Production Of Antitoxin Research Articles

The influence of the thyroid gland on antitoxin production in different species.

1. The influence of the thyroid gland on the production of antitoxins to two alum-precipitated bacterial exotoxins has been investigated in different animal species.2. Rats, rabbits and mice produce more circulating antitoxin when thyroidectomized, and less when made hyperthyroid by thyroxine administration, than control animals.3. Guinea-pigs produce slightly less antitoxin when thyroidectomized, and markedly more when thyroxine treated, than control animals.We are grateful to Dr J. S. F. Niven for histological examination of tissues, and to Miss M. V. Mussett, B.Sc., for statistical analyses. The alum-precipitated tetanus toxoid XW 2044, and tetanus toxin XW 1322, were kindly supplied by Miss Mollie Barr, M.Sc., of the Wellcome Research Laboratories, Beckenham, Kent, and we are also grateful to Mr P. Rodican of the Lister Institute, Elstree, for a supply of tetanus toxin 829.

In Vitro Production of Diphtheria Antitoxin by Tissues of Immunized Animals

Summary Addition to various basal synthetic media of dialyzed or undialyzed homologous or heterologous serum, serum ultrafiltrate, chick embryo extract, purines, pyrimidines, vitamins, coenzymes, lipids and carbohydrates did not consistently enhance in vitro antibody synthesis by lymphoid tissues from immunized rabbits. The failure of paraamino benzoic acid and thiamin to produce an effect on these short-term experiments was contrasted with their effects in longer-term cultures and an explanation of the different findings advanced. It was found that optimal concentrations of certain amino acids must be present in the medium for maximal in vitro antibody synthesis and incorporation of radioactive amino acids into antibody. These data provide further evidence for the importance of amino acids as precursors of antibodies. The complete function of the amino acids in the medium is unknown because the medium containing the smallest concentration of each amino acid still contains 5–10 times the amount of each acid required for antibody synthesis. The failure of previous investigators to observe an amino acid requirement for incorporation of radioactive amino acids into antibody was attributed to their use of much larger amounts of tissue than in the present study. As evidenced by the incorporation data, antibody synthesis in vitro is a rapid process, which is in accord with the findings of other studies. These observations also indicate that antibody is rapidly secreted into the medium. As a result of this study, a completely synthetic medium has been developed which promotes appreciable antibody synthesis and incorporation of radioactive amino acids into antibody.

The Depressant Effect of Continuous Cobalt-60 Radiation on the Secondary Tetanus Antitoxin Response in Mice

The radiosensitivity of the secondary tetanus antitoxin response in mice was demonstrated after rather low doses of continuous gamma -radiation given at a dose rate of 4 rep/hr. Accumulated doses of 48 to 288 rep depressed antitoxin formation. Comparable doses of acute gamma radiation did not depress antitoxin production. Acute doses of 350 to 650 rep sharply depressed the secondary antibody response, however. Extended periods of continuous gamma -radiation from 10 to 28 days to accumulated doses of 960 to 2688 rep markedly depressed the secondary antibody response. An accumulated dose of 2688 rep was needed to depress antitoxin formation to the level observed after an acute dose of 650 rep. When the secondary stimulus of fluid tetanus toxoid was given prior to 10 days of continuous exposure to an accumulated dose of 860 rep, the secondary antibody respense was not depressed. Irradiated mice recovered the ability to produce a normal secondary antitoxin response during the second week after an accumulated dose of 1248 rep. The secondary antitoxin response was depressed in mice given long-continued gamma -radiation at a dose rate of 1 rep/hr. (auth)

