Production Of Antitoxin Research Articles

Antigen-specific T-cell factors and drug research.

The central feature of the immune system is the specificity of antigen-binding proteins for the ligand that induced their synthesis. It is this specificity that has intrigued medical scientists for the last 100 years and has led to repeated attempts to use this feature to combat disease. One of the earliest, successful uses of immune specificity involved the production of specific anti-toxins to combat a variety of ills from bacterial infections to snake bites [1]. In addition, advantage was taken of the fact that by providing appropriate antigens in the proper form (eg. viruses, bacteria), specific immune responses could be elicited [2]. Thus, over the last 80 years or so the primary focus of the therapeutic use of antigen-specific proteins has been the antibody molecule [3]. With the development of monoclonal antibody methodology, there has developed a renewed interest in the use of specific single antibodies as therapeutic agents; “magic bullets” as they are often termed [4]. In addition, the capacity to produce monospecific reagents is providing invaluable tools to probe the structure and function of other proteins; to use in the specific detection of cellular or tissue specific antigens and for purifying and identifying a wide variety of normal body components (eg. peptides, lipids, etc.) and/or ingested drugs [5]. In the early 1970s several groups of investigators reported that, in addition to antibodies, there exist other antigen-binding moieties that appear to function primarily as immune regulators.KeywordsDrug ResearchHelper FactorTissue Specific AntigenImmune Response ModelModulate Factor ActivityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Protection against cholera toxin after oral immunization is thymus-dependent and associated with intestinal production of neutralizing IgA antitoxin.

We studied whether gut mucosal IgA antitoxin production as well as the acquired protection against cholera toxin (CT) after oral immunization with CT are both thymus-dependent immune manifestations. In contrast to normal BALB/c mice, nude, athymic mice did not respond to oral immunizations with CT with either IgA antitoxin-producing cells (SFC) in the lamina propria or protection against challenge with CT in ligated intestinal loops. However, when nude mice were first reconstituted by grafting of syngeneic thymus glands, both IgA antitoxin SFC in the lamina propria and protection were stimulated by oral immunizations with CT and the response were of similar magnitude to those of normal mice after immunizations. During in vitro culture, isolated lamina propria lymphocytes from immunized but not from control mice concomitantly and proportionally produced IgA antitoxin and CT-neutralizing activity. We conclude that intestinal antitoxin formation and protection against toxin challenge after oral immunization with CT are both critically thymus-dependent and therefore likely to be under T-cell control.

Production of Antitoxins to Two Toxins of Clostridium difficile and Immunological Comparison of the Toxins by Cross-Neutralization Studies

Production of Antitoxins to Two Toxins of <i>Clostridium difficile</i> and Immunological Comparison of the Toxins by Cross-Neutralization Studies

