Secondary responsein vitro to antigen ΦX 174 phage

In a model secondary reactionin vitro, a correlation was demonstrated between the size of the antigen dose used for the prestimulation of spleen tissue donors and the type of antibodies formed in the anamnestic reaction. After a small dose of antigen ΦX 174, the antibody response three days after prestimulation was of the 19 S (IgM) type, but later secondary contactin vitro (after four months) did not produce a 19 S anamnestic reaction. After large primary doses of antigen, a short interval between primary and secondary contact led to the formation of 19 and 7 S type antibodies, while after a long interval only 7 S (IgG) type antibodies were formed. The results are discussed in relation to differences in the size of the antigen dose needed to induce short-term 19 S and long-term 7 S immunological memory.     

In Vitro Studies on the Immunological Memory for Antibody Response to Bovine Serum Albumin

Immunological memory for T and B cells was studied in an in vitro culture system with spleen cells from mice primed with bovine serum albumin (BSA). Spleen cells taken from mice immunized at various times previously with a single intravenous injection of alum-precipitated (AP) BSA and bacterial endotoxin (ET) were cultured in Marbrook's system with dinitrophenylated (DNP) BSA as the in vitro antigen. In the cultures of spleen cells obtained from mice primed more than 14 days previously, an IgG-predominant anti-BSA response was generated. However, no anti-BSA response was observed in the culture of spleen cells taken from mice primed 7 days previously (day 7 spleen cells). The failure of day 7 spleen cells to generate an antibody response in vitro was shown to be attributable to both the lack of B memory cells and the effect of “suppressive” macrophages induced by ET. On the other hand, anti-BSA memory in the spleen of mice primed with AP-BSA plus ET and 2 months later challenged with AP-BSA matured within 7 days and declined rather quickly by 30 days after the challenge. The difference in the time course of the generation of memory between the spleen cells from primary and from secondary immunized mice might be attributable to the difference in the maturation of memory B cells, since the time course of the development of memory T cells after the secondary immunization was similar to that observed after primary immunization.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Study was made to clarify the experimental conditions for the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to exhibit maximum adjuvant effect on antibody production to bovine serum albumin (BSA) in mice. To obtain the maximum primary antibody response and also the strongest priming for a secondary response to BSA, 1000 μg of CPS-K had to be injected intramuscularly into the same or adjacent site of BSA injection within the period of 1 hr before to 6 hr after the BSA injection. The optimum amount of BSA giving the maximum antibody response and also the strongest priming under these experimental conditions was 15 mg. In mice thus primed, an extremely high secondary response was induced by injecting 0.5 mg of BSA 30 days after the initial injection. The minimum amount of CPS-K, to exhibit a strong adjuvant action, was 100 μg, which was equal to the minimum amount to induce immunologic paralysis to a homologous antigen. Extremely large amounts, such as 100 to 300 mg per mouse of BSA, were also strongly immunogenic when injected together with paralyzing doses of CPS-K. In vitro admixture of BSA and CPS-K before injection did not strengthen adjuvant action of CPS-K. Alum-precipitated BSA mixed with CPS-K was not more immunogenic than native BSA mixed with CPS-K. Addition of Freund's complete adjuvant to an injection of BSA and CPS-K mixture did not enhance the adjuvant effect of CPS-K.     

In vitro immune response of primed mouse spleen cells as affected by the time of antigenic stimulation

Normal and primed spleen cells stimulated in vitro with sheep RBC showed a different behaviour in the 19 S PFC response. The immunologic capacity of primed cells, unlike that of unprimed cells, was impaired if the antigen addition to culture had been delayed for more than six hours. The requirement of primed cells for immediate reexposure to antigen varied with the time of preimmunization and was maximum when spleen cells had acquired in the donor the ability to display the highest response in vitro upon optimal restimulation. This phenomenon of antigen requirement of primed cells in vitro is at variance with the known immunologic behaviour of primed cells grown in vivo, and can be attributed to cellular changes unmasked by in vitro cultivation.     

