Sidney Shulman

Studies on thyroid proteins: II. Purification of human thyroid proteins by gel filtration and the isolation of 19-S, 7-S, and 4-S components

Abstract A procedure is described by which the major soluble proteins of normal human thyroid tissue can be separated. Gel filtration on Sephadex G-200 yields three fractions. The first eluted fraction (A) contains samples of well-purified thyroglobulin. Each of these samples also contains varying proportions of the 34-S, 27-S and 12-S thyroid proteins. Some of the samples from Fraction A contain only the 27-S and heavier proteins. The second fraction (B) contains nearly pure 7-S thyroid protein, as judged by ultracentrifugal analysis. By this same criterion, the third fraction (C) has essentially pure 4-S material, designated thyralbumin. The components in Fraction A cannot be further separated by paper curtain electrophoresis. The 7-S protein in Fraction B is, however, separated into nearly equal amounts of two subfractions by curtain electrophoresis. Likewise, the 4-S protein in Fraction C is separated into two nearly equal amounts. These procedures have separated eight major protein fractions in human thyroid extract. Furthermore, one of the 7-S protein subfractions and both of the 4-S subfractions have been shown to contain thyroid-specific antigenic activity.

The allergic response to stinging insects: IV. Cross-reactions between bee, wasp, and yellow jacket

Abstract The cross-reaction between bee and yellow jacket is not detected by tanned cell hemagglutination, inhibition of hemagglutination, or agar gel double diffusion. Only by immunoelectrophoresis is it possible to demonstrate a common antigen contained only in the body of these insects. Bees and wasps share one common antigen which is not present in their venom sacs. Wasps and yellow jackets have two common antigens, one of which is localized in the body and the other in the venom sacs of both insects. The implications of these results in regard to human hypersensitivity to stinging insects is discussed.

Cryo-Immunology A Method of Immunization to Autologous Tissue.

Summary By means of a blind test, in which serum samples from a group of rabbits were relabeled with code designations, it was shown clearly that definite antibody production had occurred as a result of freezing of tissue in the animal. Freezing was done by means of a liquid nitrogen probe and the target tissue was the coagulating gland and seminal vesicle of the urogenital tract. Antibodies were detected by tanned cell hemag-glutination and were found to be present within 7 days after cryosurgery. No antibody was detected in any of 15 sera from 5 control animals, whereas 6 out of 7 rabbits exposed to freezing showed positive results. Titers as high as 1:4096 were obtained, and there was a progressive increase with time.

The allergic response to stinging insects: I. Preparation of extracts and their biochemical characterization

Abstract Potent allergenic extracts of three different species of stinging insects were prepared by a simple procedure. The types of Hymenoptera studied were the bee ( Apis mellifera ), the wasp ( Polistes exclamans ), and the yellow jacket ( Vespula pennsylvanicus ), and extracts of each were prepared from the whole body, from the sacless body, and from the venom sac. In order to facilitate the routine determination of protein concentration, light absorption methods were developed and an extinction coefficient for each type of extract was determined for both ultraviolet absorption, at 280 mμ, and for the biuret reaction at 540 mμ. The allergenicity of these preparations was determined by direct skin tests. Positive intradermal skin tests were elicited in sensitive patients with as little as a 1:10,000 dilution of the preparation of insect antigen. Most of the patients reacted to more than one type of insect. Commercial preparations gave similar results. Zone electrophoresis, by filter paper strip and by starch block, revealed two or three separate components. It was noted that the best pH for optimal resolution, over the range of 8.6 to 4.5, was 6.5 for bee and wasp and 4.5 for yellow jacket. This method seemed to have limited value for the possibilities of fractionation of these extracts. The extensive heterogeneity of these extracts was better shown by analytical ultracentrifugation. All three types of whole body extracts had characteristic patterns of five homologous sedimenting boundaries, although the proportions varied for each insect. The venom sac extracts showed varying patterns of minor fast components and major slow ones. At least some of the venom constituents seemed to be absent from the whole body extracts. It will require further study to identify any of these components with the allergenic substance.

The allergic response to stinging insects: II. Immunologic studies of human sera from allergic individuals

Abstract Specific antibodies in sera of patients sensitive to stinging insects were demonstrated by two immunologic methods. These were tanned-cell hemagglutination and passive transfer skin testing. Two different groups of patients were clearly demonstrated; one group reacted to only one type of insect, while the other group reacted to two or three different types of insects. The interpretation of the apparent cross-reactions was studied by in vivo neutralization experiments. The results indicated that antigens of one of the insects can stimulate the formation of antibodies that also react with constituents in the extracts of one or both of the other insects. The antigens eliciting hypersensitivity were not solely localized in the venom, but were also present in the insect body. The existence of specific venom antigens, in addition to those that are common to the whole body, has also been demonstrated.

