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A new strategy for the hapten design of natural glycoside and application for the preparation of antibody is reported in this work. With astragaloside IV (AGS-IV) as an example, C6"-CH 2 OH on a glucosyl group was selectively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation to C6"-COOH, which was subsequently condensed with -NH 2 on bovine serum albumin to get artificial antigen. Then, the successful preparation of artificial antigen was verified by TCL, SDS-PAGE, UV, and MALDI-TOF-MS. Finally, rabbits were immunized with artificial antigen to obtain an antibody against AGS-IV. After tests of the titer, IC 50 , and cross-reactivity, the results showed that the antibody prepared by TEMPO oxidation in this work had higher specificity than that the antibody prepared by conventional sodium periodate (NaIO 4 ) oxidation. The hapten, as a carboxylic acid derivative of AGS-IV, has better water solubility than AGS IV, which is more suitable for the synthesis of the hapten-carrier protein conjugate in aqueous phase, achieving another virtue of TEMPO oxidation over NaIO 4 oxidation. This new strategy provides new ideas for the design of haptens of other natural glycosides, as well as the preparation of their antibodies., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gut mucosal immune responses are known to act as the first line of defense against invasion of pathogenic microorganisms. Piglets have an incompletely developed gut mucosal immune system, making them sensitive to intestinal infections. Promoting the development of the mucosal immune system will increase the pathogen resistance of piglets. The aim of the present study was to investigate the effect of carotenoid (4,4'-diaponeurosporene)-producing Bacillus subtilis (B.s-Dia) on intestinal mucosal immunity in piglets. We showed that oral administration to piglets of B.s-Dia remarkably improved the development of Peyer's patches (PPs) (P < 0.01), and increased villus height (P < 0.01) and colon crypt depth (P < 0.01). In addition, B.s-Dia also increased the number of intraepithelial lymphocytes (P < 0.01), while Bacillus subtilis (B.s) had no significant influence on it (P > 0.05). Moreover, B.s-Dia also increased the number of SIgA + cells (P < 0.01). Oral administration of either B.s or B.s-Dia increased the number of CD4 + and CD8 + cells in ileum lamina propria (P < 0.01). These results indicate that B.s-Dia contributes to a higher extent to porcine mucosal immune system development than B.s, and might serve as an immunopotentiator candidate. Anat Rec, 302:1800-1807, 2019. © 2019 American Association for Anatomy., (© 2019 American Association for Anatomy.)

The Neisseria meningitidis factor H binding protein (FHbp) is an important virulence factor and vaccine antigen contained in both USA licensed serogroup B meningococcal vaccines. Recent studies in human factor H (hFH) transgenic mice suggest that hFH-FHbp interactions lower FHbp-elicited immunogenicity. To provide tools with which to characterize and potentially improve FHbp immunogenicity, we developed an FHbp-cholera holotoxin-like chimera vaccine expression system in Escherichia coli that utilizes cholera toxin B (CTB) as both a scaffold and adjuvant for FHbp. We developed FHbp-CTB chimeras using a wild-type (WT) FHbp and a low hFH-binding FHbp mutant R41S. Both chimeras bound to G M1 ganglioside and were recognized by the FHbp-specific monoclonal antibody JAR4. The R41S mutant had greatly reduced hFH binding compared to the WT FHbp-CTB chimera. WT and R41S FHbp-CTB chimeric antigens were compared to equimolar amounts of FHbp admixed with CTB or FHbp alone in mouse immunogenicity studies. The chimeras were significantly more immunogenic than FHbp alone or mixed with CTB, and elicited bactericidal antibodies against a panel of MenB isolates. This study demonstrates a unique and simple method for studying FHbp immunogenicity. The chimeric approach may facilitate studies of other protein-based antigens targeting pathogenic Neisseria and lay groundwork for the development of new protein based vaccines against meningococcal and gonococcal disease., (Published by Elsevier Ltd.)

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 μg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

Ricin is one of the most potent and lethal toxins known to which there is no available antidote. Currently, the most promising therapy is based on neutralizing antibodies elicited by active vaccination or given passively. Here, detailed protocols are provided for the production of two ricin holotoxin-based vaccines: monomerized subunit-based vaccine, and a formaldehyde-based ricin toxoid vaccine. Both vaccines were found to be stable with no toxic activity reversion even after long-term storage while eliciting high anti-ricin antibody titers possessing a potent neutralizing activity. The use of these vaccines is highly suitable for both the production of sera that can be used in passive protection experiments and immunization aimed to isolate potent anti-ricin monoclonal antibodies.

