Immunological memory to tetanus toxoid is established and maintained in the vitamin A-depleted rat

We have previously shown that vitamin A deficiency severely impairs the young rat's ability to produce specific antibodies after primary immunization with tetanus toxoid (TT). In the present studies, we asked whether immunologic memory to TT is established even in the vitamin A-depleted animal, and if so, whether such memory can be elicited after subsequent repletion with retinol. Vitamin A-depleted rats produced very low concentrations of TT-specific IgM and IgG antibodies in both the primary and secondary responses; however, the ratios of secondary to primary IgM anti-TT and of IgG anti-TT were normal. When rats were repleted with retinol 1 day after immunization, IgM and IgG anti-TT concentrations in both the primary and secondary responses were at least as great as those of control rats. For rats repleted with retinol 2 days before the booster immunization, secondary IgM and IgG anti-TT concentrations were equal in magnitude to those of vitamin A-sufficient controls. For all groups, the kinetics of the antibody response were similar. We conclude that immunological memory is intact in the vitamin A-depleted animal, as shown by 1) the normal ratio of its secondary to primary antibody responses, 2) the restoration of a quantitatively normal secondary antibody response in previously vitamin A-depleted animals repleted with retinol just before boosting with TT, and 3) a normal class switch from IgM to IgG. Retinol deficiency is also characterized by an abnormal elevation of total plasma IgG, despite the inability of the vitamin A-depleted animal to produce normal quantities of specific antibodies after challenge with antigen.     

Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens

The repertoire of natural anti-glycan antibodies in naive chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization.     

Splenic cathepsin L is maturated from the proform by interferon-gamma after immunization with exogenous antigens

The processing of foreign protein antigens into peptides requires the participation of various endo/lysosomal proteases in antigen-presenting cells (APCs). In this study, a proenzyme of cathepsin L, procathepsin L, was found to be present in the spleens of naive mice, as demonstrated by immunoblotting. Interestingly, the maturation of cathepsin L from procathepsin L was strongly induced when the host BALB/c mice were immunized with ovalbumin or soluble leishmanial antigen, despite the fact that mouse albumin, a kind of self-antigen, did not have such a potential. Furthermore, foreign antigens, but not self-antigens, could increase the activity of cathepsin L, probably being mediated by interferon-gamma, as demonstrated by in vivo and in vitro experiments. As cathepsin L matured, the efficiency of antigen processing was increased in APCs. These results suggest that endo/lysosomal cathepsin L plays an important role in the immune regulation via antigen processing even in peripheral lymphoid tissues as well as in the thymus.     

Detection of polysaccharide, teichoic acid, and protein antigens in bacterial colonies on an agar surface

An improved method of using fluorescein-labeled antibody for the detection of polysaccharide, protein, and teichoic acid antigens synthesized by streptococcal colonies on an agar surface is described. The bacteria were grown on the surface of an agar medium contained in the shallow well of an immunodiffusion slide. An agar overlay containing the fluorescein antiserum was dispensed over the colonies, excess antiserum was washed out of the overlay agar, and the fluorescent colonies were observed under an ultraviolet microscope. The shallow well in the immunodiffusion slide prevented the agar from floating loose during washing, and the agar overlay prevented the fragmentation and loss of colonies. The thin layer of agar facilitated microscopic examination and the counting of fluorescent and nonfluorescent colonies. Colonies producing an antigen against which the antiserum was directed could readily be distinguished from colonies not producing the antigen. The specificity of the method was shown by using mixtures of streptococci representing six serological groups and five types. Those not known to possess cross-reacting antigens were specific in their reaction to the fluorescein antibody. Cross-reactions between the group antigens of A, C, and G, as reported previously by fluorescent staining of streptococcal suspensions, were also seen. Group A colonies reacted weakly with fluorescent E antibody and vice versa. The extraction of this antigen with cold trichloroacetic acid indicates it was related to the teichoic acids. Colonies possessing polysaccharide, protein, and teichoic acid antigens gave equally strong fluorescent reactions. This procedure permits detection of the synthesis of antigen which could not be observed by the use of a selective medium; it also eliminates the necessity for subculture of each colony and testing by appropriate serological means. Such a technique has value for studies in classification and biochemical genetics, and should be applicable to other genera of bacteria.     

