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Lipopolysaccharide (LPS) ¡s an important structural component of the outer cell membrane complex of gram negative microorganisms. Its causative role in gram negative bacteria-induced diseases and broad applications in different binds of cell stimulation experiments provided a conceptual basis for studies directed at the isolation, purification, and detailed chemical characterization of LPS. The main problem with LPS purification protocols is the contamination of the end product with nucleic acids and proteins in variable proportions which could potentially interfere with downstream applications. In this study, a simple procedure for purification of LPS from Escherichia coli (E.coli) and Salmonella typhi (S.typhi) with high purity and very low contaminating nucleic acids and proteins based on the hot phenol-water extraction protocol has been introduced. The purity of extracted LPS was evaluated by silver and coomassie blue staining of SDS-PACE gels and HPLC analysis. Limulus Amebocyte Lysate (LAL) coagulation activity and rabbit pyrogen assay were exploited to monitor the functionality of purified LPS. The results showed that DNase and RNase treatment of the sample is essential r-' after the sonication step to eliminate nucleic acid contamination in the LPS fraction. Silver staining demonstrated ladder pattern which is characteristic of LPS. No contaminating protein was found as assessed by coomassie blue staining. HPLC fractionation revealed high degree of purity comparable with commercial LPS. Parenteral administration of purified LPS resulted in substantial increase of rabbits' body temperature (mean: 1.45 °C). LAL coagulation assay confirmed the functional activity of the purified LPS. In conclusion, the protocol presented here could be employed for isolation of LPS with high purity and functional activity. [ABSTRACT FROM AUTHOR]

Background: Toll-like receptor (TLR)-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells (ESCs) and whole endometrial cells (WECs) to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods: Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and production of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. Results: WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene expression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr (p<0.05). At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs (p<0.05). LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dose-dependent manner (p<0.05). Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-α in response to LPS activation (p<0.05). Conclusion: Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus. [ABSTRACT FROM AUTHOR]

Increased expression levels of KCNJ3 have been correlated with lymph node metastases and poor prognosis in patients with breast cancer, suggesting a prognostic role of KCNJ3 We aimed to establish protocols for the detection of KCNJ3 in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue. Several antibodies were tested for sensitivity and specificity by western blot, followed by optimisation of the immunohistochemistry (IHC) procedure and establishment of KCNJ3 mRNA in situ hybridisation (ISH). Methods were validated by processing 15 FFPE breast cancer samples for which microarray data were available. Spearman's rank correlation analysis resulted in borderline significant correlation for IHC versus ISH (r S : 0.625; p<0.05) and IHC versus microarray (r S : 0.668; p<0.01), but in significant correlation for ISH versus microarray (r S : 0.861; p<0.001). The ISH method was superior to IHC, regarding robustness, sensitivity and specificity and will aid to further study expression levels of KCNJ3 in both malignant and physiological conditions., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)