Introduction: Production of nitric oxide (NO) is one of the main responses elicited by a variety of immune cells such as macrophages (e.g. microglia, resident macrophages of brain), during inflammation. Evaluation of NO levels in the inflammatory milieu is considered important to the understanding of the intensity of an immune response; and has been performed using different methods including the Griess assay. To assay NO in culture, an appropriate number of cells are stimulated into an inflammatory phenotype. Common stimuli include lipopolysaccharide (LPS), IFN-γ and TNF-α. However, overt stimulation could cause cell cytotoxicity therefore an ideal concentration of LPS should be used. Objective: To set-up a model of BV-2 cell activation that allows the assay of detectable levels of NO. Optimization of BV-2 microglia cell density and LPS concentrations after stimulation by bacterial lipopolysaccharide (LPS) for the Griess assay is demonstrated in this study. Methods: BV-2 microglia were cultured at different cell densities, and treated with LPS at three concentrations (1, 5, 10 µg/ml). NO production in culture supernatants were then measured at 18, 24, 48 and 72 hours. Moreover, methyl tetrazolium assay (MTT) was also performed to ensure that NO measurement is performed at no-cytotoxic concentrations of LPS. Results and Conclusions: NO production follows a temporal pattern. The density of 25000 cells/ well was the ideal seeding density for NO evaluation in BV-2 cells. BV-2 stimulation by LPS is dose dependent, and NO levels are increased proportional to the LPS concentration up to 1.0µg/ml, whereas the higher LPS concentrations are associated with decreased cell viability may be caused by the high toxic levels of LPS or NO. Although Griess assay has been commonly used by the scientists, however, optimization of its parameters on BV-2 cells will be useful for the experiments which will be performed on this particular cell line. The optimized pattern of Griess assay on BV-2 cells was achieved in this study, hence easier and more practical for the future scientists to perform Griess assay on BV-2 cells.
Nitric Oxide

Introduction: Group B Streptococcus (GBS), infection and recurrence in newborns and pregnant women can lead to chronic medical illness resulting in significant morbidity, and mortality. Pathogenesis of GBS may be due to reasons such as activation of the immune system, followed by the production of inflammatory markers and toxic components by immune cells including macrophages. Methods: The studies on invasive and colonizing GBS strains inoculated either with peripheral or brain macrophages, the expression of nitric oxide (NO), cell viability, and CD40 were also measured by Griess assay, methyl tetrazolium assay (MTT), and flow cytometry, respectively. Furthermore, the clinical manifestations of the selected patients were also assessed for this study. Results: Outcome of inflammatory markers studies, after GBS inoculation indicated that, invasive GBS strains induced higher inflammatory markers in comparison to colonizing GBS strains. Furthermore, patients’ clinical data showed that patients with invasive GBS infections had severe condition unlike among patients with colonizing GBS strains. The fatality rate in patients with invasive GBS strain were 30.8% while there was no death among carriers. Conclusion: This study, aimed to understand the immune response to GBS, and strengthen the knowledge on GBS pathogenesis. It was concluded that invasive GBS strains not only showed higher expression of inflammatory markers on immune cells but also had higher pathogenesis effect in comparison to colonizing GBS strains.
Streptococcus agalactiae ; Pregnancy