Topics on Bordetella pertussis vaccine

1. As reported previously, histopathological changes of adrenal glands in Wistar Rats (male, 100-200g) were observed with the injection of purified histamine-sensitizing-factor (HSF): Zona glomerulosa, the outer layer of zona fasciculata, and their lipid content were constant. Marked cytolysis in zona glomerulosa. Extraordinary depletion of lipids in the inner layer of zona fasciculata. Zig-zag formetion from the beneath the capsule reaching the deeper region of the inner layer of zona fasciculata (Fig. 1 and 2).It is well-known that diphtherial exotoxin induces marked hemorrhages of adrenal gland. In refference to this, histamine-sensitizing-factor (an exotoxin) of Bordetella pertussis might cause any change in the adrenal gland. Under this hypothesis, this experiment was carried out. Consequently, the changes mentioned above (Fig. 1 and 2) were recognized. These changes imply disturbance of secretion of hormones (Cortisol, Aldosteron, Adrenal Androgens). I drew inferences from these facts that these were reported as the results of “stress”. I got a lot of letters from everywhere in the world. Unexpectely, among them, the comment of Prof. M.S. Zakharova at Moscow, USSR, was valuable. She critisized that my findings are not only results of stress, but also they are functional changes of the whole host body. It is of the very importance for establishment of prevention against whooping cough infection. This suggestive comment gave me a hint.No doubt, the changes (Fig. 1 and 2) express only static stage, followed by rapid repairement, accompanying hyperfunction and production of antitoxin. So that, the forced definition “preventive antigen” is completely useless.Recently, Arai & Munoz could crystallize HA (Fimbrial Hemagglutinin), whick lacks completely preventivity. Also, Arai & Munoz could succeed in crystallization of “pertussigen” (Package of HSF, LPF & thermo-labile lethal antigen). The future of pertussigen deserves the keen interest.2. Is IC (INTRACEREBRAL CHALLENGE) of mice the one and only as a test?IC method is spread all over the world like the Bible. Oblivious experiments by Standfast whose work I have learned from Dr. Margeret Pittman of NIH, Md, NSA. Standfast's experiments (1) Usual pertussis vaccine gave good effect on IC mouse protection as well as field trial. (2) Contrary to this, pertussis vaccine heated at 100C for one hour do not any effect on IC mouse protection at all. However, this heated vaccine gave good effect on IN (INTRANASAL CHALLENGE) mouse protection as well as field trial (human being). This undeniable facts are of the very importance, because packkage-pertussigen (a group of exotoxins-thermolabile) was vanished away by heating. Hence, one can obtain the most safe pertussis vaccine without any side effect. It reminded of my heat-stable neurotoxin.I do not know whether it is an exotoxin or not. In spite of unknown character, it shows charactristic whooping cough syndromes. My working hypothesis suggests that “heat-stable antigen” by Standfast might coincident with my “heats-table neurotoxin”.

Reinterpretation of the Dick test: role of group A streptococcal pyrogenic exotoxin

Because of the association of the group A streptococcal pyrogenic exotoxins (SPEs) with erythrogenic toxin used in the classical Dick test, the involvement of the SPEs in production of erythematous skin reactions was assessed. Unless they had been presensitized, young adult rabbits failed to show skin reactions after intracutaneous challenged with SPEs. Rabbits presensitized to purified protein derivative exhibited enhanced skin reactivity when given purified protein derivative plus SPE C; the enhancement was neutralized by antiserum to SPE C. Rabbits sensitized to bovine serum albumin showed extensive red rash development resembling scarlet fever rashes when given bovine serum albumin containing SPE C. Desquamation occurred 5 to 10 days after injection. Animals sensitized to one SPE type showed enhanced skin reactivity to challenge with homologous or heterologous SPE types, indicating the presence of a cross-reactive determinant within the SPE molecules. Repeated challenge of SPE-sensitized animals with homologous toxin resulted in concomitant antitoxin production with reduction of the enhanced skin reactivities, until typical delayed-hypersensitivity skin reactions remained. The data indicate that, in addition to the toxic reaction previously described, SPEs enhance Arthus and delayed-hypersensitivity skin reactions. It follows that erythrogenic toxin represents the enhancement of acquired skin reactivity to streptococcal antigens by one or more SPE types. Therefore, the Dick test measures SPE-enhanced hypersensitivity to streptococcal products.

Immunity to Diphtheria in an Urban Population

Immunity against diphtheria is poorly understood since protection is the result of a complex interaction between host and environment, and technical problems with the present antitoxin assays exist. A radioimmunoassay which permits a reproducible estimation of antitoxin concentration and relative avidity was employed to characterize the immunological response to diphtheria of 195 healthy urban children. The antitoxin concentration in cord serum was equal to or greater than the concentration in maternal serum and decreased rapidly during the first two months of life. The first diphtheria-tetanus-pertussis inoculation was not immunogenic enough to initiate significant antitoxin production in infants without maternal antitoxin or in older children. A significant increase in antibody concentration and affinity was found after the second, third, and fourth toxoid administrations. More than four injections did not alter concentration, affinity, or duration of active immunity. Correlation with recent epidemiologic studies suggested that a serum contained a "protective" concentration of antitoxin if it bound more than 100 ng of diphtheria toxin nitrogen per milliliter of serum (approximately 0.1 IU of antitoxin). By this criterion, among the study group only 74% of the healthy children who had received three immunizations and 84% of the children who had received four or more inoculations were "protected." Antitoxin concentrations observed in serum from children with chronic diseases who had received appropriate immunizations were lower than in serum from appropriately immunized healthy children. These observations suggest that the immunologic status of urban children against this disease is not optimal and should serve as a stimulus to improve both patient education and immunization practices.