Ricin, one of the most potent and lethal toxins known, is classified by the Centers for Disease Control and Prevention (CDC) as a select agent. Currently, there is no available antidote against ricin exposure, and the most promising therapy is based on neutralizing antibodies elicited by active vaccination or that are given passively. The aim of this study was to characterize the repertoire of anti-ricin antibodies generated in rabbits immunized with ricin toxoid. These anti-ricin antibodies exhibit an exceptionally high avidity (thiocyanate-based avidity index, 9 M) toward ricin and an apparent affinity of 1 nM. Utilizing a novel tissue culture-based assay that enables the determination of ricin activity within a short time period, we found that the anti-ricin antibodies also possess a very high neutralizing titer. In line with these findings, these antibodies conferred mice with full protection against pulmonary ricinosis when administered as a passive vaccination. Epitope mapping analysis using phage display random peptide libraries revealed that the polyclonal serum contains four immunodominant epitopes, three of which are located on the A subunit and one on the B subunit of ricin. Only two of the four epitopes were found to have a significant role in ricin neutralization. To the best of our knowledge, this is the first work that characterizes these immunological aspects of the polyclonal response to ricin holotoxin-based vaccination. These findings provide useful information and a possible strategy for the development and design of an improved ricin holotoxin-based vaccine., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Aim: Ursolic acid (UA) is a pentacyclic triterpenoid found in most plant species, which has been shown anti-inflammatory and anti-oxidative activities. In this study, we examined the effects of UA on collagen-induced arthritis (CIA) in mice, and to identify the mechanisms underlying the effects., Methods: CIA was induced in mice. Two weeks later, the mice were treated with UA (150 mg/kg, ip, 3 times per week) for 4 weeks. The expression of cytokines and oxidative stress markers in joint tissues was measured with immunohistochemistry. The numbers of CD4+IL-17+, CD4+CD25+Foxp3+ and pSTAT3 cells in spleens were determined using confocal immunostaining or flowcytometric analyses. Serum antibody levels and B cell-associated marker mRNAs were analyzed with ELISAs and qRT-PCR, respectively. CD4+ T cells and CD19+ B cells were purified from mice spleens for in vitro studies., Results: UA treatment significantly reduced the incidence and severity of CIA-induced arthritis, accompanied by decreased expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-21 and IL-17) and oxidative stress markers (nitrotyrosine and iNOS) in arthritic joints. In CIA mice, UA treatment significantly decreased the number of Th17 cells, while increased the number of Treg cells in the spleens, which was consistent with decreased expression of pSTAT3, along with IL-17 and RORγt in the splenocytes. In addition, UA treatment significantly reduced the serum CII-specific IgG levels in CIA mice. The inhibitory effects of UA on Th17 cells were confirmed in an in vitro model of Th17 differentiation. Furthermore, UA dose-dependently suppressed the expression of B cell-associated markers Bcl-6, Blimp1 and AID mRNAs in purified CD19+ B cells pretreated with IL-21 or LPS in vitro., Conclusion: UA treatment significantly ameliorates CIA in mice via suppression of Th17 and differentiation. By targeting pathogenic Th17 cells and autoantibody production, UA may be useful for the treatment of autoimmune arthritis and other Th17-related diseases.

Astragaloside IV is the chief ingredient of Radix Astragali, which has been used in the Traditional Chinese Medicine as a major component of many polyherbal formulations for the repair and regeneration of injured organ and tissues. We tested the anti-asthmatic effects of AST IV and the possible mechanisms. BALB/c mice that were sensitized and challenged to ovalbumin (OVA) were treated with AST IV (40mg/kg and 20mg/kg) 1h before they were challenged with OVA. Our study demonstrated that AST IV inhibited OVA-induced increases in eosinophil count; interleukin (IL)-4 level were recovered in bronchoalveolar lavage fluid increased IFN-γ and IL-10 levels in bronchoalveolar lavage fluid. Histological studies demonstrated that AST IV substantially inhibited OVA-induced eosinophilia in lung tissue. Flow cytometry studies demonstrated that AST IV substantially increased CD4(+)CD25(+)Foxp3 T cells (Treg). Furthermore quantitative real-time (qPCR) studies demonstrated that AST IV substantially enhanced Foxp3 mRNA expression in lung tissue. These findings suggest that AST IV may effectively ameliorate the progression of airway inflammation and could be used as a therapy for patients with allergic inflammation., (Copyright © 2014. Published by Elsevier GmbH.)

In our search for immune stimulating materials from natural source, bioassay-guided fractionation of a methanol extract of Panax ginseng leaves led to the isolation of three dammarane triterpenes (1-3), including two previously unknown compounds 27-demethyl-(E,E)-20(22),23-dien-3β,6α,12β-trihydroxydammar-25-one (1) and 3β,20(S)-dihydroxydammar-24-en-12β,23β-epoxy-20-O-β-D-glucopyranoside (2). Their structures were elucidated on the basis of spectroscopic methods, chemical transformation, and by the comparison with those of literature data. Compounds 1-3 significantly increased interleukin-12 expression in LPS-activated mouse peritoneal macrophage at a concentration of 100 ng/mL. Furthermore, compound 1 strongly increased the Th1 response-mediated cytokine IL-2, and decreased Th2 response-mediated cytokines IL-4 and IL-6 expression at 100 ng/mL on ConA-activated splenocytes. This study indicated that compound 1 showed a better effect on cellular immunity, and provided new chemical entities as promising lead compounds for the treatment of cellular immunity-related diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Centella asiatica (L.) Urban consists of two major triterpene glycosides, asiaticoside (AS) and madecassoside (MA), as active components used for wound healing and enhancing memory., Objective: To produce a polyclonal antibody against madecassoside (MA-PAb) and develop enzyme-linked immunosorbent assay (ELISA) and Eastern blotting methods for quantitative analysis of triterpene glycosides in Centella asiatica., Methods: An ELISA method was developed using polyclonal antibody against MA. An Eastern blotting method on the PES membrane was established for determination of MA and AS. The immunoassays were validated for sensitivity, precision, specificity and accuracy., Results: The prepared MA-PAb shows specificity to MA and AS. The measuring range of triterpene glycosides was 0.39-50 µg/mL using the ELISA method. An Eastern blotting method was developed for determining individual MA and AS, which could be detected in the range of 62.5-500 ng. The limit of detection for MA and AS was 31.25 ng. The two methods developed showed good specificity, precision, and accuracy, and also correlated with high-performance liquid chromatography., Conclusion: These immunoassays have several advantages that include high sensitivity as well as being rapid and facile for determination of the triterpene glycosides in C. asiatica., (Copyright © 2012 John Wiley & Sons, Ltd.)

