Guinea pigs were injected in the footpads with either purified diphtheria toxoid or recrystallized egg albumin in Freund adjuvant without mycobacteria. Each guinea pig was then skin-tested only once with the specific antigen and bled for antibody determination. After injection of the sensitizing antigen, a latent period occurred during which neither sensitivity nor circulating antibody could be detected. A period of delayed sensitivity followed wherein circulating antibody could not be discerned and which could be transferred by lymph node cells. Ultimately, the Arthus type sensitivity developed, accompanied by circulating antibody. The duration and severity of reactions to homologous antigens during the last 2 phases varied with the antigen and with the dose. An increase in the sensitizing dose decreased the duration of the delayed type of allergy, a decrease in the dose prolonged the delayed type. Inclusion of mycobacterium in the sensitizing inoculum tended to introduce delayed sensitivity earlier and delay the onset of Arthus type sensitivity. When specific precipitate in antibody excess was included with the toxoid in the sensitizing dose, the onset of the Arthus phase was hastened. When lymph nodes from a large number of sensitized donors were removed during the latter part of the latent period, recipients of the cells showed a delayed type sensitivity.

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A peptide, parathymosin a, containing %105 amino acid residues, has been isolated from rat thymus, and the sequence of the first 30 residues at the NH2 terminus has been determined. In this region, it shows 43% structural identity with thymosin a, and prothymosin a. The common sequences do not include residues 2-9, which accounts for the poor reactivity of parathymosin a with an antibody directed against this epitope in thymosin a1. Parathymosin a appears to modulate the action of prothymosin a in protecting sensitive strains of mice against opportunistic infection with Candida albicans.We have recently reported (1) a procedure for the isolation from rat thymus of three previously identified peptides, thymosin 834 (2), thymosin 6310 (3), and prothymosin a (4, 5). Prothymosin a, identified as a larger polypeptide containing the thymosin a1 sequence (6) at its NH2 terminus, was shown to account for most, if not all, of the immunoreactivity detected with an antibody directed against the NH2-terminal sequence in thymosin a1 (4).In this paper, we report that the isolation procedure also yields a fourth peptide, named parathymosin a because it shows some structural homology to prothymosin a and because it has a similar size and amino acid composition. Parathymosin a can also be isolated from other rat tissues, and in some tissues, such as liver, kidney, and brain, the concentrations of parathymosin a are much higher than the concentrations of prothymosin a. Preliminary results suggest that parathymosin a may act to modulate the immunoenhancing activity exhibited by prothymosin a.EXPERIMENTAL PROCEDURES Materials. Unless otherwise indicated, materials used were as described (1, 4, 5).Methods. The procedures for processing and extracting tissues and separating peptides from the extracts were as described (4, 5). Briefly, fresh rat thymuses were frozen in liquid N2 and pulverized in the frozen state. The powdered frozen tissue was quickly brought to 95°C-100°C in a relatively large volume of boiling 0.1 M sodium phosphate buffer (pH 7.0) and, after cooling, the suspension was homogenized with a Polytron homogenizer (Brinkmann) and the soluble fraction was collected. The clear extracts were desalted on Sep-Pak C-18 cartridges (Waters Associates), and the peptides were eluted with 1 M HCOOH/0.2 M pyridine, separated by chromatography on a column of Sephacryl S-200, and purified by reversed-phase HPLC (1, 4).Amino acid analyses, automated sequencing of peptides, and isoelectric focusing were carried out as described (4). (10 /11) were dried, hydrolyzed with alkali, and analyzed with fluorescamine (4). For subsequent purification by HPLC, the fractions corresponding to peak a, as indicated by the bar, were pooled and combined with similar fractions from three other gel filtration separations.

When mice infected with Mycobacterium tuberculosis strain BCG were inoculated intravenously with old tuberculin (OT) or living BCG cells, both migration inhibitory factor (MIF) and interferon appeared in the circulation within a few hours. In such animals, which showed delayed hypersensitivity by footpad tests, as little as 1.5 mg of OT or as few as 1.7 x 106 bacteria per mouse were capable of eliciting circulating MIF and interferon. Uninfected animals inoculated with large doses of OT or living BCG cells did not produce MIF or interferon. When nonspecific stimuli such as bacterial lipopolysaccharide (LPS; from Salmonella typhimurium strain LT-2), heat-killed Brucella abortus, Newcastle disease virus (NDV), and polyinosinic acid: polycytidilic acid (poly I: C) were inoculated intravenously into BCG-infected mice, MIF was produced in the circulation of animals challenged with LPS or Brucella but not in those challenged with NDV or poly I: C, although all the stimuli were capable of eliciting an interferon response. The interferon elicited in BCG-infected mice by specific antigen differed in at least one important property from the viral inhibitor produced by the nonspecific stimuli. The interferon which appeared after injection of OT or living BCG cells was destroyed by treatment at pH 2 for 24 hr at 4C, whereas the interferons produced after injection of the nonspecific stimuli were stable under the same conditions. The MIF activity in plasma from sensitized mice inoculated with specific antigen was also destroyed by treatment at pH 2. When mouse plasma containing both MIF and interferon activity was filtered through Sephadex G-100, both mediators were excluded in the same peak fractions. Sensitization of mice with complete Freund adjuvant instead of infection with BCG cells produces a different pattern of response. Although hypersensitive to specific antigen by footpad swelling tests, mice sensitized with complete Freund adjuvant failed to produce MIF or interferon when they were inoculated intravenously with OT or living BCG cells.

