Synthesis and immunopotentiating activity of novel isoxazoline functionalized coumarins.

A novel series (13) of isoxazoline functionalized coumarins was synthesized through 1,3-dipolar cyclization of nitrile oxides with Allylated coumarins. Synthesis of effective and target selective immunostimulators through conjugation of diversely substituted isoxazolines and 7-hydroxycoumarins is the focus of the present article. The proposed synthetic scheme was observed to be highly regiospecific yielding attempted conjugates in good yield (>90%). Kinetic resolution of the racemates was carried out by employing lipase B from Candida antarctica (CALB). The synthesized compounds were screened in vitro and in vivo for their biological activities viz. toxicity and impact on splenocyte proliferation (T- and B-cell proliferation), antibody production (HA titre), delayed-type hypersensitivity reaction (DTH), T-cell subtypes (CD4 and CD8), cytokine production (IL-2, IFN-γ, and IL-4) and NO (macrophage) production. Our results establish that isoxazoline functionalized coumarins exhibit excellent immune potentiating activity especially compounds 2, 4 and 8 whose activity is more than that of Levimasole as standard. The structure activity relations are explained in light of the structural/functional aspects of tested compounds. To the best of our knowledge the presented work is first of its kind and is presaged to prove very useful for the design and synthesis of bis-heterocycle based novel, therapeutically selective and effective immunopotentiators.

Design and Synthesis of Novel 1,2,3-Triazole- and 2-Isoxazoline-Based Bis-Heterocycles as Immune Potentiators.

A series of novel bis-heterocycles encompassing isoxazolines and triazoles were synthesized through a novel one-pot procedure that involves in situ generation of nitrile oxide and its 1,3-dipolar cycloaddition to variedly substituted acrylates. The synthesized bis-heterocycles (8a-l) were subjected to in vitro lymphocyte proliferation assays followed by in vivo studies of the more active compounds (8g and 8h) to assess their influence on various aspects of the immune system like ex vivo splenocyte proliferation (T- and B-cell proliferation), antibody production (HA titer), delayed-type hypersensitivity reaction, T-cell subtypes (CD4 and CD8), cytokine production (IL-2, IFN-γ, and IL-4), NO (macrophage) production, and toxic effects. The results from the in vitro and in vivo studies establish that the tested compounds 8g and 8h possess excellent immunopotentiating activity.

Immunosuppressive potential of Botryosphaeria dothidea, an endophyte isolated from Kigelia africana.

CONTEXT: For years, natural products from microbes have been used as drugs. Endophytes are the most important fungi that produce many novel metabolites for potential use in pharmacology and agriculture. OBJECTIVE: The objective of the present study was to explore new endophytes for novel natural products. MATERIALS AND METHODS: An endophyte BAK-I was isolated from the bark of Kigelia africana (Lam.) Beneth (Bignoniaceae). BAK-I was characterized morphologically and on the basis of ITS-5.8S rDNA sequences. BAK-I was fermented to yield an extract, which was evaluated for its anticancer, antimicrobial, and immunomodulatory activities, using MTT, agar well-diffusion, tube dilution method, lymphocyte proliferation, and pro-inflammatory cytokines (TNF-α) (by macrophages) evaluation assays. For lymphocyte proliferation and pro-inflammatory cytokines studies, four concentrations were evaluated 10, 30, 100, and 1000 µg/mL and the experiments were conducted for 72 and 48 h, respectively. RESULTS AND DISCUSSION: The BAK-I showed pink cottony growth. SEM studies showed smooth fusoid-oblong conidia with a truncated base. Furthermore, ITS-5.8S rDNA sequence showed 99% homology with the Botryosphaeria dothidea strain suggesting that the endophyte is a strain of the genus Botryosphaeria. Less than 50% growth inhibition of SF295, Lung A-549, and THP-1 cancer cell lines after treatment with BAK-I extract suggested that it did not have significant cytotoxic potential, whereas it is bactericidal for Gram-positive pathogens MRSA and VRE with MIC value 200 and 250 µg/mL, respectively. To elucidate its immunomodulation potential, splenocyte proliferation studies showed that BAK-1 suppressed the T cell proliferation by 50%. TNF-α evaluation studies also showed that the extract inhibited TNF-α production in a concentration-dependent manner suggesting that it had immunosuppressive potential. Inhibition at 10 µg/mL was found to be 55% as against 48% using ß-methasone. CONCLUSION: The results suggested that BAK-I extract can be used as a potential immunosuppressive agent.