Whole-body basal nitric oxide production is impaired in postprandial endothelial dysfunction in healthy rats

In healthy humans, a high-saturated-fat/high-sucrose meal induces vascular endothelial dysfunction, a hallmark of atherogenesis. This transient dysfunction indicates a loss in nitric oxide (NO) production and/or bioactivity in the vasculature but it remains unknown if this is the local manifestation of a general impairment in NO pathway in the postprandial state. Here, we studied whole-body NO production and systemic NO bioactivity in postprandial endothelial dysfunction, as induced by a high-saturated-fat, high-sucrose meal.We first developed a physiological test of endothelial function on conscious rats, based on the transient fall in blood pressure after iv acetylcholine, and showed that this response was NO-dependent. As assessed with this method in healthy rats, endothelial function decreased during the postprandial state, being 60 ± 7% lower than baseline at 6 h after the meal challenge, associated with important elevations in plasma triglycerides and hydroperoxides. Aortic superoxide anion production, as assessed by oxidative fluorescent detection, was higher 6 h after the meal challenge than after the nutrients vehicle (water). During the postprandial period, plasma cGMP, but not plasma ANP, markedly decreased, indicating a general decrease in NO bioavailability, which was numerically maximal 4 h after the meal challenge. As determined 4 h after ingestion by a tracer-based method using iv [¹⁵N₂-(guanido)]-arginine, the whole-body NO production fell by 27 ± 9% postprandially.This is the first study evidencing that a meal challenge that impairs the stimulated, NO-mediated, vascular response also reduces whole-body basal NO production and bioavailability. Postprandial pathophysiology may build on this general, fundamental alteration in NO production.     

Generation of suppressor T cells after local immunization with histocompatibility antigens

Subcutaneous (sc) hind-foot immunization (HFI) of mice with allogeneic spleen cells can induce a state of delayed-type hypersensitivity (DTH) as well as a state of suppression of DTH. This paper deals with the suppression induced by HFI. The state of suppression could be adoptively transferred by spleen cells and lymph node cells between Days 3 and 7 after HFI only. However, in the hind-foot-immunized mice the state of suppression lasted at least 25 days. The suppressor cells expressed the Thy-1+, Lyt-1-2+ phenotype and suppressed DTH antigen-specifically. The suppressor cells, however, also suppressed DTH responses to unrelated third-party alloantigens, provided the latter were administered during the induction of DTH together with the same alloantigens that were used for HFI. The HFI-induced T-suppressor cells suppressed the induction phase of DTH (i.e., the proliferative activity of the draining lymph node cells after secondary sc immunization), but not the expression phase of DTH (i.e., the activity of previously activated DTH effector T cells). H-2D compatibility between the donors of the HFI-induced T-suppressor cells and the recipients was required for the adoptive transfer of suppression. The differences in effect of local immunization versus systemic immunization on the induction and functional activity of T-suppressor cells are discussed.     

Delayed hypersensitivity and granulomatous response after immunization with protein antigens associated with a mycobacterial glycolipid and oil droplets.

A myocardial glycolipid (P3) mixed with protein antigens in oil-in-water emulsion induced lasting delayed hypersensitivity (DH) and granulomatous inflammation after intradermal injection into guinea pigs. This did not occur when P3 and bovine serum albumin (BSA) were given in Freund's incomplete adjuvant. The oil-in-water emulsions consisted of microscopic oil droplets suspended in aqueous medium. By separating oil and aqueous phases from BSA + P3 emulsion it was shown that antigen retained with oil droplets led to DH and granuloma formation. The association of antigen with oil droplets was P3 dependent and was quantitated with 125I-labeled BSA. The same phenomenon occurred with 125I-labeled rabbit gamma-globulin (RGG) + P3 emulsion. Fluorescein-conjugated RGG was observed in a particulate state within or on oil droplets in emulsion containing P3. These physical characteristics of antigen + P3 emulsion appeared to be important for immunogenicity.     