Two triterpenoids ursolic acid (1) and lupeol (2) isolated and characterized from Eucalyptus tereticornis and Gentiana kurroo were subjected to in silico QSAR modeling and docking studies and later the predicted results were confirmed through in vivo experiments. QSAR modeling results showed that both the triterpenoids possess immunomodulatory and anti-inflammatory activity comparable to boswellic and cichoric acids, but were less active than levamisol. Docking results suggested that both the triterpenoids (1 and 2) showed immune modulatory and anti-inflammatory activity due to high binding affinity to human receptors viz., NF-kappaB p52 (-50.549 kcal/mol), tumor necrosis factor (TNF-alpha) (-47.632 kcal/mol), nuclear factor NF-Kappa-B P50 (-16.798 kcal/mol) and cyclooxygenase-2 (-55.244 kcal/mol). Further both the triterpenoids (1 and 2) were subjected to in vivo immunomodulatory activity in female Swiss albino mice. The experimental mice were divided into nine groups, each comprised of six mice. These received oral treatment for a period of 28 days. The triterpenoids (1 and 2) showed significant increased in humoral immune function, but no significant changes were observed in cell mediated immune response and hematological parameters. The in silico and in vivo experimental data suggested that both the triterpenoids 1 and 2 may be considered as potential immunomodulatory drug-like molecules., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A triterpene acid mixture consisting of oleanolic, ursolic and betulinic acid isolated from a standardized rose hip powder (Rosa canina L.) has been shown to inhibit interleukin (IL)-6 release from Mono Mac 6 cells. The present study examined the effects of the triterpene acid mixture on the cytokine production and proliferation of CD4⁺ T cells and CD19⁺ B cells induced by a self-antigen, human thyroglobulin and by lipopolysaccharide in cultures of normal mononuclear cells. The triterpene acid mixture inhibited the production of tumor necrosis factor-α and IL-6 with estimated IC₅₀ values in the range 35-56 µg/mL, the Th1 cytokines interferon-γ and IL-2 (IC₅₀ values 10-20 µg/mL) and the antiinflammatory cytokine IL-10 (IC₅₀ values 18-21 µg/mL). Moreover, the mixture also inhibited CD4⁺ T-cell and CD19⁺ B-cell proliferation (IC₅₀ value 22 and 12 µg/mL, respectively). Together, these data demonstrate that oleanolic, ursolic and betulinic acid are active immunomodulatory constituents of the standardized rose hip powder. However, since the estimated IC₅₀ values are in the µg/mL range, it is questionable whether the content of the triterpene acids in the standardized rose hip powder, alone, can explain the reported clinical effects., (Copyright © 2011 John Wiley & Sons, Ltd.)

The adjuvant activity of Chenopodium quinoa (quinoa) saponins on the humoral and cellular immune responses of mice subcutaneously immunized with ovalbumin (OVA) was evaluated. Two quinoa saponin fractions were obtained, FQ70 and FQ90, and 10 saponins were determined by UPLC/Q-TOF-MS. Mice were immunized subcutaneously with OVA alone or adjuvanted with Quil A (adjuvant control), FQ70, or FQ90. FQ70 and FQ90 significantly enhanced the amount of anti-OVA-specific antibodies in serum (IgG, IgG1, and IgG2b) in immunized mice. The adjuvant effect of FQ70 was significantly greater than that of FQ90. However, delayed type hypersensitivity responses were higher in mice immunized with OVA adjuvanted with FQ90 than mice treated with FQ70. Concanavalin A (Con A)-, lipopolysaccharide-, and OVA-stimulated splenocyte proliferation were measured, and FQ90 significantly enhanced the Con A-induced splenocyte proliferation. The results suggested that the two quinoa saponin fractions enhanced significantly the production of humoral and cellular immune responses to OVA in mice.

Ginsenosides, the major active component of American ginseng, were analyzed using eastern blotting with anti-ginsenoside Rb(1) and Rg(1) monoclonal antibodies (MAbs). Immunoassay-guided fractionation of the methanol extract of American ginseng and column chromatography led to the isolation of two new minor dammarane-type saponins, named quinquenosides Ja (1) and Jb (2). Their structures were elucidated to be 6-O-[α-L-rhamnopyranosyl(1→2)-β-D-glucopyranosyl]-20-O-[β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl]-3β,6α,12β,20β-tetrahydroxydammar-24-ene (1) and 3-O-[β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl]-20-O-{[α-L-arabinofuranosyl(1→6)-β-D-glucopyranosyl](1→6)-β-D-glucopyranosyl}-3β,12β,20β-trihydroxydammar-24-ene (2) on the basis of chemical and spectroscopic methods.

Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA. Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity., Results: The cucumarioside A2-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times., Conclusions: We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.

Introduction: In Ayurvedic medicines, Bacopa monnieri (L.) Wettst. (brahmi) is known as a medicinal plant used for memory enhancement. Its active compounds are classified as pseudojujubogenin and jujubogenin glycosides. Owing to the lack of chromophore in the saponin glycoside structures, HPLC-UV-vis gives low sensitivity for determination of such compounds. In the case of the detection of small amounts of saponin glycosides, immunological assay could be a suitable method., Objective: To develop and validate a sensitive enzyme-linked immunosorbant assay (ELISA) using monoclonal antibody (MAb) against bacoside A₃, the major jujubogenin glycoside found in brahmi., Methodology: An immunogen was prepared by conjugating bacoside A₃ with a bovine serum albumin (BSA). To determine its immunogenicity, the ratio of hapten in bacoside A₃-BSA conjugate was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). After immunisation in mice, hybridomas secreting MAbs against bacoside A₃ were produced by fusing the immunised splenocytes with SP2/0- Ag14 myeloma cells. The antibody was raised specifically against jujubogenin glycosides. The ELISA using anti-bacoside A₃ MAb was developed., Results: Bacoside A₃ in the range of 3.05-97.70 ng mL⁻¹ could be detected by ELISA using anti-bacoside A₃ MAb. The assay showed a detection limit of 0.48 ng mL⁻¹ (0.517 nm). The validation study showed that the method was precise, accurate and sensitive. Interestingly, the MAb showed cross-reactivity with the other jujubogenin glycosides, bacopaside X and IV. However, it did not show cross-reactivity with any of pseudojujubogenin glycosides., Conclusion: The study demonstrated that ELISA using anti-bacoside A₃ MAb can be used for determination of total jujubogenin glycosides in brahmi., (Copyright © 2011 John Wiley & Sons, Ltd.)

The 95 % ethanol extract of Astragalus has been demonstrated to have potent activity as an immunological adjuvant when administered with vaccines of various types. We endeavor here to identify the components of this extract that are responsible for this adjuvant activity. Mice were immunized with KLH conjugated to cancer carbohydrate antigens globo H and GD3 and cancer peptide antigen MUC1 combined with different Astragalus fractions or with commercially available Astragalus saponins and flavonoids. The antibody responses against cancer antigens and KLH were quantitated in ELISA assays, and toxicity was calculated by weight loss. Astragalosides II and IV were the most active components, but the toxicity of these two differed dramatically. Astragaloside II was the most toxic Astragalus component with 5-10 % weight loss at a dose of 500 µg while astragaloside IV showed no weight loss at all at this dose, suggesting that astragaloside IV might be utilized as an immunological adjuvant in future studies. Several flavonoids also had significant adjuvant activity. However, when the activities of these known immunologically active components of Astragalus (and of endotoxin) are calculated based on the extent of their presence in the 95 % ethanol extract, they provide only a small proportion of the immunological activity. This raises the possibility that additional uniquely active components of Astragalus may contribute to adjuvant activity, or that the adjuvant activity of Astragalus is greater than the activity of the sum of its parts., (© Georg Thieme Verlag KG Stuttgart · New York.)

Asiaticoside (AS), the major active component of Centella asiatica (L.) Urban, is used as a memory enhancer and for wound healing. We have successfully prepared monoclonal antibodies (MAbs) against AS, and developed an enzyme-linked immunosorbent assay (ELISA) system for its determination. AS was conjugated to the carrier protein bovine serum albumin (BSA), which acted as an immunogen. In order to confirm its immunogenicity, the ratio of hapten in the AS-BSA conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting MAbs against AS were produced by fusing splenocytes with the mouse myeloma cell line, SP2/0-Ag14. After the screening, anti-asiaticoside MAb 2B4 was obtained. Weak cross-reactivities occurred with madecassoside (7.08%), but no cross-reactivities were observed with other related triterpenoid glycosides (<0.01%). The assay was suitable for quantitating AS in the range of 0.78 to 50 µg mL(-1). A good correlation of AS concentrations in crude extracts of C. asiatica between ELISA and HPLC methods was obtained (r(2) = 0.999). The contents of AS in various cultivated C. asiatica samples were assayed by the newly established ELISA. The recovery rates of AS in the samples were in the range of 95-103% with coefficients of variation of <10%. The intra- and inter-assay variations were 3.9 and 4.5%, respectively. The ELISA method described should prove useful as an analytical tool for quality control and standardization of medicinal plants and pharmaceutical products containing AS.

Introduction: Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts., Objective: The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody., Methodology: The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane., Results: The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL., Conclusion: The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants.

A novel antigen carrier has been formulated on the basis of a cucumarioside-A2-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDG(At)) and Ulva fenestrate (MGDG(Uf)). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1:2:3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and gamma-interferon and delayed hypersensitivity reaction when compared to Freund's complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.