Pyrano-isochromanones as IL-6 inhibitors: synthesis, in vitro and in vivo antiarthritic activity.

Bergenin (1), a unique fused C-glycoside isolated from Bergenia species, possesses interesting anti-inflammatory and antipain activities. To study SAR of this scaffold, first-generation derivatives were synthesized and evaluated for inhibition of lymphocyte proliferation and production of pro-inflammatory cytokines. The C-7 substituted derivatives showed inhibition of IL-6 as well as TNF-α production. Bergenin and its most potent IL-6 inhibitor derivatives 4e and 4f were then investigated in a panel of in vitro and in vivo inflammation/arthritis models. These compounds significantly decreased the expression of NF-kB and IKK-ß in THP-1 cells. In in vivo study in BALB/c mice, a dose-dependent inhibition of SRBC-induced cytokines, reduction in humoral/cell-mediated immunity, and antibody titer was observed. The CIA study in DBA/1J mice indicated that compounds led to reduction in swelling of paws, cytokine levels, and anticollagen IgG1/IgG2a levels. The significant in vivo immunosuppressive efficacy of pyrano-isochromanones demonstrates the promise of this scaffold for development of next-generation antiarthritic drugs.

Protective efficacy of piperine against Mycobacterium tuberculosis.

Piperine a trans-trans isomer of 1-piperoyl-piperidine was evaluated for its immunomodulatory activity to enhance the efficacy of rifampicin in a murine model of Mycobacterium tuberculosis infection. In-vitro immunomodulation of piperine was tested on mouse splenocytes for lymphocyte proliferation, cytokine production and macrophage activation. Protective efficacy of piperine was tested in a mice infection model of M. tuberculosis for the activation of Th-1 response and synergistic combination efficacy with rifampicin. Murine splenocytes exposed to piperine exhibited proliferation of T and B cell, increased Th-1 cytokines and enhanced macrophage activation. Piperine (1 mg/kg) in mice infected with M. tuberculosis activated the differentiation of T cells into Th-1 sub-population (CD4+ / CD8+ subsets). There was an increase in secretion of Th-1 cytokines (IFN-γ and IL-2) by these cells. The qRT-PCR studies revealed corresponding increases in the mRNA transcripts of IFN-γ and IL-2 in the infected lung tissues. Combination of piperine and rifampicin (1 mg/kg) exhibited better efficacy of and resulted in additional 1.4 to 0.8 log reduction in lung cfu as compared to rifampicin alone. The up-regulation of Th1 immunity by piperine can be synergistically combined with rifampicin to improve its therapeutic efficacy in immune-compromised TB patients.

Immunomodulatory potential of a bioactive fraction from the leaves of Phyllostachys bambusoides (bamboo) in BALB/c mice.

In order to evaluate the role of ethyl acetate fraction (PB-EtAC) obtained from the Phyllostachys bambusoides leaves in the modulation of immune responses, detailed studies were carried out using a panel of in vivo assays. Oral administration of PB-EtAC (50-200 mg/Kg) stimulated the IgM and IgG titre expressed in the form of haemagglutination antibody (HA) titre. Further, it elicited a dose related increase in the delayed type hypersensitivity reaction (DTH) after 24 and 48 h in BALB/c mice. Besides augmenting the humoral and cell mediated immune response, the concentration of cytokines (IFN-γ, IL-2, and IL-4) in serum with respect to T cell interactions also increased significantly. It also induced macrophage phagocytosis, and nitric oxide (NO) production which resulted in a high degree of protection against Candida albicans and carbon clearance. Moreover, the enhancement in CD4 and CD8 cell populations as revealed by flow cytometry. Taken together this in vivo and ex vivo preclinical data, our results suggested that PB-EtAC acts as an effective immunostimulator eliciting both Th1 and Th2 immune responses. We are reporting first time the immunostimulatory potential of P. bambusoides and it might be regarded as a biological response modifier.

Synthesis of novel lipidated iridoid glycosides as vaccine adjuvants: 6-O-palmitoyl Agnuside elicit strong Th1 and Th2 response to ovalbumin in mice.

