Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorfen

AIM： To evaluate the production of reactive oxygen species （ROS） and the expression of inducible nitric oxide synthase （iNOS） in rat isolated Kupffer cells （KCs） stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS： Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Pudfied Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS： B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION： Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.

Non-invasive panel tests for gastrointestinal motility monitoring within the MARS-500 Project

AIM:To develop an integrated approach for monitoring gastrointestinal motility and inflammation state suitable for application in long-term spaceflights.METHODS:Breath tests based on the oral administration of 13 C-labeled or hydrogen-producing substrates followed by the detection of their metabolites(13 CO 2 or H 2) in breath were used to measure gastrointestinal motility parameters during the 520-d spaceflight ground simulation within the MARS-500 Project.In particular,the gastric emptying rates of solid and liquid contents were evaluated by 13 C-octanoic acid and 13 Cacetate breath tests,respectively,whereas the orocecal transit time was assessed by an inulin H 2-breath test,which was performed simultaneously with the 13 C-octanoic acid breath test.A ready-to-eat,standardized pre-packaged muffin containing 100 mg of 13 C-octanoic acid was used in the 13 C-octanoic acid breath test to avoid the extemporaneous preparation of solid meals.In addition,a cassette-type lateral flow immunoassay was employed to detect fecal calprotectin,a biomarker of intestinal inflammation.Because no items could be introduced into the simulator during the experiment,all materials and instrumentation required for test performance during the entire mission simulation had to be provided at the beginning of the experiment.RESULTS:The experiments planned during the simulation of a manned flight to Mars could be successfully performed by the crewmembers without any external assistance.No evident alterations(i.e.,increasing or decreasing trends) in the gastric emptying rates were detected using the 13 C-breath tests during the mission simulation,as the gastric emptying half-times were in the range of those reported for healthy subjects.In contrast to the 13 C-breath tests,the results of the inulin H 2-breath test were difficult to interpret because of the high variability of the H 2 concentration in the breath samples,even within the same subject.This variability suggested that the H 2-breath test was strongly affected by external factors,which may have been related to the diet of the crewmembers or to environmental conditions(e.g.,the accumulation of hydrogen in the simulator microenvironment).At least in closed microenvironments such as the MARS-500 simulator,13 C-breath tests should therefore be preferred to H 2-breath tests.Finally,the fecal calprotectin test showed significant alterations during the mission simulation:all of the crewmembers were negative for the test at the beginning of the simulation but showed various degrees of positivity in at least one of the subsequent tests,thus indicating the onset of an intestinal inflammation.CONCLUSION:Breath tests,especially those 13 Cbased,proved suitable for monitoring gastrointestinal motility in the 520-d isolation experiment withinMARS-500 project and can be applied in long-term spaceflights.