Platycodin D2 (PD2), a saponin isolated from the root of Platycodon grandiflorum, was evaluated for its haemolytic activity and adjuvant potential on the cellular and humoral immune responses to ovalbumin (OVA) in ICR mice. The HD(50) values of PD2 and Quil A were 18.57+/-1.37 and 5.76+/-0.23 microg/ml against 0.5% rabbit red blood cell, respectively, implicating the less haemolytic activity for PD2 than Quil A. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), or PD2 (25, 50, 75 or 100 microg) on Day 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS-) and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PD2 significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice (P<0.05, P<0.01 or P<0.001). OVA-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in serum were also significantly enhanced by PD2 compared with OVA control group (P<0.05, P<0.01 or P<0.001). Further, the effects of PD2 on mRNA expression of cytokines and transcription factors in Con A-stimulated mice splenocytes were evaluated by RT-PCR analysis. PD2 significantly promoted the mRNA expression of cytokines IL-2, IFN-gamma, IL-4, and IL-10 and transcription factors T-bet and GATA-3 (P<0.05, P<0.01 or P<0.001). In conclusion, the results suggest that PD2 could be safely used as adjuvant eliciting Th1 and Th2 immune responses.

New lupane beta-enaminoketones were synthesized by interaction of methyl 2-hydroxymethylene-3-oxolup-20(29)-en-28-oate with aliphatic amines. Immunotropic activity was found for some of these compounds.

Bacopa monnieri (L.) Wettst. (Brahmi) is an Ayurvedic medicinal plant used for centuries as a memory enhancer. Dammarane-type triterpenoid saponins classified as pseudojujubogenin and jujubogenin glycosides are reported as the active components in this plant. In this study, the monoclonal antibody (MAb) against bacopaside I, a major pseudojujubogenin glycoside found in Brahmi, was produced and characterised. Bacopaside I was conjugated with bovine albumin serum to prepare an immunogen. Hybridomas secreting a monoclonal antibody against bacopaside I was produced by fusing splenocytes from an immunised mouse and SP2/0-Ag14 myeloma cells. The results showed that the antibodies were raised specifically against pseudojujubonin glycosides. An enzyme-linked immunosorbant assay (ELISA) using anti-bacopaside I MAb was performed in the range of 1.95-62.5 ng/mL of bacopaside I, with a limit of detection of 0.5 ng/mL. The method was validated and the practical use of the ELISA for analysing saponin glycosides from Brahmi was demonstrated., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Bacopa monnieri (L.) Wettst. (Brahmi) is a medicinal plant used as a memory enhancer in Ayurvedic medicines. Its active components are triterpenoid glycosides namely pseudojujubogenin and jujubogenin glycosides. In order to analyze these saponin glycosides, an enzyme-linked immunosorbant assay (ELISA) was developed using polyclonal antibodies against bacopaside I, one of the pseudojujubogenin glycosides found in the plant. Bacopaside I was conjugated with a bovine albumin serum (BSA) to prepare an immunogen. The bacopaside I-BSA conjugate was immunized to a rabbit for producing polyclonal antibodies (PAbs). The results showed that the antibodies were raised specifically against pseudojujubogenin glycosides. An ELISA using anti-bacopaside I PAbs was performed in the range of 1.95-62.5 ng mL(-1) of bacopaside I and the limit of detection was 0.1 ng mL(-1). The method was validated and the applicability of the ELISA for analyzing saponin glycosides from Brahmi was demonstrated.

Protopanaxadiol saponins (Rg3, Rd, Rc, Rb1 and Rb2) and protopanaxatriol saponins (Rg1, Re and Rg2) isolated from the root of Panax ginseng C.A. Meyer were evaluated for their adjuvant effects on the immune responses to ovalbumin (OVA) in mice. BALB/c mice were subcutaneously injected twice at a 3-week interval with 10 microg of ovalbumin or 10 microg of OVA plus 50 microg of ginsenosides Rg3, Rd, Rc, Rb1, Rb2, Rg1, Re or Rg2 or Quil A (n=5). Blood samples were collected for measuring specific total-IgG, IgG1 and IgG2a, and splenocytes were harvested for determining lymphocyte proliferation as well as IFN-gamma and IL-5 production 2 weeks after the boosting. The results indicated that OVA-specific antibody responses were significantly higher in mice immunized with OVA co-administered with Rg1, Re, Rg2, Rg3 and Rb1 but not with Rd, Rc and Rb2 when compared with the control (immunized with OVA only). Significantly enhanced splenocyte proliferative responses to Con A, LPS and OVA as well as the production of both IL-5 and IFN-gamma stimulated by OVA were also detected in mice immunized with OVA co-administered with Rg1 but not with Rb1, Re and Rg3. Of the ginsenosides studied, Rg1, Re, Rg2, Rg3 and Rb1 have more potent adjuvant properties than the others, indicating that they are the major constituents contributing to the adjuvant activities of total ginseng saponins. Varieties of ginsenosides in adjuvant activity might be attributed to the varieties of molecular conformations determined by the side sugar chains attaching to their dammarane skeleton.

