uhlenhuth, 1 who, by testing bovine lens antiserum with lens extracts from animals of different species, noticed a cross reaction between this antiserum and all lens extracts used. He therefore concluded that the lens antigens in various species are identical. This antigenic identity has since been confirmed repeatedly by other investigators, such as Woods and Burky, 19 van Dold, Flossner, and Hutscher, 18 and several others. Recently developed immunologic procedures, such as the agar-diffusion technique of Ouchterlony, have provided us with new tools to analyze the species specificity of organ and tissue antigens. To determine the number of identical antigens in chick and rabbit lens, therefore, the latter was tested with chick lens antiserum, with the use of the agar-diffusion test. When chick lens extract was tested with chick lens antiserum prepared in the rabbit, 6 to 9 precipitin bands became visible. This indicates that 6 to 9 antibodies against the chick lens were circulating in the rabbit blood and even had penetrated into the yolk-sac fluid. However, to be effective, these antibodies must find corresponding antigens in the lens of the rabbit embryo. This apparently was not the case, because, when rabbit lens extract was tested with chick lens antiserum, only one precipitin band was formed, 13 indicating that rabbit and chicken lens have only one antigenic fraction in common. Further experiments showed that this fraction was alpha crystallin and that it was only partially identical to that of the chick. Hence, of all lens antibodies circulating in the rabbit, only one will be able to interfere with lens formation in the rabbit, whereas the others, having no relation to rabbit lens antigens, are idle. Since the concentration of antibodies is of great importance to the production of abnormalities (Table I), and the titer of the lens antibodies in the fetal circulation was low (1:640), this may imply that the concentration of the specific antibody was too low to effect formation of the embryonic lens in the treated rabbits. By comparison of the antibody treatment in both series of experiments, it is evident that there is an important difference. Chick embiyos were treated with antiserum prepared by injecting chick lens extract into rabbits. The rabbit embryos, however, were treated with chick lens extract likewise prepared in the rabbit. In order to make antibody treatment in both experiments similar, rabbit embryos should be treated with an antiserum prepared by injection of rabbit lens extract into chicken. Surprisingly, this brings us back to the experiments of Guyer and Smith, ' s who immunized rabbits passively with an antiserum prepared by injecting rabbit lens into chicken and observed ocular malformations in the offspring. Huxley and Saunders, 9 however, repeating this experiment, did not succeed in obtaining eye anomalies in mammalian embryos by antibody action. Recently, Miller 14 immunized a large series of rabbits with an antiserum prepared by injecting rabbit lens extracts into chicken. When the eyes of 460 offspring of the treated does were examined, only 4 had eye defects. The latter experiments, therefore, seem to indicate that circulating lens antibodies may interfere with eye development. In general terms, however, considering the low percentage of eye defects obtained in Miller's experiment, there does not seem to be much evidence that lens antibodies can interfere with eye development in mammals. We wish to thank Miss Jane Townsend for the preparation of the histologic sections and technical assistance in the experiments.

10 Volume 1 Number 3 Lens antibodies and eye development 405 REFERENCES 1. Barber, A. N., Willis, J., and Afeman, C: Changes in the lens induced by maternal hypersensitivity in mice 51: 949, Boyd, W. C: Fundamentals of immunology, London, 1956, Interscience Publishers, Ltd. 3. Brent, R. L., Averich, E., and Drapievvski, V. A.: Production of congenital malformations using tissue antibodies. I. Kidney antisera, Proc. Soc. Exper. Biol. & Med. 106: 523, Clarke, W. M., and Fowler, I.: The inhibition of lens-inducing capacity of the optic vesicle with adult lens antisera, Develop. Biol. 2: 155, Fowler, I., and Clarke, W. M.: Development of anterior structures in the chick after direct application of adult lens anitsera, Anat. Rec. 136: 194, Gluecksohn-Waelsch, S.: The effect of maternal immunization against organ tissues on embryonic differentiation in the mouse, J. Embryol. & Exper. Morph. 5: 83, Guyer, M. F., and Smith, E. A.: Studies on cytolysin. I. Some prenatal effects of lens antibodies, J. Exper. Zool. 26: 63, Guyer, M. F., and Smith, E. A.: Further studies on inheritance of eye defects induced in rabbits, J. Exper. Zool. 38: 449, Huxley, J. S., and Saunders, A. M. Carr: Absence of prenatal effects of lens antibodies in rabbits, J. Exper. Biol. 1: 216, Langman, J.: The first appearance of specific antigens during induction of the lens, J. Embryol. & Exper. Morph. : 193, Langman, J.: The effect of lens antiserum on chick embryos, Anat. Rec. 13: 135, Maisel, H., and Langman, J.: An immunoembryological study on the chick lens, J. Embryol. & Exper. Morph. 9: 191, Maisel, H., and Langman, J.: Chick lens proteins in various tissues of the eye and in the lens of animals throughout the vertebrate series, Anat. Rec. 140: 183, Miller, W. J.: Antilens sera as a mutagen in rabbits, J. Exper. Zool. 13: 463, Ouchterlony, O.: Antigen-antibody reactions in gels, Acta path, et microbiol. scandinav. 32: 231, Ouchterlony, O.: Antigen-antibody reactions in gels and the practical application of this phenomenon in the laboratory diagnosis of diphtheria, Thesis, Karolinska Institutet, Stockholm, Uhlenhuth, P. T.: Zu Lehre von der Unterscheidung verschiedener Eiweissarten mit Hilfe spezifischer Sera, Festschrift zum 60. Geburtstage v. Robert Kock, Jena, 1903, Gustave Fischer Verlag, pp Van Dold, H., Flossner, D., and Hutscher, F.: Biologische Untersuchungen iiber die Linsen-Eiweisskorper, Ztschr. Immunitats forschung u. exper. Therap. 46: 36, Woods, A. C, and Burky, E. L.: Lens protein and its fractions. Preparations and imraunological and chemical properties, J. A. M. A. 89: 102, 192.