Antibody Synthesis by Homotransplanted Cells and Tissues

Summary By attention to technical details, particularly the number of spleens employed and the time of removal of the donor spleens, the transplantation of splenic cells from toxoid-immunized rats into recipients consistently resulted in antitoxin production. Splenic fragments caused greater antibody formation than homogenates or cells. Antigen could be added to splenic cells from primarily stimulated donors, the cells washed and antibody be formed upon transfer. Transfers into pantothenic acid deficient recipients of cells removed from 1 donor 7 days after the booster eventuated in antibody synthesis, whereas transfers into normal rats did not. Similar cell transplants from donors 7 day-s after secondary injection resulted in equivalent antibody production in deficient and normal recipients. Usually there was a drop in titer 3 days after the booster inoculation, and an increase 7 days after this inoculation. The rat produced abundant amounts of diphtheria antitoxin, but no antibody was detected by precipitation or hemagglutination methods when bovine γ globulin was injected. The organ sites of antibody formation as related to the routes of injection of donor were similar in rat and rabbit. In no instance did a recipient which produced antibody after transfer show an anamnestic reaction to reinjection of toxoid 3 to 4 weeks after transfer. Most of the transplanted cells were lymphocytes. Interrelations, explanations and implications of these data were discussed.

Experimental enterotoxæmia of sheep : The effect of the permeability of the intestine and the stimulation of antitoxin production in immune animals

Experimental enterotoxæmia of sheep : The effect of the permeability of the intestine and the stimulation of antitoxin production in immune animals

A Misleading “Reaction of Identity” in Agar Diffusion Precipitin Studies of Tetanus Antitoxin

Summary The displacement of zone growth by apparently nonspecific antibody—a phenomenon previously reported—is shown to be illusory. Comparable effects may be produced by specific antibody when used in sub-threshold concentrations. At the same time, evidence is offered to show that the presence of heterospecific antibody in a particular region of agar does not significantly change the path of zone growth through that region. The quantitative analysis of several antibodies in a polyvalent serum is therefore not complicated by possible additive concentration effects between physically similar molecules. Internal evidence as to the relative antigen-antibody concentrations used in the present experiments suggests that the phenomenon was produced by qualitative, rather than quantitative relationships in this particular case. It is suggested that antibody formed in horses inoculated with toxoid for prophylactic purposes may have differed in specificity from antibody formed in horses used for tetanus antitoxin production. If so, the former was directed toward a portion of the toxin molecule which was altered or masked during the detoxification process, while the latter was specific for a determinant group which remained unchanged during the exposure of the antigen to formaldehyde. Attention is called to the fact that, on logical grounds, the reaction of identity cannot be used in ascertaining the specificity of an unknown antibody unless it can be shown that antibodies from two different sources necessarily choose the same determinant group for their site of attachment to the antigen molecule. Since antibody from the scarlatinal antiserum is shown to compete with the antibody in tetanal antiserum for the same antigen, this principle may be said to have presumptive experimental support.

In Vitro Production of Diphtheria Antitoxin by Tissues of Immunized Animals

Summary Lymph node and spleen fragments and slices from immunized animals appeared to produce diphtheria antitoxin when placed in a suitable medium. Disruption of the tissues or inclusion in the medium of various inhibitors of oxidative metabolism such as cyanide or dinitrophenol resulted in complete inhibition of antibody production. Moreover, the in vitro process of antibody formation appeared to parallel both the in vivo process and the passive transfer reaction whereby antitoxin appeared in the blood of normal rabbits following the injection of lymph node or splenic cells from immunized animals. In fact, the striking parallels between the in vitro and passive transfer reactions suggested that these two reactions might be fundamentally similar. It appears that under the conditions employed the tissue fragments and slices continued in vitro the net synthesis of antitoxin initiated in vivo.

Participation of the popliteal lymph node and spleen in the production of diphtheria antitoxin in the rabbit.

The objective of this study was the development of an experimental system for the elucidation of the mechanisms of antibody formation. It was first considered worthwhile to attempt to localize the antigen and possibly, therefore, the antibody-producing mechanism in as few organs as feasible. Under these conditions the possibilities for the study of antibody synthesis appeared great. Restriction of antigen to a few organs might limit antigenic stimulation of the host to these organs. Residual antigen and antibody might be expected to be present most regularly and in highest concentration in these organs where initial fixation of antigen occurred. Extirpation of one or more of the organs might then remove the antigenic stimulus or the actual mechanism involved in antibody synthesis. In addition to the attempt to localize the process of antibody formation, it was considered advantageous to employ the booster response to a second injection of diphtheria toxoid1 as a phenomenon which further increased the range of experimental possibilities. Since a great deal of evidence pointed

Effect of ACTH on antitoxin production in guinea pigs.

