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PubMed1963

Properties of a bacteriophage derived from Escherichia coli K235.

A temperate bacteriophage was isolated from the colicinogenic strain of Escherichia coli K235 and characterized. This phage, termed PK, is related to P2 virus morphologically, serologically, and, possibly, genetically and it bears no relationship to the T-even phages. It was also demonstrated that PK virus and colicine K differ both in their host range and in their immunological specificity, and that PK prophage does not induce the colicinogenesis in its host bacterium. It was concluded that the formation of colicine K. and PK phage in E. coli K235 are controlled by different genetic determinants.

Anti-Bacterial Agents↗
PubMed1963

Antibody formation. IV. Formation of rapidly and slowly sedimenting antibodies and immunological memory to bacteriophage phi-X 174.

Injection of a sufficient dose of bacteriophage phiX 174 into guinea pigs results in the formation of rapidly sedimenting antibody molecules (19S), and later, slowly sedimenting molecules (7S). Above a threshold dose of antigen, the relative rate of 19S formation is maximal and dose-independent; below this dose, slower relative rates are obtained. The time for doubling the serum 19S level is as short as 6 to 8 hours, suggesting that the absolute rate of antibody formation per cell is increasing in addition to proliferation of antibody-producing cells. Synthesis of 19S after injection of 10(10) phiX virtually ceases at 10 days after which 19S antibody activity disappears from the circulation with a half-life of approximately 24 hours. A second injection of phiX on day 5 or 9 prolongs 19S synthesis, indicating that antigen not only can regulate the relative rate, but also is essential for continued synthesis of 19S. 19S synthesis is also prolonged in guinea pigs by injection of phiX with endotoxin or by 400 r whole body x-irradiation 24 hours after injection of phage into rabbits. The primary 7S response is not detected until approximately 1 week after immunization and relative rates are antigen-dependent. Primary 7S synthesis can continue for many months and leads to preparation for a secondary antibody response (immunological memory) during which only 7S is detected. In contrast, in animals that form precipitating 19S without detectable 7S, a second injection of phage 1 month later results in a second 19S response which closely resembles the first. These findings have led to the suggestion that formation of 19S does not lead to persisting immunological memory.

Animals↗
PubMed1963

Antibody formation initiated in vitro. II. Antibody synthesis in x-irradiated recipients of diffusion chambers containing nucleic acid derived from macrophages incubated with antigen.

The diffusion chamber technique permitted the demonstration of specific antibody formation in x-irradiated recipients of such chambers filled with normal lymph node cells and a cell-free homogenate of macrophages which had been incubated in intro with T2 bacteriophage. The activity of the cell-free homogenate was retained in its RNA fraction isolated by means of the phenol method. No antibody formation occurred if such RNA was treated with RNAase. On sucrose gradients (5 to 20 per cent), the active RNA was found to be present in the top third layer. The question of the possible presence of antigen complexed to the RNA is discussed.

Antibodies↗
PubMed1963

Sequences of synthesis of gamma-1 macroglobulin and gamma-2 globulin antibodies during primary and secondary responses to proteins, salmonella antigens, and phage.

The nature of the antibodies produced by the rabbit during the primary and secondary responses to T(2) phage, proteins, and the O and H antigens of Salmonella typhosa has been determined. Immune sera have been fractionated by zone electrophoresis, sucrose density ultracentrifugation, and anion exchange chromatography. The resulting fractions have been assayed by phage neutralization or hemagglutination (antisera to proteins) or bacterial agglutination. In confirmation and extension of earlier work from this laboratory, the primary response to these antigens, with the exception of the O antigen of the Salmonella, included the early synthesis of 19S, gamma-1 globulin antibody, and the later synthesis of 7S, gamma-2 globulin antibody. The primary response to the O antigen consisted of the synthesis of only a macroglobulin agglutinin. The secondary response to the proteins, including the H antigen of the Salmonella, comprised the early synthesis of large amounts of the 7S gamma-2 globulin antibody to the same level attained during the primary response. The secondary response to the phage consisted in the synthesis of 7S, gamma-2 globulin antibody alone. Treatment of the macroglobulin phage-neutralizing antibody with mercaptoethanol resulted in complete loss of its neutralizing activity. A working hypothesis to explain these observations was presented. A salient feature of this hypothesis was the suggestion that different cells synthesized the two distinct molecular forms of antibody. The significance of the sequential synthesis of the two forms of antibody is not known. It was proposed that the system for synthesis of macroglobulin antibody is an auxiliary system for antibody synthesis, perhaps the first to develop phylogenetically and ontogenetically. It is felt that the present observations indicate a clear-cut qualitative distinction between the primary and secondary responses to immunization whereby these responses might be identified in various experimental situations. It is also felt that these findings with the primary and secondary responses to various antigens in the rabbit may be of widespread occurrence in nature among a variety of species.