Some biological characteristics of lipid conjugates of protein antigen that selectively induce delayed-type hypersensitivity in mice

Covalent coupling of lipid to protein antigen, BSA, modifies the immunogenicity leading to selective induction of delayed type hypersensitivity in mice with no or very little concomitant antibody production. The mode of linkage of lipid to protein, however, controls its tissue distribution and retention in the body and cell uptake in vitro. Whereas D-BSA accumulates in the draining lymph node after foot pad inoculation, DA-BSA stays at the site. Further, DA-BSA is eliminated much more slowly and D-BSA more quickly from the body than the native antigen. Although both lipid-conjugates are taken up by lymphoid cells in vitro more than the native antigen, DA-BSA binds significantly more than D-BSA. On the basis of in vitro blastogenic response and enumeration of antigen sensitive cells and in vivo tests of delayed type hypersensitivity, DA-BSA appears superior to D-BSA but neither was as potent as BSA in CFA.     

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.     

The passive anaphylactic reaction in guinea pigs elicited by whole and cathepsin D-degraded antigen

The systemic anaphylactic reaction and thein vitro anaphylactic contraction of the terminal segment of the ileum performed according to the Schultz-Dale technique, were elicited in guinea pigs passively sensitized with rabbit antibodies to human serum albumin, using whole and cathepsin D degraded antigen. The intensity of the systemic anaphylactic response that was evoked by degraded antigen was lower; a highly significant suppression of the response was obtained provided an antigen degraded more than by 70% was injected. With an increasing degradation of antigen, more animals responded with lower or even with no reaction; the number of animals developing severe or lethal shock, decreased at the same time. The number of animals that developed a medium anaphylactic response remained at the same level. The degraded antigen did not evoke the anaphylactic contraction of the terminal segment of the ileumin vitro, and moreover, it blocked the contraction after addition of the whole antigen.     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Application of <Emphasis Type="Italic">in vivo</Emphasis>microscopy: evaluating the immune response in living animals

The initiation of an immune response requires that professional antigen-presenting cells, such as dendritic cells, physically interact with antigen-specific T cells within the complex environment of the lymph node. Although the way in which antigen is presented to T cells and in particular the cellular associations involved in antigen-specific stimulation events have been extensively investigated, data on antigen presentation have come primarily from studies in vitro or examination of the late consequences of antigen presentation in vivo. However, there is increasing recognition that events defined in vitro might not correspond entirely to the physiological situation in vivo. Recent developments in imaging technology now allow real-time observation of single-cell and molecular interactions in intact lymphoid tissues and have already contributed to a more detailed picture of how cells coordinate the initiation or suppression of an immune response.     

Mouse Uterine 24p3 Protein as a Suppressor of Sperm Acrosome Reaction

Under in vitro conditions, incubation with 0.3% bovine serum albumin (BSA) and 1.8 mM CaCl₂ induces mouse sperm capacitation and increases the consequential acrosome-reaction. The effect of mouse uterine 24p3 protein on such stimulated sperm has been investigated to understand the biological function of the 24p3 protein. Variations in the intracellular pH (pHi), calcium concentration, cAMP levels and tyrosine phosphorylation in cytosol were determined and on in vitro mouse fertilization was evaluated. The presence of 24p3 protein reduced the response of sperm to BSA and calcium by suppressing the elevation of intracellular pH, calcium uptake, cAMP accumulation and protein tyrosine phosphorylation of BSA/calcium-stimulated sperm and showed inhibitory effect on mouse in vitro fertilization. The results indicated the inhibition of the BSA-stimulated sperm acrosome reaction by 24p3 protein then suppressed sperm fertilization. We suggested that the 24p3 protein acts as an in vitro inhibitor of the acrosome reaction in BSA stimulated sperm and this might be an anti-fertilization factor in vitro.     

THE DENDRITIC CELL: ITS POTENT ROLE IN THE RESPIRATORY IMMUNE RESPONSE

The importance of the dendritic cell for the capture of antigens and initiating an immune response is now well recognized. Whereas much is known about their structure and function, their lineage is still not clear. Studiesin vitrohave demonstrated that the regulated maturation of function that occurs in culture explains many of thein vivoevents relating to antigen capture and presentation. The control over maturation and migration of these cells to the immune system is decisive as to whether an immune response is mounted or not. ‘Danger ’ signals provided by conserved bacterial products or by microenvironmental cytokines are important regulators. Dendritic cells have been clearly involved in the development of respiratory disease and our understanding of their involvement will have an impact on our future therapeutic strategies.     