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Two peptides related to thymosin a1 have been isolated from preparations of calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, is designated des-(25-28)-thymosin al. The other, named thymosin all, contains seven additional amino acid residues at the COOH terminus. The sequence of this peptide is: AcSer-Asp-Ala-Ala-Val-Asp-ThrSer-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-ValVal-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH. Thymosin all, in doses of <300 ng per mouse, protects susceptible inbred murine strains against opportunistic infections with Candida albicans. It is m30 times as potent as thymosin fraction 5 and approximately equal in potency to thymosin al.

In the previous paper (1) it was reported that guinea pigs infected intraderreally with a living toxigenic strain of C. diphtheria~ developed delayed hypersensitivity to diphtheria toxin even when treated with antitoxin before infection. The present paper describes experiments which show that small amounts of diphtheria toxoid or of ovalbumin are capable of inducing a high degree of tuberculin type hypersensitivity in guinea pigs ff the antigen is injected intradermally in the form of a complex with excess homologous antibody. Maximum sensitization is achieved several weeks before circulating antibody can be detected. Materials and MethodsA ntigens.--Schick test materials and purified toxoid KP28 were the same as described in the preceding paper (1). Ovalbumin, three times recrystallized, was supplied by Dr. Milton Levy in the form of a dry powder. It was dissolved in saline or in phosphate buffer, filtered, and the protein concentration determined by measuring the absorption at 277 m/~ of aliquots diluted in 0.25 N acetic acid.AnHsem.--Rabbit antitoxin 379-380, horse antitoxic gamma globulin 5353AD, and human precipitating antitoxic gamma globulin were the same materials used in the preceding study (1). Human skin-sensitizing antitoxin ltu (2) was used without fractionation. This serum was from a recent bleeding taken from subject Hu who had received an immunizing dose of alum toxoid 5 years previously. The serum still contained 20 units of non-precipitating, skin-sensitizing antitoxin per ml.

Injection of foreign protein into a guinea pig may induce delayed hypersensitivity, followed by circulating antibody and Arthus-type hypersensitivity (1,2). If the antigenic dose is sufficiently minute, delayed hypersensitivity is not followed by the appearance of detectable amounts of circulating antibody (3). If a guinea pig with delayed hypersensitivity is, however, again stimulated with the specific homologous antigen, an anamnestic response appears, wherein circulating antibody and Arthus-type hypersensitivity develop.The suggestion has been made that delayed hypersensitivity is a step in the formation of circulating antibody (2). The question then arises whether delayed hypersensitivity has a more primitive type of specificity than Arthus reactions and circulating antibody. The present paper describes experiments which indicate that the specificity governing delayed hypersensitivity is different from that governing circulating antibody. The delayed response, as illustrated by experiments with protein conjugates and avian albumins as antigens, is produced in response to a broad general area of the antigen molecule, whereas the reactions of circulating antibody are controlled by small, specific groupings of the antigen. These studies also indicate that delayed hypersensitivity is an intermediate stage in the formation of circulating antibody. Materials and MethodsAnimals.--Guinea pigs of the Hartley strain weighing 400 to 500 gin. were used for studies on sensitization and immunization. White or albino guinea pigs weighing from 300 to 400 gin. were employed for studies on passive cutaneous anaphylaxis (PCA).Antigens.--Hen egg albumin (HEA) : Five times recrystallized hen egg albumin was obtained from the K & K Laboratories, Inc., Jamaica, New York.Duck egg albumin (DEA) : Whites of four dozen duck eggs were separated, diluted with an equal volume of water, and strained through cheese cloth. Globulins were precipitated with an equal volume of saturated (room temperature) ammonium sulfate and filtered out. Albumin was then precipitated by acidification of the supernatant to pH 4.7. Repeated attempts at crystallization were unsuccessful. 465 on