Animals↗
PubMed1964

EVOLUTION OF THE IMMUNE RESPONSE. I. THE PHYLOGENETIC DEVELOPMENT OF ADAPTIVE IMMUNOLOGIC RESPONSIVENESS IN VERTEBRATES.

1. The California hagfish, Eptatretus stoutii, seems to be completely lacking in adaptive immunity: it forms no detectable circulating antibody despite intensive stimulation with a range of antigens; it does not show reactivity to old tuberculin following sensitization with BCG; and gives no evidence of homograft immunity. 2. Studies on the sea lamprey, Petromyzon marinus, have been limited to the response to bacteriophage T(2) and hemocyanin in small groups of spawning animals. They suggest that the lamprey may have a low degree of immunologic reactivity. 3. One holostean, the bowfin (Amia calva) and the guitarfish (Rhinobatos productus), an elasmobranch, showed a low level of primary response to phage and hemocyanin. The response is slow and antibody levels low. Both the bowfin and the guitarfish showed a vigorous secondary response to phage, but neither showed much enhancement of reactivity to hemocyanin in the secondary response. The bowfin formed precipitating antibody to hemocyanin, but the guitarfish did not. Both hemagglutinating and precipitating antibody to hemocyanin were also observed in the primary response of the black bass. 4. The bowfin was successfully sensitized to Ascaris antigen, and lesions of the delayed type developed after challenge at varying intervals following sensitization. 5. The horned shark (Heterodontus franciscii) regularly cleared hemocyanin from the circulation after both primary and secondary antigenic stimulation, and regularly formed hemagglutinating antibody, but not precipitating antibody, after both primary and secondary stimulation with this antigen. These animals regularly cleared bacteriophage from the circulation after both the primary and secondary stimulation with bacteriophage T(2). Significant but small amounts of antibody were produced in a few animals in the primary response, and larger amounts in the responding animals after secondary antigenic stimulation. 6. Studies by starch gel and immunoelectrophoresis show that the hagfish has no bands with mobilities of mammalian gamma globulins; that the lamprey has a single, relatively faint band of this type; and that multiple gamma bands are characteristic of the holostean, elasmobranchs, and teleosts studied. By this method of study, the bowfin appeared to have substantial amounts of gamma(2) globulin. 7. We conclude that adaptive immunity and its cellular and humoral correlates developed in the lowest vertebrates, and that a rising level of immunologic reactivity and an increasingly differentiated and complex immunologic mechanism are observed going up the phylogenetic scale from the hagfish, to the lamprey, to the elasmobranchs, to the holosteans, and finally the teleosts.

Adaptation, Physiological↗
PubMed1964

IMMUNOLOGIC INCOMPETENCE OF IMMUNOLOGICALLY RUNTED ANIMALS.

1. Adult (A x C(57)Bl/1)F(1) hybrids regularly show runt disease when injected with adult spleen cells from A strain donors. This also occurs when A strain spleen cells are administered to adult C(3)H mice made tolerant of A strain tissue in the neonatal period. 2. Mice undergoing the graft versus host reaction fail to form antibodies to an intraperitoneal challenge of T(2) bacteriophage. This phenomenon was observed well before any of the other overt signs of runting had occurred. Further, inhibition of antibody production to T(2) phage by graft versus host reaction initiated at an interval following antigenic stimulation is demonstrated. 3. The basis for the immunologic incompetence of the host with respect to T(2) phage is presumed to be the attack of immunologically competent donor cells on the lymphoid cells of the recipient. 4. The failure of the injected parent strain cells to respond to the antigen used may imply immunologic commitment of these cells.

Animals↗
PubMed1964

ELECTRON MICROSCOPY OF IN VITRO ENDOCYTOSIS OF T2 PHAGE BY CELLS FROM RABBIT PERITONEAL EXUDATE.

Macrophages from rabbit peritoneal exudate cells incubated in vitro with T(2) bacteriophage from 10 up to 120 minutes show phage particles adsorbed to cell membranes, in the process of being engulfed by means of rhopheocytosis, micropinocytosis, and phagocytosis, and localized within dense vacuoles, semi-dense vacuoles, and clear vacuoles of the cytoplasm. The electronmicrographs suggest that newly formed endocytic vacuoles containing phage particles fuse with one another and also fuse with dense bodies of the cytoplasm as they migrate towards the cell interior, thus yielding larger vacuoles of varying densities containing higher concentrations of phage. The polymorphonuclear cells present in a small proportion in the peritoneal exudate cells also endocytosed phage particles. The T(2) particles are found in large cytoplasmic vacuoles surrounded by an electron-opaque material presumably derived from cytoplasmic granules. No disintegration of T(2) phage within the macrophage following incubations up to 120 minutes could be demonstrated; however, disrupted phage particles were noted within cytoplasmic vacuoles of polymorphonuclear leucocytes after 15 minutes' incubation.

Animals↗