The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked immunosorbent assay. Immunoglobulins from selected antisera were immobilised on CNBr-activated Sepharose 4B. The immunoaffinity column was used for the purification of plant and plant cell culture extracts prior to their analysis by HPLC. Immunoaffinity chromatography enabled the selective concentration of taxoids and enhanced sample clean-up.

Endo-maleopimaric acid (MPA) is a contact allergen formed when colophonium is "modified" with maleic anhydride or fumaric acid. Previous patch testing showed a higher allergic response to petrolatum (pet.) preparations of MPA in amorphous form compared to MPA in crystalline form. In the present study, the impact of the physical form of MPA on the allergic response was investigated. Since the amorphous form is difficult to standardize, crystalline MPA mechanically incorporated or dissolved in pet. was used. A lower eliciting capacity was obtained from crystalline MPA, compared to that obtained from dissolved MPA, in guinea pigs intradermally induced with MPA. Using 3H-MPA a 3X difference in the dissolution into synthetic sweat from MPA dissolved in pet., compared to MPA mechanically incorporated, was demonstrated. A difference in bio-availability between dissolved and crystalline MPA could therefore be assumed. Crystalline MPA had a low sensitizing capacity compared to that seen for amorphous MPA in previous studies. The amorphous form of MPA is likely to have a larger surface area than crystalline MPA, with less ordered molecules, resulting in a higher dissolution rate and a greater bio-availability. Modified colophonium exists as amorphous solids and as viscous liquids. Thus, exposure will probably be to non-crystalline MPA and cases of contact allergy could be overlooked when screening with crystalline MPA.

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 microg) was intranasally (i.n.) inoculated with influenza vaccine (10 microg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3-14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 microg) was i.n. inoculated with the vaccine (10 microg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27-50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 microg) and influenza HA vaccine (1 microg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 microg) each and diphtheria-pertussis-tetanus (DPT) vaccine (10 microg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 microg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.

The influence of the triterpenoid saponins 1-10 has been investigated on murine spleenocytes in the lymphocyte transformation test and on murine macrophages in an phagocytosis assay. The lymphocyte transformation test and the phagocytosis assay showed that the tested compounds have no stimulating effect. However, a significant inhibition of lymphocyte proliferation by the triterpenoid saponins 2, 6 and 10 was demonstrated.

An enzyme-linked immunosorbent assay (ELISA) was developed for azadirachtin (aza), a biopesticide from the neem tree (Azadirachta indica A. Juss). The immunogen was synthesized by epoxidation using the furan ring in the aza molecule. Rabbits were immunized with either bovine serum albumin (BSA)-azadirachtin or ovalbumin (OA)-azadirachtin conjugate. Evaluation of the antisera by antibody capture assay showed that the antibody titer of antisera raised against OA-aza was 1:30,000. An indirect competitive ELISA was developed with BSA-azadirachtin as coating antigen and aza-specific antibodies raised against OA-aza immunogen. The immunoassay showed an inhibitory concentration (IC50) value of 75 ppb, with a range of detection from 0.5 to 1,000 ppb for azadirachtin [based on regression analysis, y= 85.87 (-18.89x); r2 = -0.97]. Cross-reactivity of the antibodies with 2 aza- derivatives (22,23-dihydro-23beta-methoxy azadirachtin and 3-tigloylazadirachtol) was 33 and 29%, respectively. The indirect competitive ELISA was validated and evaluated by quantitating aza in spiked agricultural commodities and from neem formulations. Azadirachtin was spiked into 5 different agricultural commodities: tomato, brinjal, coffee, tea, and cotton seed at 500 and 1,000 ppb and recovered at 62-100%. In samples drawn from 6 lots, the aza content in neem-seed kernels ranged from 0.1 to 0.15%; in commercial neem formulations the content ranged from 200 to 2,000 ppm. The method developed may be applied to environmental monitoring of aza and quality assurance studies of aza-based commercial formulations.

The crystal structure of the verotoxin 1 (VT1) B subunit complexed with a globotriaosylceramide (Gb(3)) analogue showed the presence of three receptor binding sites per monomer. We wished to study the effects of altering the three sites, singly or in combination, on animal toxicity and cytokine induction in vitro. We found that while the site 1 and 2 mutants were modestly (two- to sevenfold) reduced in their ability to cause disease in BALB/c mice, the site 3 mutant, W34A, was as toxic as VT1. However, all the double-mutant proteins, irrespective of which two sites were mutated, exhibited approximately a 100-fold reduction in their 50% lethal doses for mice. These results suggest that multivalent receptor binding is important in vivo and that all three binding sites make a similar contribution to the latter process. The triple-mutant holotoxin, F30A G62T W34A, administered intraperitoneally without adjuvant, stimulated a strong antibody response in BALB/c mice, and the immune sera neutralized the activity of VT1 in vitro. Induction of tumor neurosis factor alpha release from differentiated human monocytes (THP-1 cells) was relatively impaired for site 1 and site 2 but not site 3 mutants, suggesting an auxiliary role for the latter site in mediation of cytokine release in vitro. Cytotoxicity assays on undifferentiated THP-1 cells have also demonstrated the importance of sites 1 and 2 and the relatively small role played by site 3 in causing cell death. These data suggest an association between the cytotoxicity of the protein and its ability to induce cytokine release.