Titration of tetanus antitoxin by passive hemagglutination. II. Serological characteristics of antitoxin production in rabbits and monkeys.

1) Production of tetanus antitoxin in rabbits and monkeys was followed by passive hemagglutination (HA) and toxin-neutralization (TN) tests. The HA activity was observed in both IgM and IgG in both animal species. 2) In rabbits, IgM antitoxin was detected as early as in 7 days, reached the maximum titer in 10--14 days, and disappeared in 3 weeks after the primary immunization. Antitoxin of IgG class was detected in 10 days, and increased gradually. The ratio of HA/TN titers ("serum ratio") was high at an early stage of primary immunization and approached the unity in 3--4 weeks. Unlike the case of guinea pigs, IgM was found to contribute greatly to this high level of ratio. Besides, most rabbits produced IgG antitoxin of high ratios at early stages of immunization. 3) The immune response of monkeys showed a pattern very similar to that of rabbits except a few days' delay in the time course of antitoxin titers. No IgG antitoxin with a high serum ratio was demonstrated. Therefore, the high serum ratio of early sera could be accounted for mainly by IgM. 4) In response to the secondary immunization, no IgM antitoxin was detected in either animal species. 5) No definite correlation between serum ratio and avidity in terms of "dilution ratio" was demonstrated. However, both the dilution ratio and serum ratio were high at an early stage of immunization and gradually decreased, though the magnitudes of the ratios were variable depending on individual animals.

IMMUNITY TO DIPHTHERIA IN AN URBAN POPULATION

Immunity against diphtheria (D) is poorly understood since protection is a complex interaction between host and environment and there are technical problems with existing antitoxin assays. Using a radioimmunoassay, antitoxin was detected in sera from premature infants after 33 weeks of gestation and the maternalcord serum ratio of 18 term infants was 0.8 ±0.3 (1 SD), Following the first DTP (7.5 Lf units D toxoid) significant antitoxin production was not detected. This relatively poor antigenicity was found in the absence as well as presence of maternal antibody. A rise in antibody concentration and affinity was found after the 2nd, 3rd, and 4th injections. More than four injections did not alter concentration and affinity of the antitoxin or the rate of decline in concentration. Correlation with recent epidemiologic studies suggests that a serum contained a protective concentration of antitoxin if it bound more than 150 ng D toxin N/ml of serum. By this criteria only 74% of children who received 3 immunizations and 84% of children who received 4 or more were protected. Among the 188 children studied 34% had appropriate immunizations for age. These observations suggest that the immunologic status of urban children to D is inadequate and an improvement in patient education and immunization practices is needed, (supported by AI-07854)

Enterotoxigenic Escherichia coli and Diarrheal Disease in Mexican Children

Enterotoxigenic strains of Escherichia coli were isolated from eight (16%) of 50 Mexican children admitted to the hospital with diarrhea and from one of 50 children hospitalized for nonenteric disorders. All of the toxigenic strains tested elaborated a heat-labile enterotoxin, and in seven of nine patients no E. coli capable of concomitant production of heat-stable enterotoxin were found. None of the strains of E. coli with classical enteropathogenic serotypes isolated from nine patients with diarrhea produced either heat-labile or heat-stable enterotoxin. Although the results of this study strongly suggest that enterotoxigenic strains of E. coli are probably responsible for a significant number of cases of diarrhea in an indigenous Mexican pediatric population, further proof will require demonstration of in vivo production of enterotoxin and/or antitoxin.