Interferon- or interleukin-10 production is induced by related Trypanosoma cruzi antigens

The purpose of this study was to evaluate the effects of a crude Trypanosoma cruzi antigen (TCA) and its partially purified subfractions TCF1, TCF2 on peripheral blood mononuclear cells (PBMC) of normal donors and chagasic patients. TCFI and TCF2 stimulated cells from normal donors and chagasic patients in association with a significant production of interleukin (IL)-10. Only PBMC from chagasic patients multiplied after incubation with TCA and released mainly interferon-y but also IL-10. Neither the production of IL-2 and IL-4 nor CD4/CD8 ratios were changed after culture with antigens. These data suggest that some antigens active during the acute phase of T. cruzi infection would stimulate the production of cytokines that promote progression of infection, and the immune system can produce a desired cytokine(s) once the appropriate antigenic stimulus is used.     

Polyclonal antisera production by immunization with mixed cell antigens of different rhizobial species

Somatic antigens of Bradyrhizobium japonicum, Rhizobium sp. (Cicer arietinum) and Rhizobium sp. (Leucaena leucocephala) were prepared as standard, single-species type from cultured cells. Equal numbers of the cells of these rhizobia were then combined to obtain a mixed-rhizobial-species antigen preparation. Rabbits were immunized either with the standard, single-species type or with the mixed-rhizobial-species antigen preparations. The antisera developed from the mixed antigen immunization contained antibodies for all three rhizobial species, detectable at agglutination titres of over 800. The mixed-rhizobial-species antisera were made species specific by cross-absorption. The cross-absorbed and the mixed-rhizobial-species antisera were generally similar in quality for strain identification by agglutination, fluorescent antibodies, immunoblot and ELISA. A 66% reduction in cost was estimated for the production of antisera by immunization with mixed-rhizobial-species antigen.H.J. Hoben and P. Somasegaran are with the NifTAL Center and MIRCEN, University of Hawaii, 1000 Holomua Road, Paia, Maui, HI 96779-9744, USA: N. Boonkerd is with the School of Agricultural Technology, Suranaree University of Technology, University Avenue, Nakorn Racharsima, Thailand. Y.D. Gaur is with the Division of Microbiology, Indian Agricultural Research Institute, New Delhi-110012, India.     

Low IgG response of the mouse strain C57BL/10ScSn after immunization with protein antigens

The low IgG response of the strain C57BL/10ScSn is not restricted to the reaction to sheep red blood cells; but it can be demonstrated even after immunization with ARS, DNP or FITC haptens, coupled to various heterologous (BGG, RSA) or autologous (MGG) protein carriers. The level of the IgG response is - using the same immunization schedule - influenced both by the bound hapten and the carrier. In both strains tested (i.e. in the high-responding A/J and the low-responding C57BL/10ScSn), the highest IgG response is elicited by FITC-BGG. The response of the C57BL/10ScSn strain is, similarly as after immunization with SRBC, approximately ten times lower. The IgG response to other antigens tested was lower in both strains and therefore the quantitative differences were less pronounced. The affinity of antibodies against the ARS and TNP determinant, detected by inhibition of plaque-forming cells, is similar in the two strains. Thus the low reactivity of the strain C57BL/10ScSn is not caused by the absence of suitable VH and VL genes, but it rather indicates a defect of some general regulatory mechanism, involved in the synthesis of IgG antibodies. After repeated administration of ARS-BGG, the antigen and the antigen - antibody complexes accumulate in high concentrations primarily in the liver of mouse strain A/J. The amount of antigen accumulated in the liver of strain C57BL/10ScSn is significantly lower.     

Intranasal immunization with liposomes containing IL-2 enhances bacterial polysaccharide antigen-specific pulmonary secretory antibody response.