Studies of antigen-RNA and immunity

Summary This review is about the involvement of antigen, and more specifically that of antigen-RNA complexes, in the inductive phase of an immune response. Early studies are reviewed that were begun by the senior author more than twenty years ago to probe the question of the role of antigen in antibody production in rabbits. It was shown that the quantity of antigen declined with time after injection but persisted at significant levels for very long intervals of time after injection, suggesting that all the antigen may never be lost. Since the liver yielded more detectable antigen than any other tissue it was the main source of in vivo antigen for biological and chemical characterization. As more extensive experiments were undertaken these showed stability of the radioactive hapten labels used to detect antigen and also the dynamic involvement of the liver in handling antigen. A body of evidence was thus accumulating that implied, at least indirectly, an immunological role for the liver-retained antigen. As for the antigen findings, one of the earliest was that antigen persisted, not in the original form of the injected protein, but as antigen fragments associated with ribonucleic acid, from whence originated the term antigen-RNA complexes. In later studies that utilized deproteinizing techniques, aimed at obtaining pure ribonucleic acid, antigen peptide was found to persist as a moiety of the isolated nucleic acid, thus providing convincing evidence for the in vivo origin of these complexes. The antigen-RNA complexes were immunologically active by both in vivo and in vitro tests. Recent experiments, using rats injected with aniline-azo-BSA have extended the earlier findings and it is these studies upon which attention will be focused in the experimental section. Liver polysomes and isolates of pure hepatocytes have been found to contain antigen material associated with RNA, and studies are in progress using these sources of material for elucidation of antigen-RNA function. It seems timely to suggest that an equally important application may be the use of these products of in vivo metabolism as probes to study the many inductive phenomena in an immune response.Abbreviations Ag antigen - BGG bovine gamma globulin - BSA bovine serum albumin - cFA complete Freund's adjuvant - ³H-BGG ³H-aniline-azo-BGG - ³H-BSA ³H-aniline-azo-BSA - ³H-RSA ³H-aniline-azo-rat serum albumin - ip intraperitoneal - iv intravenous - KLH keyhole limpet hemocyanin - np nucleopeptide - RSA rat serum albumin - [³⁵S]-BSA [³⁵S]-sulfanilate-azo-bovine serum albumin - S-BSA sulfanilate-azo-bovine serum albumin     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was performed to clarify the roles of primary and secondary injections of antigen and adjuvant (capsular polysaccharide of Klebsiella pneumoniae, CPS-K) in induction of antibody responses and in development of immunological memory in mice to bovine serum albumin (BSA). A primary injecion of BSA alone neither induced significant primary antibody response nor increased immunological memory for a secondary antibody response but, if primary injections of BSA and CPS-K were performed simultaneously, high antibody responses were induced. Moreover, a prior injection of BSA alone or CPS-K alone decreased the level of primary antibody response and the degree of increase in memory following the subsequent injection of BSA mixed with CPS-K. In contrast, a secondary injection of BSA alone into mice once primed with a mixture of BSA and CPS-K elicited very high secondary type antibody response and increased secondarily the memory for a tertiary antibody response. Injection of CPS-K simultaneously with or shortly before or after the secondary injection of BSA did not increase the level of the secondary antibody response and the degree of the secondary increase in memory. Augmentation of the secondary antibody response was elicited by simultaneous injection of CPS-K only when the secondary response was induced inadequately by a suboptimum or supraoptimum dose of antigen.     