It is known that antibody production can take place without the adrenal cortical hormones. This was shown some years ago by several investigators (Jaffe & Marine, 1924, Perla & Marmorston-Gottesman, 1928, Marmorston-Gottesman & Perla, 1929, Khorazo, 1931) and has recently been confirmed by others (Murphy & Sturm, 1947, Eisen et al., 1947, Roberts & White, 1951), who all found that antibody production with different antigens in adrenalectomized animals, chiefly rats and rabbits, was of the same order of magnitude as in normal animals. It can thus scarcely be expected that adrenal cortical hormones should play any predominant rôle in the synthesis of antibodies.

Antigens in Scarlatinal Erythrogenic Toxin Demonstrable by the Oudin Technic

Summary Dick toxin contains a mixture of antigens for which specific antibodies exist in the sera of horses immunized for the production of scarlatinal antitoxin. At least eight distinct precipitin systems are represented in a mixture of commercial antitoxin and standard erythrogenic toxin employed in this laboratory. Several methods of identifying individual precipitin systems, based on the Oudin technic, have been examined: Specific adsorption of one antibody from the serum gives the most unequivocal results. Replacement of the antigen layer on agar in which zones have been established is a reliable method, but produces some unexplained effects. Agreement between the slopes of lines representing the relation between concentration of antigen and rate of migration should be used only as supportive evidence when dealing with ill-defined preparations. At least one antigen present in Dick toxin, antigen A, is derived from the heart-infusion broth used in toxin production. The erythrogenic toxin proper is represented by the fourth zone (D) in tubes containing our standard toxin and antitoxin.

The Effect of Added Vaccines on Diphtheria Antitoxin Production

The Effect of Added Vaccines on Diphtheria Antitoxin Production

Antibody formation in early infancy against diphtheria and tetanus toxoids

Summary A study was made of the development of antitoxin after two injections of combined diphtheria and tetanus toxoids in a group of young infants. It was found that the production of tetanus antitoxin is as good in the early months of life as later, and this finding denotes that the mechanism for the production of antibodies is well developed in young infants. With diphtheria antigen, the development of antitoxin is definitely defective in a considerable number during the first 6 months of life. This impairment of antibody formation is related to the presence of passive immunity in the resistant infants and appears specific for diphtheria. The application of these findings to the practical immunization of infants to diphtheria, tetanus, and whooping cough was discussed.

Combined pertussis-diphtheria prophylactic antigens: An experimental study to determine the specific immunizing value of these antigens used in combination

1. Experiments in laboratory animals are described, which were made to determine the specific antibody production following injections of diphtheria and pertussis antigens separately or in combination.2. Agglutinins to H. pertussis vaccine were not increased or otherwise changed by combining H. pertussis vaccines with either diphtheria anatoxine or A.P.T.3. A striking enhancement of diphtheria antitoxin production occurred when diphtheria prophylactics were used combined with pertussis vaccine. Such improvement in antitoxin production was most marked in the case of diphtheria anatoxine, but was also shown following the use of A.P.T. in combination with pertussis vaccine.4. Diphtheria anatoxine combined with H. pertussis vaccine gave no better antitoxin response than did a.p.t. alone.

Studies on C. Pyogenes Antitoxin in Relation to Immunity: I.—The Production of C. Pyogenes Antitoxin in the Serum of Normal Dairy Cows Following the Inoculation of Alum-Precipitated Toxoid and of a Commercial Toxoid

Summary A good antitoxin titre was obtained by the use of C. pyogenes A.P.T. when given in a dose of 5, 10, and 10 c.c. at ten- and sevenday intervals. Bi-monthly rejections of 10 c.c. helped to maintain the level of antitoxin. Various doses were tried and all gave a satisfactory result after three series of inoculations given at 86- and 160-day intervals. It appeared a maximum level of antitoxin production in cattle was reached. The “commercial toxoid” was not antitoxinogenic. No ill effects resulted from the injections.

