Tabasum Sidiq

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 IC50 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.

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.

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.

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.

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-1β, 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-1β, 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.

Ammonium chloride mediated synthesis of alkyl glycosides and evaluation of their immunomodulatory activity

Therapeutic immunoadjuvants have been derived through a straightforward glycosylation of various alcohols with unprotected and non-activated monosaccharides, under solvent free conditions using ammonium chloride as a catalyst. Sugar acids such as glucuronic acid can be glycosylated without esterification and after acetylation yielded a novel 3,6-anhydro derivative. All the synthesized sugar glycosides were screened for immunomodulatory activities to be classified as immunostimulator, immunosuppressor, or immunoadjuvant against the weak antigen oval albumin (OVA). Most of the compounds revealed immunosuppressive (↓) or immunostimulatory (↑) activity with reference to lymphocyte proliferation and possess potential as vaccine adjuvants.

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.

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.

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-1β 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-1β 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.

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.