Development and validation of an HPLC-DAD method for Rapid quantification of vasicine in Adhatoda vasica leaves and commercial products.

Leaves from Adhatoda vasica Nees, Acanthaceae (synonym Justicia adhatoda L.) have been widely used in traditional medicine for their beneficial effect in the treatment of respiratory diseases. Vasicine, the main quinazoline alkaloid in A. vasica, has been linked to its medicinal properties. The purpose of this work was to develop and validate a reliable analytical method for the quantification of vasicine in A. vasica leaves and commercially available products. For this purpose, a high-performance liquid chromatography method coupled to diode array detection (HPLC-DAD) was used. After optimization of the extraction process and the HPLC conditions, linearity, precision, accuracy, and specificity were checked. During the validation, six commonly available food supplements and dosage forms were tested using the validated method. The calibration model was found to be linear in the concentration range of 5.125-205 µg/mL. The average vasicine content at different concentration levels was 0.99 g/100 g with an RSD% of 0.05%. The average recovery was found to be 102.3% with an RSD of 4.3%. Additionally, it was confirmed that the validated method was still precise and accurate for quantifying vasicine in other matrices like the tested preparations. In summary, the validated method was suitable for the determination of vasicine in leaves of Adhatoda vasica, as well as for investigating the quality and the prescribed intake of several commercial products.

Inflammatory monocytes regulate Th1 oriented immunity to CpG adjuvanted protein vaccines through production of IL-12.

Due to their capacity to skew T cell responses towards Th1 oriented immunity, oligonucleotides containing unmethylated CpG motifs (CpG) have emerged as interesting adjuvants for vaccination. Whereas the signalling pathways in response to CpG mediated TLR9 activation have been extensively documented at the level of the individual cell, little is however known on the precise identity of the innate immune cells that govern T cell priming and polarisation to CpG adjuvanted protein antigens in vivo. In this study, we demonstrate that optimal induction of Th1 oriented immunity to CpG adjuvanted protein vaccines requires the coordinated actions of conventional DCs and of monocytes. Whilst conventional DCs were required for antigen presentation and initial T cell priming, monocytes constitute the main source of the Th1 polarising cytokine IL-12.

Neutralizing TNFα restores glucocorticoid sensitivity in a mouse model of neutrophilic airway inflammation.

Asthma is a heterogeneous disorder, evidenced by distinct types of inflammation resulting in different responsiveness to therapy with glucocorticoids (GCs). Tumor necrosis factor α (TNFα) is involved in asthma pathogenesis, but anti-TNFα therapies have not proven broadly effective. The effects of anti-TNFα treatment on steroid resistance have never been assessed. We investigated the role of TNFα blockade using etanercept in the responsiveness to GCs in two ovalbumin-based mouse models of airway hyperinflammation. The first model is GC sensitive and T helper type 2 (Th2)/eosinophil driven, whereas the second reflects GC-insensitive, Th1/neutrophil-predominant asthma subphenotypes. We found that TNFα blockade restores the therapeutic effects of GCs in the GC-insensitive model. An adoptive transfer indicated that the TNFα-induced GC insensitivity occurs in the non-myeloid compartment. Early during airway hyperinflammation, mice are GC insensitive specifically at the level of thymic stromal lymphopoietin (Tslp) transcriptional repression, and this insensitivity is reverted when TNFα is neutralized. Interestingly, TSLP knockout mice displayed increased inflammation in the GC-insensitive model, suggesting a limited therapeutic application of TSLP-neutralizing antibodies in subsets of patients suffering from Th2-mediated asthma. In conclusion, we demonstrate that TNFα reduces the responsiveness to GCs in a mouse model of neutrophilic airway inflammation. Thus antagonizing TNFα may offer a new strategy for therapeutic intervention in GC-resistant asthma.