Secretory IgA (sIgA) present at mucosal surfaces such as the lungs and intestine plays an important role in resistance to infection occurring at these anatomic sites. Because IL-2 and IL-4 can augment B cell proliferation and Ig production, we investigated possible adjuvant effects of these cytokines on bacterial polysaccharide-specific pulmonary sIgA generation. As shown in previous studies, intranasal immunization with liposomes containing bacterial polysaccharide from Aerobacter levanicum and Pseudomonas aeruginosa resulted in increased numbers of bacterial polysaccharide-specific pulmonary plasma cells and sIgA titers, compared with those found in unimmunized mice. Inclusion of IL-2, but not IL-4, into the intranasally administered liposomes further increased titers of bacterial polysaccharide specific sIgA and pulmonary plasma cells. Intranasal vaccination with liposomes containing bacterial polysaccharide and 10 micrograms/kg IL-2 increased bacterial polysaccharide-specific pulmonary plasma cell numbers by more than 80-fold compared with the response in mice immunized with liposomes containing bacterial polysaccharide, but without IL-2. The percentage of pulmonary plasma cells producing antibody to polysaccharide from A. levanicum rose from 0.14% in mice intranasally immunized with liposomes containing only polysaccharide to 4.1% in animals vaccinated with liposomes containing polysaccharide and IL-2. Intranasal immunization with liposomes containing P. aeruginosa polysaccharide and IL-2 significantly reduced mortality from P. aeruginosa pneumonia. These results demonstrate that IL-2 has potent adjuvant effects on bacterial Ag-specific sIgA production in the lungs when included in intranasally administered liposomes.     

Effect of bacterial polysaccharide on the primary and secondary immunologic response following immunization with sheep erythrocytes

The effect of typhoid bacterial polysaccharide on the primary and secondary immune response to SRBS was studied. The polysaccharide was shown to have both stimulating and depressive effect on the population of antibody-producing cells. This effect depended on the time and the number of polysaccharide injections. Thus, a single polysaccharide injection made on the day preceding immunization resulted in the maximum stimulation in the system of IgM- and IgG-producing cells, while the maximum depression of these cells could be observed after 2 polysaccharide injections: on the day preceding immunization and on the day of immunization. In the secondary immune response considerable stimulation of the populations of antibody-producing cells was observed after polysaccharide injections made on days 2 and 3 after reimmunization.     

The association between EAE development in mice and the production of autoantibodies and abzymes after immunization of mice with different antigens

We have previously shown that immunization of C57BL/6 mice, prone to spontaneous development of experimental autoimmune encephalomyelitis (EAE), with three antigens (MOG_35-55, DNA-histone complex or DNA-methylated BSA complex), alters the differentiation profiles of bone marrow haematopoietic stem cells (HSCs). These are associated with the production of autoantibodies (auto-Abs) against these antigens and the formation of abzymes hydrolysing DNA, MOG, myelin basic protein (MBP) and histones. Immunization of mice with antigens accelerates the development of EAE. This work is the first to analyse the ratio of auto-Abs without and with catalytic activities at different stages of EAE development (onset, acute and remission phases) after immunization of mice with the three specific antigens. Prior to immunization and during spontaneous in-time development of EAE, the concentration of auto-Abs against MBP, MOG, histones and DNA and activities of IgG antibodies in the hydrolysis of substrates increased in parallel; correlation coefficients = +0.69-0.94. After immunization with MOG, DNA-histone complex or DNA-met-BSA complex, both positive (from +0.13 to +0.98) and negative correlations (from −0.09 to −0.69) were found between these values. Our study is the first showing that depending on the antigen, the relative amount of harmful auto-Abs without and abzymes with low or high catalytic activities may be produced only at onset and in acute or remission phases of EAE. The antigen governs the EAE development rate, whereby the ratio of auto-Abs without catalytic activity and with enzymatic activities of harmful abzymes hydrolysing MBP, MOG, histones and DNA varies strongly between different disease phases.