The immune responses to immunostimulating complexes (iscoms) containing recombinant Epstein-Barr virus (EBV) gp340 envelope protein was evaluated in BALB/c (H-2(d)) and CBA (H-2(k)) mice. Gp340-iscoms were used either with a low content of Quillaja triterpenoid adjuvant (L-iscoms) or supplemented with additional Quillaja adjuvant in the form of iscomatrix (S-iscoms). Class and subclass distribution of anti-gp340 antibodies, EBV-neutralizing antibodies, antigen-specific T cell proliferation and cytokine production were determined and these results compared to those obtained by immunization with non-adjuvated gp340. The H-2(d) and H-2(k) mice were characterized as low or high responders in respect to the level of specific anti-gp340 antibodies, secretion of IgG2a isotype, antigen-specific lymphoproliferative capacity, interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) production in the basic immunizations with gp340. While presentation of the antigen in iscom formulations with low levels of Quillaja triterpenoids induces a moderate enhancement of the immune responses in the low responder H-2(d) mice, supplementation with high levels of iscomatrix immunomodulator was required to enhance the immune responses in the high responder H-2(k) mice. In both mouse strains subcutaneous immunization with S-iscoms resulted in a significant increase of IgG1- and IgG2a-specific antibodies, as well as in strong antigen-specific proliferative response confirmed by the simultaneous cytokine production. The enhanced antigen-specific secretion of IL-2 and IFN-gamma together with the abrogation of IL-10 and the absence of IL-4 indicates that the responses were driven towards a Th1-type rather than Th2-type immune response. The S-iscom formulations minimized the differences in immune responses between the two mouse strains, but the capacity of immune sera to neutralize EBV transformation in vitro remained completely strain-dependent. These data indicate that immune responses generated by iscoms can be manipulated by altering the triterpenoid composition of the iscoms and that the levels of triterpenoids can determine whether or not a Th1-type response is made.

Evaluation of the efficacy of nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit (LTB) containing a trace amount of the holotoxin (LT) in inducing antibody responses among volunteers, which was conducted during the winter season of 1993-1994, is reported. A trivalent inactivated vaccine, composed of A/Yamagata/32/89 (H1N1), A/Kitakyusyu/159/93 (H3N2) and B/Bangkok/163/90 influenza virus strains, was used alone or together with the adjuvant, recombinant LTB supplemented with 0.5% recombinant LT (LTB*). The volunteers were divided into two groups: 73 volunteers (mean age 35.0 +/- 12.0 years) inoculated intranasally (i.n.) with LTB*-combined vaccine and 49 volunteers (37.9 +/- 11.3) inoculated i.n. with the vaccine alone. Vaccination was done twice 4 weeks apart. Salivary secretory IgA and serum hemagglutination-inhibiting (HI) antibodies were measured before and 8 weeks after the primary vaccination. For the sake of convenience, more than a 1.4-fold rise in IgA antibody response (units of specific IgA antibody per microgram of total IgA) and a fourfold or greater rise in HI antibody titer after vaccination were regarded as a positive antibody response. Thirty-seven (50.3%) and 36 (49.3%) of the 73 vaccinees, respectively, given the nasal LTB*-combined vaccine showed positive IgA and HI antibody responses to one or more of the three vaccine strains. In comparison, positive antibody responses in the group given vaccine alone were 32.7% for IgA and 30.6% for HI antibody. There was a significant difference between these two groups. These results suggest that the nasal LTB*-combined vaccine could enhance the production of higher levels not only of serum HI antibody but IgA antibodies in the respiratory tract than do the nasal vaccine alone.

QS-21, a purified Quillaja saponaria saponin immunologic adjuvant, contains two functional groups that we hypothesized to be involved in the adjuvant mechanism of action through charge or Schiff base interaction with a cellular target. Derivatives, prepared by modification of these sites, were prepared and tested for their ability to augment the immunogenicity of the antigen ovalbumin (OVA) in C57BL/6 mice. QS-21 derivatives that were modified at the carboxyl group on an anionic sugar, glucuronic acid, retained adjuvant activity for antibody stimulation, inducing relative increases in antibody titers similar to those induced by QS-21, although the minimum adjuvant dose required for this stimulation was increased several fold relative to the dose of unmodified QS-21. One of these derivatives also retained significant activity for induction of OVA-specific cytotoxic T-lymphocytes. In contrast, QS-21 derivatives modified at an aldehyde on the triterpene did not show adjuvant activity for antibody stimulation or for induction of cytotoxic T-lymphocytes, suggesting that this functional group may be involved in the adjuvant mechanism.

Escherichia coli heat-labile enterotoxin B subunit (LTB) (2 micrograms), supplemented with a trace amount of the holotoxin (LT) (0.02-20 ng), was examined for the adjuvant effect on antibody (Ab) responses against influenza inactivated haemagglutinin (HA) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation with the vaccine (1.5 micrograms), prepared from PR8 (H1N1) virus, together with LT-containing LTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The inoculation of the vaccine with the LT-containing LTB induced significantly high primary and secondary anti-HA IgA and IgG Ab responses in the nasal wash and the serum, while the vaccine with LTB or less than 2 ng of LT induced little response. The synergistic adjuvant effect was maximal in the concentration of LTB supplemented with 0.2-2 ng of LT. Under these conditions, the augmented IgA and IgG Ab responses, which are cross-protective to PR8 HA molecules, provided complete cross-protection against PR8 virus challenge in mice immunized with heterologous vaccine within the same subtype. These results suggest that LTB containing a trace amount of LT can be used as a potent adjuvant for nasal vaccination of humans against influenza.