Alternate Changes of Surface Antigen(s) in Adenovirus Type 12-Transformed and Tumor Cells1

Alternate changes of specific surface antigen(s) (S antigen) were examined in transformed and tumor cells induced by human adenovirus type 12. All of the hamster and mouse cells transformed in vitro showed ring-form membrane fluorescence staining by anti-S antigen rabbit sera, whereas tumor cells, either induced by the virus in vivo or produced by inoculation with the S(+)-transformed cells, did not show any fluorescence. When the S(–) tumor cells were serially subcultured in vitro, all of them converted to S(+) cells, although more than ten subcultures were necessary. For the S(+) cells to form tumors in hamsters about ten times as many cells were necessary as the S(–) cells. This difference became greater when tumor formation was tested in preimmunized hamsters, while little, if any, when tested in X-irradiated hamsters. In addition, immunogenicity of the S(+) cells was suggested to be higher than that of the S(–) cells. These findings indicate that the S(+) cells are more immunosensitive and immunogenic than the S(–) cells, and that in vivo conversion from S(+) to S(–) may be due to selection of S(–)-mutant cells. In vitro conversion from S(–) to S(+) was also suggested to be due to the appearance of S(+)-mutant cells.     

Toxicity testing of polymer materials for dialysis equipment: is there any need forin vivo testing?

In an earlier work, slightly more than 650 plastic materials, intended for use in medical devices, were tested with a battery of chemical, as well asin vitro andin vivo biological tests. An analysis showed that only a limited number of the tests used were actually necessary to obtain the same pass or fail decision as that obtained using the full test battery. This prompted us to prescreen all new materials with a small test battery consisting of the two most discriminating chemical tests and anin vitro cell growth inhibition test. The present work is a report of our findings after testing another 155 materials using this prescreen system.For each single one of the 155 tested materials the same decision on whether or not to use the material in the intended medical device would have been reached without anyin vivo testing. In no single case in a total of 851in vivo tests did an eluate that had passed thein vitro cell test give rise to a reactionin vivo. Thus, among the tests on living systems the cell test alone seems to be sensitive enough to provide sufficient information. Nothing appears to be gained from thein vivo animal tests. However, some of the materials that passed the prescreening tests later failed in one or several of the chemical tests. Both nonspecific chemical tests and tests for specific molecules seem to detect undesirable levels of leachable substances not detected by the prescreening system. Therefore these tests should not be abandoned. Abandoning unnecessaryin vivo testing, on the other hand, would save considerable costs.     

The histochemical demonstration of aniline hydroxylase activity in rat liver

Synopsis A procedure is described for the histochemical demonstration of aniline hydroxylase activity in cryostat sections of rat liver. Tissue sections are incubated in a medium containing aniline; thep-aminophenol formed as a result of enzymatic action is coupledin situ with Fast Blue RR. The staining reaction is found to be confined to the cytoplasm of the hepatocytes. Confirmatory tests for true enzymatic staining reaction include the incubation of sections in medium from which aniline is omitted, and under conditions of enzyme inhibibition. A method for the quantitation of the histochemical staining reaction is also described.The histochemical reactions have been investigated on rat livers subjected to conditions eliciting microsomal enzyme stimulation and inhibition, bothin vitro andin vivo. A close correlation was found between the staining reactions observed and the results of the quantitative histochemical method and the biochemical estimations of aniline hydroxylase activity in liver microsomal fractions obtained by differential centrifugation.     

Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro

Thermally aggregated, endogenous proteins of Escheri-chia coli form a distinct fraction, denoted S, which is separable by sucrose-density-gradient centrifugation. It was shown earlier that DnaK, DnaJ, IbpA and IbpB heat-shock proteins are associated with the S fraction. Comparison of the rise and decay of the S fraction in mutants defective for heat-shock proteases Lon (La), Clp, HtrA (DegP, Do) and in wild-type strains made studies of proteolysis and the function of the heat-shock response possible in vivo. Different timing and the extent of action of particular proteases was revealed by the initial size and decay kinetics of the S fraction. The proteases Lon, Clp, and HtrA all participated in removal of the aggregated proteins. Mutation in the gene encoding ClpB caused the most prominent effect (47% stabilization of the S fraction). The correlation between the disappearance of the S fraction and proteolytic activity was supported by the result of the in vitro reaction. Approximately one third of the isolated S fraction was converted to trichloroacetic acid-soluble products by the purified HtrA protease. Mg²⁺ ions stimulated the reaction, in contrast to the reaction of the HtrA protease with casein. The digestion of the aggregated proteins, unlike the digestion of casein, by HtrA protease in vitro was inhibited by added DnaJ, which might reflect protection of the aggregated proteins in vivo by DnaJ from excessive degradation. One might expect that such an activity of DnaJ would promote denatured protein renaturation versus proteolysis. Moreover, among the aggregated proteins that are discernible by electrophoresis, none could be identified as being more susceptible than any other to HtrA degradation. The separation pattern of these proteins before and after the in vitro digestion did not show a difference corresponding to the loss of about 30% of constituting proteins. This was interpreted as recognition by the HtrA protease of a state of protein denaturation rather than specific amino acid sequences in particular proteins. We conclude that the fraction consisting of proteins heat- aggregated in vivo (i.e. the S fraction) contains endogenous substrates for the heat-shock proteases tested. Their use for in vitro reaction reveals information that is in some respects different from that obtained with exogenous substrates such as casein.     

Relationships between nitrate level,nitrate reductase activity and anaerobic nitrite production inPisum sativum leaf tissue

Anaerobic nitrite production (thein vivo NO₃-R activity) in an incubation medium lacking exogenous nitrate but containing 0.5%n-propanol and 0.1% Triton X-100 showed higher correlation (y - ax ^b) with the level of endogenous nitrate inPisum sativum L. leaves than thein vitro nitrate reductase activity. Thein vivo NO₃-R activity correlated well with thein vitro activity up to the 50 ppm NO₃-N level of endogenous nitrate. The ratioin vivo: in vitro activity slightly decreased with increasing level of endogenous nitrate in leaf tissue.     

In VitroRearing ofTrichogramma minutumRiley (Hymenoptera: Trichogrammatidae) for Ten Generations, with Quality Assessment Comparisons ofin Vitroandin VivoReared Adults

Trichogramma minutumRiley were reared for 10 generations on an artificial diet containing a yeast extract, FreeAmine III, nonfat dry milk, chicken egg yolk, chicken embryo extract, andManduca sexta(L.) egg liquid. Quality control parameters, including adult longevity, sex ratio, pupation rate, percentage of pupae to emerge as adults, adult female body length, number ofHelicoverpa zea(Boddie) eggs parasitized by a female, and percentage of deformed females were assessed and compared to insects rearedin vivoon irradiatedH. zeaeggs. The development time was longer forin vitroreared insects, but there were more deformed females in thein vitroculture. The sex ratio, however, was generally not significantly different between thein vitroandin vivocultures. Thein vitroreared females generally were larger, lived longer, and parasitized moreH. zeaeggs. Emergence of adults was in excess of 75% in all but the firstin vitrogeneration and was generally not significantly different from adult emergence in thein vivoculture. These findings will be of value in the development of a practical system forin vitromass rearing ofTrichogrammafor use in biological control.     

Diphtheria toxin inhibits the synthesis of myelin proteolipid and basic proteins by peripheral nerve in vitro

Abstract— Diphtheria toxin (DT) did not produce measurable degradation of myelin proteins or sulphatide in sciatic nerves of chick embryos after incubation in vitro for 4 h. In contrast, DT inhibited the in vitro incorporation of L-[U-¹⁴C]leucine into myelin proteins by the nerves after a delay of 1 h. Separation of the myelin proteins by SDS-polyacrylamide gel electrophoresis indicated that the synthesis of Wolfgram proteins and proteins not entering the gel was inhibited by 21–22 per cent, whereas synthesis of myelin proteolipid and basic proteins was inhibited by 79–88 per cent. Incorporation of ³⁵SO₄ into myelin [³⁵S]sulphatide was also inhibited by DT after a delay of 2 h. The inhibition of [³⁵S]sulpha-tide incorporation into myelin caused by DT differed from that observed with puromycin in that it did not depend on depletion of an intracellular transport lipoprotein. Instead, the inhibition seemed to be secondary to the decreased synthesis of myelin proteolipid and basic proteins.