Novel lipidated analogs of iridoids viz., Agnuside and Negundoside with different chain length were synthesized and tested for immune adjuvant activity in the presence of weak antigen ovalbumin. Based on in vitro assay, 6-O-palmitoyl Agnuside (AG-3) was further taken up for detailed in vivo activity and found to significantly enhance the production of anti OVA IgG titer, neutralizing antibody (IgG1 and IgG2a) titer as well as soluble mediators of a Th1 (IL-2, IFN-γ)/Th2 response (IL-4) and proliferation of T lymphocyte subsets (CD4/CD8) and co stimulatory molecules CD80/CD86. Furthermore, the SAR studies revealed that presence of acyl group at aglycon moiety of these iridoid glycosides is crucial for immune adjuvant activity.

Anti-inflammatory and immunomodulatory flavones from Actinocarya tibetica Benth.

Herein, we report the isolation and immunomodulatory activity of 11 phytoconstituents, viz. 7 flavonoids, 3 pentacyclic triterpenes and 1 phytosterol of an unexplored plant Actinocarya tibetica Benth. Three flavones, 5-methoxy-6,7-methylenedioxyflavone (6), mosloflavone (7) and negletein (8), showed promising anti-inflammatory activity via inhibition of TNF-α and IL-1ß with IC50 values of 0.22, 0.71, 16.4 µM and 10.8, 7.8, 6.4 µM, respectively. These flavones also showed dose-dependent inhibition of TNF-α, IL-1ß and iNOS levels in the supernatant of mouse macrophage cell line J774A. Molecular modelling studies revealed orientation and interactions of flavones 6-8 in the active site of TNF-α. These flavones can be used as a starting point to discover lead structures for treatment of inflammatory and immunomodulatory diseases.

Efficacy evaluation of two synthetic lysine lipidated tripeptides as vaccine adjuvants against HBsAg.

In the present investigation, adjuvant potential of two novel lipidated tripeptide lysine derivatives (KKSM and KKSMB) was evaluated using various in vitro and animal-derived models of humoral and cell-mediated immune events in response to hepatitis B surface antigen (HBsAg). The results were compared with alum adjuvanted with HBsAg. Both these molecules were found to stimulate anti-HBsAg IgG and neutralizing (IgG1 and IgG2a) antibody titres in mice sera. The two molecules stimulated the proliferation of T-lymphocyte sub-sets (CD4/CD8) as well as the production of soluble mediators of Th1 (IL-2 and IFN-γ) and Th2 response (IL-4) in spleen cell culture supernatant. Furthermore, the two lipidated tripeptides enhanced the CD4, CD8, CD3 and CD19 cell populations as well as CD4/CD8 derived IL-2, IL-4, IFN-γ and TNF-α in whole blood of treated mice. There was found to be the significant enhancement in the release of IL-12, IFN-γ and nitrite content in macrophage supernatant. Moreover, the two lipidated tripeptides enhanced the population of CD80 and CD86 in spleen-derived macrophages and did not show any hemolytic effect on rabbit RBCs. Taken together, these results suggest that both these molecules are the potent enhancers of anti-HBsAg immune response via augmenting Th1/Th2 response in a dose dependent manner.

Identification and bioactive potential of endophytic fungi isolated from selected plants of the Western Himalayas.

This study was conducted to characterize and explore the endophytic fungi of selected plants from the Western Himalayas for their bioactive potential. A total of 72 strains of endophytic fungi were isolated and characterized morphologically as well as on the basis of ITS1-5.8S-ITS2 ribosomal gene sequence acquisition and analyses. The fungi represented 27 genera of which two belonged to Basidiomycota, each representing a single isolate, while the rest of the isolates comprised of Ascomycetous fungi. Among the isolated strains, ten isolates could not be assigned to a genus as they displayed a maximum sequence similarity of 95% or less with taxonomically characterized organisms. Among the host plants, the conifers, Cedrus deodara, Pinus roxburgii and Abies pindrow harbored the most diverse fungi, belonging to 13 different genera, which represented almost half of the total genera isolated. Several extracts prepared from the fermented broth of these fungi demonstrated strong bioactivity against E. coli and S. aureus with the lowest IC(50) of 18 µg/ml obtained with the extract of Trichophaea abundans inhabiting Pinus sp. In comparison, extracts from only three endophytes were significantly inhibitory to Candida albicans, an important fungal pathogen. Further, 24 endophytes inhibited three or more phytopathogens by at least 50% in co-culture, among a panel of seven test organisms. Extracts from 17 fungi possessed immuno-modulatory activities with five of them showing significant immune suppression as demonstrated by the in vitro lymphocyte proliferation assay. This study is an important step towards tapping the endophytic fungal diversity from the Western Himalayas and assessing their bioactive potential. Further studies on the selected endophytes may lead to the isolation of novel natural products for use in medicine, industry and agriculture.