Anti-10 deacetylbaccatin III (DAB) antibodies (IgY) were elicited in hens immunized with a succinyl-DAB/BSA conjugate and extracted from egg yolk. As shown by indirect competitive inhibition enzyme immunoassay (CIEIA), the addition of free-DAB competitively inhibited the binding of affinity purified anti-DAB IgY to DAB/BSA solid phase conjugated antigen. The assay enabled the detection of DAB in concentrations as low as 7.5ng/ml (13.7 nM DAB), whereas anti-DAB IgY did not react with taxol even at a concentration a thousand times higher. The structural requirements of the diterpenoid nucleus for binding to IgY were considered on the basis of the levels of cross-reaction found with 10 authentic taxanes. The results indicate that anti-DAB IgY represents the first high affinity antibody produced capable of recognizing the ring skeleton of taxol precursors.

A high-sensitive ELISA method was developed for the detection and semi-quantitative determination of 10-deacetylbaccatin III and its structurally related compounds in crude extract of Taxus sp. plants and tissue cultures. The antibodies were raised in rabbits using 7- or 10-succinyl-10-deacetylbaccatin III-BSA conjugate as immunogen. The working range of the assay was from 0.003 to 1.000 ng (0.09 to 31.33 nM) of 10-deacetylbaccatin III per assay. The cross-reacting material in crude plant extract was examined by chromatographic (silica gel CC, HPLC) and immunoassay methods. Study on the evaluation of cross-reacting material in crude Taxus plant extracts showed that at least 80% of the immunosignal correspond to 10-deacetylbaccatin III in the extract. The ELISA method was applied to investigate the 10-deacetylbaccatin III equivalent content in crude extracts of 19 plants species including Taxaceae, Taxodiaceae and Pinaceae species. The 10-deacetylbaccatin III-like structure was only detected in Taxus and Torreya sp. The results indicate that this immunoassay is a useful tool for the rapid screening of species, varieties or individual plants out of a wide population. The distribution of 10-deacetylbaccatin III equivalent content in 9-month old Taxus plantlets cultivated in vitro as well as in callus culture was investigated.

The sensitizing capacity of Centella asiatica (raw extract) and its triterpenic constituents asiaticoside, asiatic acid and madecassic acid has been studied in guinea pigs. The extract itself as well as the 3 acids were found to be very weak sensitizers. Centella asiatica extract is used effectively in the treatment of keloids, leg ulcers, phlebitis, slow-healing wounds, leprosy, surgical lesions, striae distensae and cellulitis. Although applied frequently to damaged skin, the risk of acquiring contact sensitivity to this plant or its constituents is low.

The influence of cucumarioside, triterpene glycoside obtained from Cucumaria japonica (Echinodermata, Holoturioidea), or sea cucumbers, on the resistance of mice to Bordetella pertussis infection (with the use experimental pertussis meningoencephalitis as a model) and on the development of immune response to corpuscular pertussis vaccine was studied. The preparation under test was shown to have greatly pronounced immunomodulating properties depending on both the concentration of cucumarioside and the route of its administration, as well as on the dose of pertussis vaccine. When administered orally in a dose of 4 micrograms per mouse and intraperitoneally in doses of 0.04 and 0.0004 micrograms, cucumarioside enhanced the protective effect of corpuscular pertussis vaccine. The use of cucumarioside in a dose of 0.001 micrograms per mouse abolished the suppressive action of large doses of pertussis vaccine in the background rosette-formation test at an early period after immunization and increased number of immune rosettes formed by lymphocytes in the spleen of mice immunized with different doses of the corpuscular vaccine.

The toxic triterpene acids lantadene A and lantadene B were isolated from Lantana camara and conjugated to bovine serum albumen or haemocyanin. The conjugates were emulsified with complete Freund's adjuvant and injected into sheep and cattle. Vaccinated animals produced antibodies against the toxic compounds. Cholestasis was less severe in vaccinated than in non-vaccinated sheep challenged with a toxic dose of lantana. The results indicated a mild protective effect of vaccination against the hepatotoxic effects of lantana toxins.

When injected intraperitoneally into mice in doses of 40.0-0.4 microgram, Cucumarioside, the preparation of triterpene glycosides obtained from sea cucumbers (Cusumaria japonica), enhanced the resistance of the animals to the subsequent challenge with Salmonella typhimurium. The study of the duration of the persistence of salmonellae in mice receiving the preparation in a dose of 0.001 microgram revealed a decrease in the contamination of their organs. The same dose of the preparation stimulated the phagocytic activity of peritoneal exudate cells with salmonellae showing decreased cytopathogenic action. This suggests that Cucumarioside enhances nonspecific protective factors, activates the macrophagal system and facilitates the development of complete phagocytosis.