Molecular characterization of BK virus detected in renal transplant patients in Sri Lanka: a preliminary study.

Background & objectives: BK virus (BKV) is a polyomavirus and cause of a common infection after renal transplantation which could be preceded to BKV-associated nephropathy. It has four main subtypes (I-IV). BKV subtypes II and III are rare, whereas subtype I shows a ubiquitous distribution. The objective of the present study was to investigate the prevailing BKV subtypes and subgroups in renal transplant patients in Sri Lanka. Methods: The presence of BKV in urine was tested through virus load quantification by real-time PCR from 227 renal transplant patients who were suspected to have BKV infection. Of these patients only 41 were found to be BKV infected (>103 copies/ml) and those were subjected to conventional PCR amplification of VP1 gene followed by BKV genotyping via phylogenetic analysis based on DNA sequencing data. Results: Persistent BK viral loads varied from 1×103 to 3×108 copies/ml. Of the 41 patient samples, 25 gave positive results for PCR amplification of subtyping region of VP1 gene of BKV. BKV genotyping resulted in detecting subtype I in 18 (72%) and subtype II in seven (28%) patients. BKV subgroups of Ia, Ib-1 and Ib-11, and Ic were identified with frequencies of 6/18 (33.3%), 6/18 (33.3%), 5/18 (27.8%), and 1/18 (5.6%), respectively. Interpretation & conclusions: Findings from this preliminary study showed a high occurrence of subtype I, while the presence of subtype II, which is rare and less prevalent, was a novel finding for this Asian region. This emphasizes the need for further molecular and serological studies to determine the prevalence of different BKV subtypes in Sri Lanka.

In vitro pro-inflammatory enzyme inhibition and anti-oxidant potential of selected Sri Lankan medicinal plants.

BACKGROUND: The extracts of the ten selected Sri Lankan medicinal plants have been traditionally used in the treatment of inflammatory mediated diseases. The extracts were investigated for anti-inflammatory and anti-oxidant potential in vitro to identify bio-active extracts for further chemical characterization. METHODS: In vitro anti-inflammatory activities of total ethanol extracts were investigated measuring the inhibitory activities of four pro-inflammatory enzymes, arachidonate-5- lipoxygenase (A5-LOX), hyaluronidase (HYL), xanthine oxidase (XO) and inducible nitric oxide (iNO) synthase. Cytotoxicity of extracts were determined by MTT assay. Oxidative burst inhibition (OBI) on human whole blood (WB) and isolated polymorphoneutrophils (PMNs) was carried out for a selected bio-active extract. Anti- oxidant activities of the extracts were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, ferric reducing antioxidant power (FRAP), ferrous ion chelation (FIC) and oxygen radical absorbance capacity (ORAC) assays. Total polyphenol and total Flavonoid contents of the extracts were also determined. The most active plant extract was analysed using Gas chromatography-Mass spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC). RESULTS: The ethanol bark extract of Flacourtia indica showed the highest A5-LOX (IC50: 22.75 ± 1.94 g/mL), XO (70.46 ± 0.18%; 250 µg/mL) and iNOs inhibitory activities on LPS- activated raw 264.7 macrophage cells (38.07 ± 0.93%; 500 µg/mL) with promising OBI both on WB (IC50: 47.64 2.32 µg/mL) and PMNs (IC50: 5.02 0.38 µg/mL). The highest HYL inhibitory activity was showed by the leaf extracts of Barathranthus nodiflorus (42.31 ± 2.00%; 500 µg/mL) and Diospyros ebenum (41.60 ± 1.18%; 500 µg/mL). The bark and leaf extracts of Callophyllum innophyllum (IC50: 6.99 ± 0.02 µg/mL) and Symplocus cochinchinesis (IC50: 9.85 ± 0.28 µg/mL) showed promising DPPH free radical scavenging activities. The GC-MS analysis of ethanol bark extract of F. indica showed the presence of two major bio-active compounds linoleic acid ethyl ester and hexadecanoic acid, ethyl ester (> 2% peak area). The HPLC analysis showed the presence polyphenolic compounds. CONCLUSION: The ethanol bark extract of F. indica can be identified as a potential candidate for the development of anti-inflammatory agents, which deserves further investigations. The bio-active plant extracts may be effectively used in the applications of cosmetic and health care industry.

Increased expression of co-stimulatory molecules and enhancement of the IgG response in rats orally administered with a polyherbal formulation.

BACKGROUND: Link Samahan® (LS) is a standardized modern formulation of a polyherbal preparation used in the indigenous system of medicine in Sri Lanka. OBJECTIVE: Evaluation of the immunostimulatory activity of LS and the molecular mechanisms that modulate the humoral immune response. MATERIAL AND METHODS: Immunostimulatory activity of LS was tested in rats following oral administration on days 1-5 and 15-19 and immunization with bovine serum albumin (BSA) on day 1 and 15. Anti-BSA IgM and IgG response in rats treated with LS, water and sugar (as controls) were compared on days 0-35, using ELISA. The expression of co-stimulatory molecules on lymphocytes was assessed on days 0-8 and days 14-22 using RT-qPCR. RESULTS: IgM and IgG levels of LS-treated rats were increased significantly by day 7 and 21 respectively compared to controls (p < 0.05). IgG response of LS-treated group reached a higher magnitude compared to its IgM response. Gene expression of CD28 and CD40L on T cells (4.9-5.1 fold) and CD80, CD86 and CD40 on APCs (2.4-3.1 fold) were induced significantly by day 2 compared to their expression on day 0 (p < 0.05). The expression levels of CD28 and CD40L on day 2-4 and 16-18 were similar while the expression of CD80, CD86 and CD40 on day 16-18 was higher (3.7-5.1 folds) compared to their levels on day 2-4 (2.4-3.2). CONCLUSIONS: These findings support an adjuvant effect of LS contributing to its immunostimulatory activity and increased expression of co-stimulatory molecules that contribute to boosting immune response.