Modulation of Macrophage Function by Lactobacillus-Conditioned Medium

Probiotics are used as microbial food supplements for health and well-being. They are thought to have immunomodulatory effects although their exact physiological mechanism of action is not clear. This study investigated the influence of probiotic Lactobacillus rhamnosus GG conditioned media (LGG-CM) on macrophage phagocytosis of non-pathogenic Escherichia coli HfrC. The gentamicin protection assay was used to study the bacterial killing phases of phagocytosis. Macrophages co-incubated with E. coli for an hour allowed them to ingest bacteria and then the rate of E. coli killing was monitored for up to 300 min to determine the killing or digestion of the bacteria by recovering them from the macrophage lysate. We found that the LGG-CM significantly increased the bacterial killing by approximately 6-fold when compared with that of controls. By contrast, this killing process was found to be associated with enhanced free radical production via the activation of NADPH oxidase, stimulated by the LGG conditioned medium. We also found that the conditioned medium had small effect on nitric oxide (NO) generation, albeit to a lesser extent. This work suggests that LGG-CM may play an important role in suppressing the total microbial load within the macrophages and hence, the extent to which pro-inflammatory molecules such as free radicals and NO are generated. The modulation of inflammation-promoting signals by LGG-CM may be beneficial as it modulates bacterial killing, and thereby prevents any collateral damage to host

Lactobacillus rhamnosus GG conditioned media modulates acute reactive oxygen species and nitric oxide in J774 murine macrophages

Phagocytes such as macrophages are capable of detecting and killing pathogenic bacteria by producing reactive oxygen and nitrogen species. Formation of free radicals in macrophages may be regulated by probiotics or by factors released by probiotics but yet to be identified. Thus, studies were carried out to determine whether cell-free conditioned medium obtained from cultures of Lactobacillus rhamnosus GG (LGG-CM) regulate production of reactive oxygen species (ROS) and/or nitric oxide (NO) in macrophages. J774 macrophages in culture were loaded with either H2DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Free radical production was measured on a fluorescence microplate reader and changes were analysed by Cumulative sum (CuSum) calculations. Low concentration of LGG-CM (10% LGG-CM) or LPS did not cause any significant change in basal levels of ROS or NO production. In contrast, high concentration of LGG-CM (75% and 100%) significantly enhanced ROS generation but also significantly reduced NO level. These findings are novel and suggest for the first time that probiotics may release factors in culture which enhance ROS production and may additionally reduce deleterious effects associated with excessive nitrogen species by suppressing NO level. These events may account, in part, for the beneficial bactericidal and anti-inflammatory actions ascribed to probiotics and may be of clinical relevance

Effects of endothelin-1 on the rat isolated heart

This study shows that bolus injections of endothelin-1 (ET-1) (1-30 pmol) produce transient vasodilator and prolonged coronary vasoconstrictor actions. The initial effect on cardiac contractility was a positive inotropic action, but with repeated doses a negative inotropic action developed. Verapamil (0.1 microM) antagonized the vasoconstrictor action of Bay K 8644 but did not affect ET-1-induced vasoconstriction. In contrast, removal of extracellular calcium did block the vasoconstrictor action of ET-1. This suggests that vasoconstriction is due to activation of receptor-rather than potential-operated calcium channels. The ET-1-induced vasoconstriction was not due to the release of platelet-activating factor (PAF) or thromboxane A2 since it was not inhibited by WEB 2086 (0.5 microM), fluribiprofen (2 microM), or BW755C (7 microM). In addition, thromboxane B2 could not be detected in the effluent from the heart. The vasoconstrictor action of ET-1 was potentiated by passage of air through the coronary vascular bed, suggesting that an intact endothelium normally opposes this vasoconstrictor effect

y+ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages

The original publication is available at www.springerlink.com--Copyright Springer --DOI : 10.1007/s00726-005-0311-9Peer reviewe