Toll-like receptors and CD40 modulate each other's expression affecting Leishmania major infection.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and results in innate immune system activation that results in elicitation of the adaptive immune response. One crucial modulator of the adaptive immune response is CD40. However, whether these molecules influence each other's expression and functions is not known. Therefore, we examined the effects of TLRs on CD40 expression on macrophages, the host cell for the protozoan parasite Leishmania major. While polyinosinic-polycytidylic acid [poly (I:C)], a TLR-3 ligand, lipopolysaccharide (LPS), a TLR-4 ligand, imiquimod, a TLR-7/8 ligand and cytosine-phosphate-guanosine (CpG), a TLR-9 ligand, were shown to enhance CD40 expression, CD40 stimulation enhanced only TLR-9 expression. Therefore, we tested the synergism between CD40 and CpG in anti-leishmanial immune response. In Leishmania-infected macrophages, CpG was found to reduce CD40-induced extracellular stress-regulated kinase (ERK)1/2 activation; with the exception of interleukin (IL)-10, these ligands had differential effects on CD40-induced IL-1α, IL-6 and IL-12 production. CpG significantly enhanced the anti-leishmanial function of CD40 with differential effects on IL-4, IL-10 and interferon (IFN)-γ production in susceptible BALB/c mice. Thus, we report the first systematic study on CD40-TLR cross-talk that regulated the experimental L. major infection.

Leishmania expressed lipophosphoglycan interacts with Toll-like receptor (TLR)-2 to decrease TLR-9 expression and reduce anti-leishmanial responses.

Two different Toll-like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR-2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR-9 has been shown to promote a host-protective response. However, whether there is a relationship between the interaction of LPG and TLR-2, on one hand, with the effect of TLR-9, on the other hand, remains unknown. In this study, we report that in-vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR-9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti-LPG as well as anti-TLR-2 antibodies prevents this reduction of TLR-9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR-9 expression, which is shown to be mediated by transforming growth factor (TGF)-ß and interleukin (IL)-10. Finally, in-vitro treatment of macrophages with anti-LPG and/or anti-TLR-2 antibodies before infection reduces the number of amastigotes in macrophages and co-treatment of mice with anti-TLR-2 antibodies and cytosine-phosphate-guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)-γ-predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR-2 reduce anti-leishmanial responses via cytokine-mediated decrease of TLR-9 expression.

Development and validation of reversed phase-high-performance liquid chromatography method for determination of paracetamol and lornoxicam in tablet dosage form.

A simple, precise, reliable, rapid and reproducible reversed phase-high-performance liquid chromatography method was developed and validated for the simultaneous estimation of Paracetamol (PCM) and Lornoxicam (LOX) present in tablet dosage forms. Chromatographic separation achieved isocratically on Luna C18 column (5 µm, 150 × 4.60 mm) and methanol/phosphate buffer (60:40, v/v, pH 7.0) as mobile phase, at a flow rate of 1 ml/min. Detection was carried out at 260 nm. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the ICH guidelines. The retention times for PCM and LOX was found to be 2.06±0.013and 4.38±0.07 min, respectively. Linearity for PCM and LOX was in the range of 10-50 mg/ml and 8-40 mg/ml, respectively. The mean recoveries obtained for LOX and PCM were 100± 0.16 and 99.50± 0.43%, respectively, and relative standard deviation (RSD) was less than 2. The correlation coefficients for all components are close to 1. The RSDs for three replicate measurements in three concentrations of samples in tablets are always less than 2%. Developed method was found to be accurate, precise, selective and rapid for simultaneous estimation of PCM and LOX in tablets.

Evaluation of wound-healing activity of Acorus calamus Linn.

The aim of the present study was to assess the wound-healing activity of ethanolic extracts of Acorus calamus leaves. A wound was induced by an excision- and incision-based wound model in rats of either sex. The mature green leaves of A. calamus were collected and authenticated. Extractions of dried leaves were carried out with 80% ethanol in a soxhlet apparatus. For wound-healing activity, the extracts were applied topically once daily in conc. of 40% w/w and 20% w/w in the form of ointment and compared with a standard drug (povidion-iodine). The healing of the wound was assessed by the rate of wound closure, period of epithelialisation, tensile strength and weight of the granulation tissue, hydroxyproline content and histopathology of the granulation tissue. The ethanolic extract of A. calamus promoted wound-healing activity significantly in both the wound models studied. The histological study of the granulation tissue with 20% A. calamus extract ointment-treated animals showed a larger number of inflammatory cells and lesser collagen when compared with the 40% A. calamus extract ointment-treated animals. However, this was better than the control group of animals. Enhanced wound contraction, decreased epithelialisation time, increased hydroxyproline content and histological characteristics suggest that A. calamus extract may have therapeutic benefits in wound healing.