Modulation of LPS induced inflammatory response by Lawsonyl monocyclic terpene from the marine derived Streptomyces sp.

In continuing research for compounds with immunosuppressive activity, Lawsonone (1), a novel Lawsonyl derivative isolated from marine-derived bacteria Streptomyces sp. was evaluated for its potent immunosuppressive activity on immune system. The effect of Lawsonone (1) was elucidated on the immune cells (splenocytes and macrophages) collected from BALB/c mice. Study was carried out to find the effect of Lawsonone (1) on Con-A and LPS stimulated splenocyte proliferation, LPS-induced NO, IL-1ß, IL-6 and TNF-α production in macrophages. Furthermore, the effect of Lawsonone (1) on T-cell subsets (CD4 and CD8) and total B-cell (CD19) population was analyzed by flow cytometry. The results obtained in the present study showed that Lawsonone (1) inhibited the proliferation of both T and B splenocytes. It inhibited the nitric oxide (NO) and pro-inflammatory cytokine (IL-1ß, IL-6 and TNF-α) production in LPS-stimulated macrophages in a dose-dependent manner. Moreover, flow cytometric analysis indicated the prominent inhibition of CD4, CD8 and CD19 cell populations in the spleen of mice treated with the variable doses of Lawsonone (1), with the maximum inhibition at the lowest dose (0.1µM). Taken together, the present results suggest that Lawsonone (1) may act as a potent molecule for immunosuppression and anti-inflammation, supporting its immunopharmacologic application to modify the immune system.

Immunomodulatory studies of a bioactive fraction from the fruit of Prunus cerasus in BALB/c mice.

In order to evaluate the role of ethyl acetate fraction (PNRS-EtOAC) obtained from the Prunus cerasus fruit in the modulation of immune responses, detailed studies were carried out using a panel of in vivo assays. Oral administration of PNRS-EtOAC (25-100 mg/kg) stimulated the IgM and IgG titre expressed in the form of hemagglutination antibody (HA) titre. Further, it elicited a dose related increase in the delayed type hypersensitivity reaction (DTH) after 24 and 48 h in BALB/c mice. Besides augmenting the humoral and cell mediated immune response, the concentration of cytokines (IFN-γ, IL-4, and TNF-α) in serum with respect to T cell interactions, i.e. the proliferation of lymphocytes were significantly increased at 50 mg/kg compared with the control. The results in these studies demonstrated the immunostimulatory effect of PNRS-EtOAC in a dose-dependent manner with respect to the macrophage activation possibly expressing the phagocytosis and nitrite production by the enhancement of TNF-α production as a mode of action.

Study of the adjuvanticity of lysine lipopeptides; carbamate analogs elicit strong Th1 and Th2 response to ovalbumin in mice.

Bacterial lipoproteins and their synthetic analogs are strong immune modulators of the early host responses. In view of the strong adjuvanticity of bacterial lipopeptide mimics bearing lysine residues, a focused library of lipidated dipeptides and tripeptides has been synthesized with a view to understand the pattern of activity vis a vis the site and extent of lipidation. Compounds 4, 5 and 14 stimulate OVA specific IgG titer, neutralization of antibodies (IgG1 and IgG2a), T lymphocyte sub-sets (CD4/CD8) and its production of soluble mediators for Th1 (IFN-γ)/Th2 (IL-4) cytokines and costimulatory molecules (CD80/CD86) which are ideal traits of immune adjuvants. The results support lipidated lysine dipeptides as potent enhancers of humoral and cell mediated immune responses and thus might become promising immune-adjuvants for self adjuvanted vaccines.

Possible role of macrophages induced by an irridoid glycoside (RLJ-NE-299A) in host defense mechanism.