The production of avid diphtheria antitoxin

The production of avid diphtheria antitoxin

Some practical applications of immunological principles

General immunological principles appear to be of wide if not univeral application, and can be applied equally to the hyperimmunization of horses for the production of therapeutic sera, or to human immunization against diphtheria and tetanus. A wider knowledge of the results obtained in animals, chiefly guinea-pigs, rabbits and horses, can help in a better understanding of all that is involved in the immunization of children against diphtheria.The response of an animal to an antigenic stimulus depends almost entirely on its previous experience of that antigen. If it has had no previous experience, antibody is produced slowly and in small amounts. If it has had previous experience, a change occurs in its antibody-producing mechanism which enables it on further stimulation to produce anti-body more rapidly and in larger amounts. To produce this effect the early experience must be adequate and followed by a sufficient period of rest. It is shown that the normal antitoxic values of animals can give considerable information on their previous experience of antigen; that this information can be made use of in the hyperimmunization of horses; and that inadequate primary stimuli and failure to provide adequate rest may lead to the production of antitoxins of low value and poor quality.Two things are necessary for defence against toxins: circulating antitoxin and a high reactivity of the antibody-producing mechanism (potential immunity). Methods of immunization should be chosen so as to provide both these essentials.

Effect of X-Ray Irradiation Upon Bacterial Toxemia in Rabbits

In a previous publication in this journal the writers presented experimental data which showed that in rabbits x-ray irradiation caused the production of non-specific antitoxin. Forty-eight hours after receiving approximately 100 r over the abdomen these animals were able to survive the intraperitoneal administration of a quantity of bacterial toxin which was constantly lethal to the untreated control animals. Two types of toxin were used, a standard diphtheria toxin (supplied by Eli Lilly & Co.) and one produced in our laboratory, a heat-killed culture of a hemolytic E. coli. The antitoxin resulting from irradiation was present in both the peritoneal fluid and blood serum. Time was required for its production or elaboration. Although present twenty-four hours following irradiation, it reached maximal concentrations only after forty-eight hours. These experimental findings suggested certain related problems for investigation. A report of these studies follows. I. Tissue Specificity Since only the abdomen had been irradiated in the previous experiments, there evolved the question whether this response to irradiation might be peculiar to the peritoneum or certain of the abdominal viscera. It seemed desirable, therefore, to determine the response of the other portions of the body—the chest and extremities—to irradiation. Twenty-four rabbits were given approximately 100 r each, one half of the animals receiving the rays over the chest and the other half over the extremities. The remaining portions of the body were protected with lead and radiation was administered through one port. It was delivered through an inherent filter of 3 mm. aluminum and an added filter of 0.5 mm. copper and 1 mm. aluminum, at 50 cm. target-skin distance, at 140 kv. p. and 15 ma. The intensity of the beam was 20 r per minute, measured in air. The dosage was determined by the duration of exposure and was varied accordingly. Forty-eight hours later one-half of the animals in each group received a minimal lethal dose of diphtheria toxin and the other one-half a minimal lethal dose of the hemolytic E. coli toxin. The latter consisted of 4 forty-eight-hour agar slant cultures which had been heat-killed. Six untreated animals were given the same dose of the toxins and acted as controls, 3 for each toxin. All controls died within forty-eight hours. All irradiated animals which received diphtheria toxin survived, as did 9 of the 12 which received the hemolytic E. coli toxin. Of the 3 that died, 2 were irradiated over the chest and 1 over the extremities. From the results of these experiments it may be concluded that antitoxin is produced or elaborated in response to x-ray irradiation irrespective of the portion of the body irradiated. If the sole benefit from irradiation in the treatment of infection is the production of antitoxin, then it is unnecessary to deliver the rays into the infected tissue.

Observations on tetanus immunization: The dosage of alum-precipitated toxoid and the use of fluid toxoid after trauma

CTIVE immunization against tetanus has established itself with surprising speed. Dunkirk, Pearl Harbor, and Libya were convincing field trials. 1~ Only military engagements could have furnished the proof of the potency of tetanus toxoid in such a short time. Since the first publication of Ramon and Zoeller, ~ many studies on the tetanus antitoxin production following different preparations, different dosages, and varying intervals have appeared. Reviews ~'1~ of the subject are recent. The optimal procedures to be used for adults in military service and for infants and children, however, are unsettled. Age and occupation certainly have an important bearing on the technique used. The following studies on infants and children may not be applicable to immunization in the Armed Forces. However, as they bear on some of the still debated potentialities of tetanus toxoids, they may interest the immuni ty officer as well as the pediatrician.

