Early Secreted Antigen ESAT-6 of Mycobacterium tuberculosis Promotes Protective T Helper 17 Cell Responses in a Toll-Like Receptor-2-dependent Manner

Despite its relatively poor efficacy, Bacillus Calmette-Guérin (BCG) has been used as a tuberculosis (TB) vaccine since its development in 1921. BCG induces robust T helper 1 (Th1) immune responses but, for many individuals, this is not sufficient for host resistance against Mycobacterium tuberculosis (M. tb) infection. Here we provide evidence that early secreted antigenic target protein 6 (ESAT-6), expressed by the virulent M. tb strain H37Rv but not by BCG, promotes vaccine-enhancing Th17 cell responses. These activities of ESAT-6 were dependent on TLR-2/MyD88 signalling and involved IL-6 and TGF-β production by dendritic cells. Thus, animals that were previously infected with H37Rv or recombinant BCG containing the RD1 region (BCG::RD1) exhibited improved protection upon re-challenge with virulent H37Rv compared with mice previously infected with BCG or RD1-deficient H37Rv (H37RvΔRD1). However, TLR-2 knockout (TLR-2-/-) animals neither showed Th17 responses nor exhibited improved protection in response to immunization with H37Rv. Furthermore, H37Rv and BCG::RD1 infection had little effect on the expression of the anti-inflammatory microRNA-146a (miR146a) in dendritic cells (DCs), whereas BCG and H37RvΔRD1 profoundly induced its expression in DCs. Consistent with these findings, ESAT-6 had no effect on miR146a expression in uninfected DCs, but dramatically inhibited its upregulation in BCG-infected or LPS-treated DCs. Collectively, our findings indicate that, in addition to Th1 immunity induced by BCG, RD1/ESAT-6-induced Th17 immune responses are essential for optimal vaccine efficacy

Sperm motility inhibiting activity of a phytosterol from <i>Alstonia macrophylla </i>Wall ex A. DC. leaf extract: A tribal medicine

1104-1109The role of methanolic extract and n-butanol fraction of A. macrophylla leaves was investigated on the forward motility of goat spermatozoa. The methanol extract (600 μg/ml) and one n-butanol fraction (Fraction A; 100 μg/ml) showed marked inhibition of sperm forward motility, tested by microscopic and spectrophotometric methods. Approximately, 50- 60% of the spennatozoa lost their motility when treated with 600 μg/ml of methanol extract or 100 μg/ml of Fraction A. The Fraction A at 400 μg/ml concentration showed complete inhibition of sperm forward motility at 0 min. The inhibitory activity increased with the increasing concentrations of the fraction. The motility inhibitory activity of the Fraction A was stable to heat treatment at 100°C for 2 min. The compound showed high inhibitory effect in the pH range 6.7-7.6. Fraction A also showed high efficacy for inhibiting human sperm motility, assessed by the microscopic method. The phytochemical analysis of methanolic extract of A. macrophylla leaves revealed the presence of sterols, triterpene, flavonoid, alkaloid, tannin and reducing sugar, while the Fraction A contains β-sitosterol, a common phytosterol. The results demonstrate that Fraction A (β-sitosterol) is a potent inhibitor of sperm motility and thus it has the potential to serve as a vaginal contraceptive

Anti-Inflammatory Activity of <i>Odina wodier</i> Roxb, an Indian Folk Remedy, through Inhibition of Toll-Like Receptor 4 Signaling Pathway

<div><p>Inflammation is part of self-limiting non-specific immune response, which occurs during bodily injury. In some disorders the inflammatory process becomes continuous, leading to the development of chronic inflammatory diseases including cardiovascular diseases, diabetes, cancer etc. Several Indian tribes used the bark of <i>Odina wodier</i> (OWB) for treating inflammatory disorders. Thus, we have evaluated the immunotherapeutic potential of OWB methanol extract and its major constituent chlorogenic acid (CA), using three popular <i>in vivo</i> antiinflammatory models: Carrageenan- and Dextran-induced paw edema, Cotton pellet granuloma, and Acetic acid-induced vascular permeability. To elucidate the possible anti-inflammatory mechanism of action we determine the level of major inflammatory mediators (NO, iNOS, COX-2-dependent prostaglandin E2 or PGE2), and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12). Further, we determine the toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), c-Jun N-terminal kinases (JNK), nuclear factor kappa-B cells (NF-κB), and NF-kB inhibitor alpha (IK-Bα) by protein and mRNA expression, and Western blot analysis in drug treated LPS-induced murine macrophage model. Moreover, we determined the acute and sub-acute toxicity of OWB extract in BALB/c mice. Our study demonstrated a significant anti-inflammatory activity of OWB extract and CA along with the inhibition of TNF-α, IL-1β, IL-6 and IL-12 expressions. Further, the expression of TLR4, NF-κBp65, MyD88, iNOS and COX-2 molecules were reduced in drug-treated groups, but not in the LPS-stimulated untreated or control groups, Thus, our results collectively indicated that the OWB extract and CA can efficiently inhibit inflammation through the down regulation of TLR4/MyD88/NF-kB signaling pathway.</p></div