The allergic response to stinging insects: III. The specificity of venom sac antigens

Abstract Common antigens were demonstrated in venom sac and sacless insect extracts of hymenoptera by hemmagglutination, gel diffusion, and immunoelectrophoresis. The insects studied were bee, wasp, and yellow jacket. Antigens specific to venom sac extracts of hymenoptera were also demonstrated by the same methods. There is at least one, and possibly two, antigens specific for the venom sac, for each of these insects. The complex antigenic nature of hymenoptera extracts was illustrated by immunoelectrophoresis. These results are discussed in the light of previous studies on human sera of patients sensitive to hymenoptera.

An experimental system for the production of antibodies in response to cryosurgical procedures

Summary Localized freezing of tissue in rabbits by means of a liquid nitrogen-cooled probe has been shown to stimulate antibody formation in certain situations. Successful induction of antibody formation has been achieved in rabbits after intensive but brief freezing of parts of the male accessory glands of reproduction. Positive results were obtained reproducibly with antibody titers that were generally high, as measured by the method of tanned cell hemagglutination. This was the result of an experimental procedure of freezing and additional cooling, in which the coagulating gland was frozen and cooled, followed by further freezing and cooling of seminal vesicle tissue. In comparison, an earlier one-step procedure stimulated lower and less reproducible responses. Temperature profiles (freeze-thaw-time curves) were obtained by feeding the thermocouple outputs into a multichannel recording instrument. Rates of freezing were established by either of two procedures, using a single temperature set point or a stepwise series of several set points. The rates were of the order of 0.1 to 1.0°C per sec. Distinct peculiarities were shown in the nature of the temperature changes with time, indicative of asymmetric freezing. The modified freezing procedure reported here has produced antibody levels of appreciable titer in 7 out of 7 rabbits, as compared with 6 out of 12 rabbits with the use of the previous, simpler procedure. The possible mechanisms that may be responsible for the induction of antibody formation by cryosurgical destruction of tissue are discussed.

The allergic response to stinging insects. VII. Fractionation of whole body and venom sac extracts of yellow jacket

Abstract Extracts of whole body and of venom sac of yellow jackets (Vespula pennsylvanicus) were fractionated by DEAE-cellulose chromatography. Ten fractions were separated from whole body, and seven fractions from venom sac preparations. Only fraction 1 of venom sac was active in direct skin testing. While several fractions of whole body were active by skin testing, only one of them, fraction 1, was significantly active in a patient who was sensitive to yellow jacket only. Other fractions, therefore, reflected the cross-reaction between the different insects. The antigenic heterogeneity of each fraction was analyzed by appropriate rabbit antisera to yellow jacket extracts. Eight of the ten body fractions gave precipitation, as did three of the seven venom sac fractions. They variously gave one or two lines of precipitation. In addition, fractions 6, 7, and 8 of whole body showed, by testing with rabbit antisera, common reactions with wasp and bee. An analysis of the studies comparing the findings of bee, wasp, and yellow jacket extracts and fractions with each other is presented at the end of this paper.

THE THYROID GLAND AS SOURCE AND TARGET IN AUTOSENSITIZATION

The proteins of the thyroid gland have been studied for several reasons. In addition to their attractiveness to endocrinologists and biochemists, they possess unusually high interest for immunologists. These antigens were first studied because they exhibited in an unambiguous fashion the phenomenon of organ specificity.*pz For example, if extracts of various hog organs are tested with rabbit antiserum elicited by thyroid extract, it is found with many of these antisera that thyroid extract gives a positive result at a much greater dilution than do other organ preparation^.^ More recently, the thyroid has acquired even greater interest with the discovery of its role in thyroiditis, and its capability of actually eliciting autountibodies. From experimental studies of animals it was established that circulating antibodies could be demonstrated by several techniques after suitable injection of thyroid extract from within the species or even from the same animal.4 This condition was frequently accompanied or followed by extensive degenerative changes in the thyroid tissuechanges that, in fact, resembled human thyroiditis.6 Subsequent studies on a large number of human sera revealed a very high degree of correlation between the occurrence of thyroid antibodies in people with chronic nonspecific thyroiditis and its absence in other conditions or in normal serum.6,7 To learn more about the character of this autosensitization response and the nature of the autoantibody, we have again immunized a group of rabbits and followed the serum changes with time. We present some of the results in this report. At the same time it has also seemed to be of importance to learn more about the antigen or antigens involved, and for this and other reasons we have been studying newer methods of purification, using hog thyroid extract as test material. Some of these results are also presented.

Immunologic studies of Caddis fly: V. Cross-reaction with other insects

Abstract 1.1. A purified fraction of Caddis fly extract has been shown to consist of two components, which can be separated in electrophoresis, but not in the ultracentrifuge. The electrophoretic mobilities are −7.7 and −6.1 × 10 −5 cm. 2 sec. −1 volt −1 . The sedimentation coefficient is 0.72 S. 2.2. The estimated molecular weight for each component is 3000, assuming the particles to be spherical and unhydrated. 3.3. Extinction coefficients were determined for this preparation and found to be 35.2 by ultraviolet (280 mμ) and 1.3 by biuret (540 mμ). 4.4. From the nitrogen content, spectral properties, hexose determination, and other data, it is concluded that the allergen is a peptide or glycopeptide.