Active immunization against tetanus by the combined subcutaneous and intranasal routes: A simple procedure for the maintenance of a protective antitoxin titer

CTIVE immunization against tetanus A is regarded by many as a safe and effxcacious procedure.‘-lo It requires the injection of a primary course of two doses of aIum precipitated tetanus toxoid or three doses of pIain toxoid, which confers a basic immunity, to be followed by the injection of a “repeat” dose of toxoid whenever an injury occurs. The repeated injection of toxoid immediateIy raises the question of sensitization. Another question of importance is the rate of antitoxin production foIIowing the injection of the repeat doses of toxoid. FinaIIy, aIthough IocaI reactions at the site of injection are sIight to moderate in severity, any procedure that requires the repeated use of “ the needIe” with subsequent sore arms is IikeIy to meet with objections on the part of the patient. The intranasa1 route has been successfuIIy used for th e administration of drugs, glandular extracts and various bioIogica1 products. 11-18 Lesne et aI., SaIvioIo,24 .Jensen2j Bousheld and KingBrown,26 and more recently Wenger, HampiI and Masucci,“7 have reported on the use of diphtheria toxoid intranasaIIy. Ramon and ZoeIIer1g-“2 have also reported on intranasal immunization against diphtheria, tetanus, scarIet fever, and bacihary dysentery through the use of singIe or combined antigens. In 1927, these authors called attention to the chief benefits to be derived from the intranasa1 method of immunization, particuIarIy with reference to diphtheria. These are simplicity of administration and absence of reactions. Thus, individuals that were strongIy sensitive to diphtheria toxoid were immunized intranasalIy by Ramon with only a transient sweIIing of the nasa1 mucosa. The chiI1, fever, maIaise and IocaI inflammatory sweIIing that usualIy foIIow the injection of diphtheria toxoid into sensitive adults were conspicuously absent. Ramon’s objection to intranasa1 immunization was its expense, due to the need of three courses of treatment, each consisting of two daiIy instiIIations repeated for eight days. Each nasa1 treatment was equivalent to 0.3 cc. of Ramon’s toxoid. AIthough systemic reactions are rarely encountered after the injection of tetanus toxoid we can definitely state from our experience with subjects that have been immunized against tetanus bythe combined subcutaneous and intranasal routes that the instiIIation of toxoid into the nose rather than its injection under the skin is preferred by both aduIts and children. We are interested particularly in the use of a product for the “repeat dose ” that wiI1 not require subcutaneous injection and which wiI1 resuIt in the deveropment of a tetanus antitoxin titer generally relied upon to protect an individual against tetanus. A purified and concentrated tetanus toxoid (tetanus toxoid topagen) for topical appIication intranasaIIy was supplied to us by the MuIford BiologrcaI Laboratories of Sharp and Dohme. * We ha\-e overcome Ramon’s objection by the manner in which tetanus toxoid topagen is prepared and used as outIined in this report. * Tetanus toxoid topagen was prepared from highI antigenic plain tetanus toxoid. The toxoid WAS then purified and concentrated to contain 25 U’s per CL. Animal tests performed on guinea pigs that had IXXII previo”sIy injected with alum toxoid, showed that tetanus toxoid topagen administered intrannsall\, served as an excellent secondary stimulus.

Tetanus Toxin Production on a Simplified Medium

The routine preparation of tetanus toxin for the purpose of antitoxin production, or for conversion into toxoid, appears to offer difficulties similar to those formerly encountered in the case of diphtheria toxin, although perhaps in lesser degree. Active immunization against tetanus by means of toxoid, now coming into general use, demands that an attempt be made to place the manufacture of this important biological product on as satisfactory a basis as it has now been possible to reach in the case of diphtheria toxin.1, 2 The traditional explanation for failures and irregularities with the latter, namely the types of peptones and meat infusions used in the medium, has been shown to be completely erroneous, although still held to be applicable in dealing with tetanus. In this country, Witte, or the very similar Berna peptone is generally held to be indispensable, together with veal infusion. Elsewhere, enzymatic digests are employed, often with the addition of even more complex materials.The writers have ...