Lymphoid cell dependence of eosinophil response to antigen. IV. Effects of in vitro x-irradiation on adoptive transfer of anamnestic cellular and humoral responses.

Recently, we have reported the detection of two types of memory cells induced by a priming injection of tetanus toxoid—one memory cell mediating the secondary eosinophil response and a separate memory cell associated with antitoxin production. It was further demonstrated that formation of both memory cells was dependent upon the presence of thymic cells at the time of priming. Memory cells involved in eosinophil responses, however, are formed earlier and become more widely distributed in lymphatic tissues than do memory cells involved in antitoxin responses. Consequently, adoptive transfer of specific lymphoid tissues at selected intervals after priming can demonstrate the presence of one type of memory without the other (1, 2). For example, at 10 days after priming, cells present in the spleen were capable of inducing a secondary rise in eosinophils, but not antitoxin. However, at 30 days, splenic cells were capable of inducing both responses. In the experiments to be described, we have demonstrated differing sensitivities of these memory cells to in vitro X-irradiation. Materials and Methods. The experimental procedure is similar to that utilized in previous studies (1–3). Briefly, adult BAFX (C57BL × A/J) female donor mice were primed with a single subcutaneous injection of 0.2 ml aluminum phosphate-adsorbed tetanus toxoid (National Drug Co., Philadelphia, PA) in an adjuvant of 0.2 ml pertussis vaccine (Eli Lilly Drug Co., Indianapolis, IN) diluted 1:10 with saline (PVTT). Thirty days after priming, the mice were sacrificed and cell suspensions made of the spleen.

SYNERGISM IN IMMUNOSUPPRESSION

SUMMARY The relative efficacy of azathioprine, chlorambucil, methylprednisolone, and total body γ-radiation, alone and as combined therapy, in suppressing tetanus antitoxin production in mice was determined. An in vivo “live-die” assay for serum antitoxin titer was developed. Increasing the number of drugs in the treatment regimen to two or three caused greater depression of the primary immune response than did only one drug. The most profound immunosuppression was produced when sublethal radiation and three drugs were used together

Primary and secondary immune responses of sheep to tetanus toxoid

After primary and secondary stimulation respectively with tetanus toxoid antitoxin appeared in the circulation of 19 sheep. The rate of antitoxin production, duration of the period of rapid production, persistence of a high titre, and the uniformity of the rate of its decline, were not significantly different after the primary and after the secondary responses. The primary response developed 9 to 15 days after injection: the secondary response occurred generally between the third and fifth days. Animals which have received a primary injection, can be differentiated from immunologically intact animals by ascertaining whether they produce antitoxin 7 days after one injection of toxoid. The difference in the magnitude of the two responses was less than would be anticipated from the generally accepted concepts of antibody production. The rate of the appearance of antitoxin in the circulation fits in with the observations of other workers using more sophisticated procedures.

Specificially increased graft-versus-host reactivity of thymus cells from immunized mice.

IT has recently been shown that thymus cells from immunized mice are capable of transferring immune reactivity. Sub-lethally irradiated recipient mice exhibit recall tetanus antitoxin production after they have been injected with thymus cells from mice immunized with tetanus toxoid1. Similarly, thymus cells from mice immunized with bovine serum albumin are capable of transferring antibody formation capacity when injected into non-irradiated recipients2. The work recorded here was undertaken to determine whether thymus cells from mice immunized to F1 hybrid histocompatibility antigens would exhibit specifically increased graft-versus-host immunological reactivity after injection into F1 hybrid mice of the immunizing strain. We will show that immunization does specifically increase the graft-versus -host reactivity of thymus cells, and that the relative increase in reactivity is similar to that observed with injection of spleen cell populations from similarly immunized animals.

FAILURE OF ACTINOMYCIN D TO INHIBIT ANTITOXIN PRODUCTION TO A CHALLENGING INJECTION OF ANTIGEN.