In order to explore the possible role of macrophages and other necessary immune competent (T and B) cells in the modulation of immune responses, an attempt was made to study the immunomodulatory effect of an irridoid glycoside (RLJ-NE-299A) isolated from the roots of Picrorhiza kurroa. Both in vitro and in vivo studies were used to evaluate the effect of RLJ-NE-299A on humoral, cellular, and phagocytic activity of macrophages. The data obtained in the present study showed that RLJ-NE-299A significantly increased sheep red blood cell (SRBC) and induced antibody (IgM and IgG) titer and delayed type hypersensitivity (DTH) reaction in mice. Besides augmenting the humoral and cell-mediated immune response, it induced macrophage phagocytosis and stimulated cytokine-induced macrophage activation and nitric oxide (NO) production, which resulted in a high degree of protection against Candida albicans and Salmonella typhimurium infections. Flow cytometric analysis indicated the enhanced expression of co-stimulatory surface molecules CD80 and CD86. The ability of RLJ-NE-299A to upregulate these cell surface antigens involved in antigen presentation may provide an explanation for the increased T-cell mediated immunity involving macrophages. Taken together this in vitro and in vivo preclinical data suggests that RLJ-NE-299A acts as an effective immunomodulator specifically to improve macrophage function during infections. The effects of this agent on these cells at concentrations relevant to in vivo therapy support its immunopharmacologic application to modify cellular immunity.

A novel sarsasapogenin glycoside from Asparagus racemosus elicits protective immune responses against HBsAg.

The aim of the present investigation was to evaluate the adjuvant potential of a novel sarsasapogenin glycoside (immunoside) isolated from Asparagus racemosus in combination with hepatitis B surface antigen (HBsAg). Various in vitro and animal derived protocols were used to determine the response of immunoside adjuvanted with HBsAg and the results were compared with alum adjuvanted with HBsAg. Several biomarkers such as antibody titre (IgG, IgG1/IgG2a) were measured in mice sera. Cell proliferation, cytokines (IL-2, IFN-γ and IL-4), and lymphocyte sub-populations (CD4/CD8, CD3 and CD19) were determined in splenocytes from mice administered subcutaneously with test substances. In these cells CD4/CD8 derived IFN-γ release was also determined. Macrophage preparations were used for the determination of IL-12, IFN-γ and nitrite content. Seroconversion potential was compared with a standard vaccine. Acute safety evaluation of immunoside was done in mice. Effect of immunoside on red blood cell haemolysis was determined. The results have suggested that immunoside potentially enhanced anti-HBsAg immune response via augmenting Th1/Th2 response in a dose dependent manner.

Development of novel lipidated analogs of picroside as vaccine adjuvants: acylated analogs of picroside-II elicit strong Th1 and Th2 response to ovalbumin in mice.

The acylated analogs of picroside-II were synthesized and tested for immune-adjuvant activity in the presence of weak antigen ovalbumin found to stimulate anti-OVA IgG titer, neutralizing antibody (IgG1 and IgG2a) titer as well as the production of soluble mediators of a Th1 response (IL-2 and IFN-γ) and Th2 response (IL-4) and proliferation of T lymphocytes sub-sets (CD4/CD8). Furthermore, these modified analogs of picroside-II were able to elicit a substantial increase in anti-OVA IgG when compared with OVA alone. These results support the use of acylated analogs particularly PK-II-3 and PK-II-4 as potent enhancer of antigen-specific Th1 and Th2 immune responses and thus are promising immune-adjuvant candidate for vaccines.

Immune modulation and apoptosis induction: Two sides of antitumoural activity of a standardised herbal formulation of Withania somnifera.