A dihydro-pyrido-indole potently inhibits HSV-1 infection by interfering the viral immediate early transcriptional events

In our continued quest for identifying novel molecules from ethnomedicinal source we have isolated an alkaloid 7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole, also known as Harmaline (HM), from an ethnomedicinal herb Ophiorrhiza nicobarica. The compound exhibited a potent anti-HSV-1 activity against both wild type and clinical isolates of HSV-1. Further we demonstrated that HM did not interfere in viral entry but the recruitment of lysine-specific demethylase-1 (LSD1) and the binding of immediate-early (IE) complex on ICP0 promoter. This leads to the suppression of viral IE gene synthesis and thereby the reduced expression of ICP4 and ICP27. Moreover, HM at its virucidal concentration is nontoxic and reduced virus yields in cutaneously infected Balb/C mice. Thus, the interference in the binding of IE complex, a decisive factor for HSV lytic cycle or latency by HM reveals an interesting target for developing non-nucleotide antiherpetic agent with different mode of action than Acyclovir

Effect of OWB extract and CA on the JNK, MAPK, IkB-α and MyD88 expression.

<p>Expression of (A) JNK, (B) MAPK, (C) IkB-α and (D) MyD88 were determined by the Western blot, using GAPDH as the internal control. The LPS (1 µg/ml) induced peritoneal macrophage(s) were treated with OWB (100 µg/ml) or CA (10 µg/ml), and after 24 h the protein extract from whole cell were harvested in buffer, containing 20 mM Tris (pH 7±0.5), 50 mM NaCl, 5% NP-40 and 0.05% DOC. The soluble fraction was separated by centrifugation, subjected to SDS-PAGE and blotted to pre-equilibrated PVDF membrane. The membrane was then blocked in 5% NFDM in 1X TBST, rinsed and incubated with specific antibody at 4°C overnight. Immunoblotting was performed with peroxidase-labelled specific antibodies and visualized by ECL Western blot detection kit. The average expression of NF-kB and MAPK was significantly higher in the LPS-induced macrophage, as compared to the control and OWB or CA co-treated group (** <i>P</i></p

Effect of OWB extract and CA on pro-inflammatory and anti-inflammatory cytokine release in LPS-induced peritoneal macrophages by sandwich ELISA and RT-PCR.

<p>Peritoneal macrophages were cultured overnight and incubated with LPS (1 µg/ml), washed after 4 h and then treated with the OWB (50 or 100 µg/m) or CA (10 µg/ml). The cells were further incubated for 24 h, and the cell-free supernatants were subjected to sandwich ELISA to determine the level of (A) TNF-α, (C) IL-1β, (E) IL-6 and (G) IL-6 (pg/mL). In a separate set similarly treated cells were cultured for 5 h, and collected in TRI Reagent for mRNA extraction and subsequent RT–PCR analysis (vide Materials and methods) to study the cytokine and β-actin mRNA expression. The data were shown for the expression of (B) TNF-α, (D) IL-1β, (F) IL-6 and (H) IL-12. The ELISA and RT–PCR data are expressed as Means ± SD from triplicate experiments, yielding similar results. Asterisks indicate statistically significant (**P, 0.05) induction of TNF-α, IL-1β, IL-6, and IL-12 release; and increase or decrease (**P, 0.05; *P, 0.001) in cytokine expression, compared to the infected macrophages.</p

Effect of OWB extract and CA on protein denaturation.

<p>Each value represents the Mean ± SD; n = 6) * p<0.001** p<0.01.</p

Effect of OWB extract and CA on TLR4 and MyD88 expression.

<p>The LPS-stimulated macrophages were treated with the OWB or CA and incubated for 24 h, following which RNA was isolated for RT–PCR analysis of the expression of MyD88 (A) and TLR4 (B) mRNA. The RT–PCR data are expressed as Means ± SD from triplicate experiments. The expression of TLR4 and MyD88 was significantly higher in the LPS-induced macrophage as compared to the control and OWB or CA co-treated group (** <i>P</i></p

Primer used in Real time-PCR assay.

<p>Primer used in Real time-PCR assay.</p