SummaryDaily injections of Actinomycin D, which were sufficiently high to produce marked weight losses and other toxic manifestations, failed to eliminate antitoxin production to a challenging injection of tetanus toxoid. Since DNA-dependent RNA synthesis was presumably inhibited, the data support the existence of a persisting inactivated mRNA, in a form which is activated by the reinjection of antigen.

Experiments designed to determine the mechanism of the adjuvant activity of gram-negative organisms upon antibody production.

The characteristics of Gram-negative organisms and some of the underlying reasons for their adjuvant action with diphtheria toxoid are described.The adjuvant effect was shown by an earlier production of antitoxin, with a maintained differential advantage over controls, but with the usual decline in titre with passage of time. The adjuvant effect only occurred with a primary stimulus. There was no adsorption between toxoid and vaccine and mixture of the two was not necessary, but the vaccine had to be given simultaneously with or within 24 hr. following injection of the toxoid. There was evidence for believing that these adjuvants decreased the minimal stimulating dose of antigen and caused hyperplasia of antibody-producing cells. No direct link could be found between the characteristic stress symptoms caused by lipopolysaccharides and their ability to enhance antibody formation.

On the Relation between diphtheric Allergy and Antitoxin production

On the Relation between diphtheric Allergy and Antitoxin production

混合ワクチンに関する研究

In the case of tetanus toxoid, only one type of the International Standard (plain) has been established. In this paper, experiments were carried out to investigate whether various kinds of tetanus toxoid can be assayed by this standard.Guinea-pigs were immunized with graded doses of several preparations of the simple or combined vaccine including T., D-T, P-T., and P-D-T.In the case of fluid toxoids, the circulating tetanus antitoxin reached maximum titer between 8 and 12 weeks after immunization, while, in the case of toxoids with adjuvants such as aluminiferous compounds or pertussis vaccine, maximum titers were obtained between 6 and 8 weeks.The dosage-response lines of fluid toxoids and combined vaccines were proved paralell, while those of precipitated toxoids were not. Therfore, it was concluded that the precipitated toxoids could not be assayed against the fluid standard toxoid. In the case of the combined vaccines, it was possible to find the relative potency to the fluid standard. However, a tendency was observed that the relative potency may be different depending on the immunization periods, probably because of the differences in the process of antitoxin production. Therefore it is indicated that this may be an obstacle in the evaluation of combined vaccine against the present standard, although further experiments should be necessary to confirm this point.

The role of lymphoid organs in the formation of antitoxic immunity.

Experiments carried out on 110 rabbits were devoted to the comparative study of the relationship between immunological reactivity of lymphoid organs and the character of the antigen administered (crude and adsorbed) as well as the immunization scheme. Toxoid was injected subcutaneously into the interior part of the posterior extremity. Antitoxin was determined in dynamics in the regional and distant lymph nodes, as well as in the spleen and blood. As shown by the investigations, primary injection of the antigen, irrespective of its character, is accompanied by a weak regional lymph node antitoxin production and complete absence of antitoxin accumulation in the distant lymphoid organs.

The Production of Antibody by Transferred Bone Marrow

Summary Bone marrow cells from mice hyperimmunized with tetanus toxoid are capable of continuing production of tetanus antitoxin after transfer to irradiated hosts. Isologous cells functioned well 25 days after transfer, whereas there was a sharp decline in production of antitoxin by homologous cells during this period.

The mechanism of immunity to tetanus toxin at early periods after active immunization

Experiments on white rats have established that after primary immunization with a single, relatively large dose of adsorbed tetanus toxoid the resistance of the animals to tetanus toxin develops prior to the appearance of antitoxin in the blood. However, the administration at that period of various substances, such as tetanus toxin andBac. perfringens toxoid, or cortisone, makes the animals react by rapid production of antitoxin in the blood in detectable amounts. This phenomenon is regarded as a nonspecific stimulation of the antitoxin-forming apparatus (regional lymph modes) periods after immunization and in the absence of antitoxin in the blood at the moment of toxin administration.