Deregulated apoptosis and suppressed tumour reactive immunity render tumour cells to grow amok in the host body. Traditionally used botanicals may offer potential anticancer chemo-immunotherapeutic leads. We report in this study a chemically standardised herbal formulation (WSF) of Withania somnifera possessing anticancer and Th1 immune up-regulatory activities. WSF produced cytotoxicity in a panel of human cancer cell lines in vitro. The molecular mechanism of cell cytotoxicity, IC(50) 48h approximately 20mug/ml, was investigated in HL-60, where it induced apoptosis by activating both intrinsic and extrinsic signalling pathways. It induced early generation of reactive nitrogen and oxygen species (RNOS), thus producing oxidative stress mediated mitochondrial membrane potential (MMP) loss leading to the release of cytochrome c, the translocation of Bax to mitochondria and apoptosis-inducing factor to the nuclei. These events paralleled the activation of caspase-9, -3 and PARP cleavage. WSF also activated caspase-8 through enhanced expression of TNF-R1 and DR-4, suggesting also the involvement of extrinsic pathway of apoptosis. WSF at 150mg/kg, i.p., inhibited >50% tumour growth in the mouse tumour models. In tumour-bearing mice, WSF inhibited the expression of pStat-3, with a selective stimulation of Th1 immunity as evidenced by enhanced secretion of IFN-gamma and IL-2. In parallel, it enhanced the proliferation of CD4(+)/CD8(+) and NK cells along with an increased expression of CD40/CD40L/CD80. In addition, WSF also enhanced T cell activation in camptothecin treated tumour-bearing mice. WSF being safe when given orally up to 1500mg/kg to rats for 6 months may be found useful in the management of malignancy by targeting at multiple pathways.

Immunomodulatory activity of biopolymeric fraction BOS 2000 from Boswellia serrata.

Oral administration of BOS 2000 (1-10 mg/kg) elicited a dose related increase in the delayed hypersensitivity reaction (early 24 h and delayed 48 h) in mice. It also stimulated the IgM and IgG titre expressed in the form of plaques (PFC) and complement fixing antibody titre. The concentration of cytokines (IL-4, IFN-gamma and TNF-alpha) in serum with respect to T cell interactions, i.e. (CD4/CD8) and the proliferation of lymphocytes were significantly increased at 10 mg/kg compared with the control. The results in these studies demonstrated the immunostimulatory effect of BOS 2000 in a dose-dependent manner with respect to the macrophage activation possibly expressing the phagocytosis and nitrite production by the enhancement of TNF-alpha and IFN-gamma production as a mode of action.

Boswellic acids and glucosamine show synergistic effect in preclinical anti-inflammatory study in rats.

The present study revealed the synergistic effect of boswellic acid mixture (BA) and glucosamine for anti-inflammatory and anti-arthritic activities in rats. Two studies were conducted, that is, acute anti-inflammatory by carrageenan edema and chronic anti-arthritic by Mycobacterium-induced developing arthritis. Five groups of animals were included in each of the study: the vehicle control, positive control (ibuprofen 100mg/kg), boswellic acids (250 mg/kg), glucosamine (250 mg/kg) and a combination of boswellic acids (125 mg/kg) and glucosamine (125 mg/kg). BA when administered at 250 mg/kg in rats, carrageenan-induced paw edema and Mycobacterium-induced developing arthritis were significantly inhibited. In comparison to boswellic acids, glucosamine when administered at 250 mg/kg showed a mild effect in carrageenan-induced edema and moderate inhibition of paw swelling against developing arthritis. Although the combination of boswellic acids and glucosamine did not affect the acute inflammation to a greater extent yet a significant anti-arthritic activity was observed in rats. In conclusion, a synergistic effect was observed in chronic inflammatory conditions when two chemical entities were administered in combination in preclinical study.

A new vaccine adjuvant (BOS 2000) a potent enhancer mixed Th1/Th2 immune responses in mice immunized with HBsAg.

Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component. In search of a potent vaccine adjuvant, the water-soluble biopolymeric fraction BOS 2000 from Boswellia serrata was evaluated for desired activity. We investigated the ability of BOS 2000 to enhance HBsAg specific immune responses. The effect was determined in the form of protective anti-HBsAg titers, neutralizing antibodies (IgG1 and IgG2a), spleen cell lymphocyte proliferation by using MTT assay, Th1 (IFN-gamma and TNF-alpha) and Th2 (IL-4) cytokines as well as T-lymphocyte subsets (CD4/CD8) and intracellular cytokines (IFN-gamma/IL-4), these responses were highest in BOS 2000 immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of adjuvant by 18.1-fold in comparison to alum, total IgG and its subtypes (IgG1 and IgG2a) antibodies titer in serum was significantly enhanced. Analysis of HBsAg specific cytokines revealed that alum was associated with a predominantly IL-4 response. In contrast, BOS 2000 was associated with production of both IFN-gamma and IL-4. We conclude that BOS 2000 is a potent enhancer of antigen-specific Th1 and Th2 immune responses in comparison to alum with Th2 limitation and is a promising adjuvant for vaccine applications.