DIAGRAMA 1. Diagrama PERT de planificación
6. PROPUESTA
6.1.1 El aprendizaje en DS-TMS
Herein, we demonstrate that plasma levels of nDNA and mtDNA are increased in septic shock patients. Furthermore, plasma levels of nDNA, but not mtDNA, are related to markers of inflammation, shock, and organ damage in these patients. Similar findings were obtained during human endotoxemia: LPS administration in healthy volunteers results in increased plasma levels of nDNA and mtDNA, although healthy volunteers exhibit lower peak levels compared with septic shock patients. Moreover, peak levels of nDNA, but not mtDNA, are related with the endotoxin-induced pro-inflammatory cytokine response.
It is generally assumed that nucleic acids are released after rupture or necrosis of cells 5. However, our results obtained in the relatively mild human endotoxemia model demonstrate that a systemic inflammatory response, which is not expected to cause significant cell rupture or necrosis, also results in release of nucleic acids. These data suggests that inflammation results in active release of nucleic acids. Such active release, both spontaneous and during cell death, has been described previously, although the exact mechanisms remain to be elucidated 6-8. Interestingly, plasma cytokine levels were higher in healthy volunteers during endotoxemia compared with septic shock patients, while levels of nucleic acids were much lower. While the pathogenesis of septic shock cannot be directly compared with experimental human endotoxemia, these differences suggest
Figure 4 – Plasma concentrations of DAMPs during endotoxemia Plasma levels of DAMPs
nDNA (panel A) and mtDNA (panel B) in healthy volunteers during endotoxemia. Data are expressed as median±IQR. * indicates p<0.05 compared with time-point T=-60.
the inflammatory response and passively as a result of cell damage.
Our data further show that, compared with nDNA, the fold increase in plasma levels of mtDNA in sepsis patients compared with healthy controls as well as the fold increase observed in healthy volunteers following LPS administration is relatively low. Furthermore, mtDNA levels showed neither a relationship with inflammatory parameters in both patients and healthy volunteers, nor with clinical parameters in patients. These findings suggest that the role of mtDNA during sepsis and/or systemic inflammation is limited. nDNA might exert immunological effects, possibly via binding of PRRs, e.g. RIG-I (retinoic acid- inducible gene-I) or DAI (DNA-dependent activator of IFN-regulatory factors), both cytosolic receptors for double-stranded DNA 10. In turn, activation of this inflammatory cascade may result in aggravation of shock and organ damage. However, the fact that higher levels of nDNA were found in septic patients than in healthy volunteers during endotoxemia, while concentrations of inflammatory cytokines were much lower might indicate that the role of nDNA in initiation and/or propagation of the inflammatory response is limited as well. This could be due to the fact that DAMP-properties of nucleic acids may be dependent on the release of other DAMPs, e.g. HMGB1, and that nucleic acids can also affect activity of other DAMPs 21. PRRs recognizing nucleic acids are generally present intracellularly, and internalization necessary for receptor-binding is stimulated by other DAMPs 21. These interactions underline the fact that multiple complex processes take place simultaneously in critically ill patients, making it difficult to distinguish causality from epiphenomena and unravel underlying mechanisms in observational studies.
This study has several limitations we want to acknowledge. First, the control group consist of young males, whereas the patient population comprises both males and females in a wide age range. With regard to gender, we investigated differences between male and female septic patients for all parameters described in this study and only found slightly, but statistically significant, higher levels of IL-6 and IL-8 on day 3 in women. No differences on other days were found. Therefore it is very unlikely that the presence of both men and women in our patient population relevantly biased our results. Concerning age, only 4 patients of our patient population fell within the age range of the healthy volunteers studied. Therefore, we could not analyze differences between young (i.e. those with comparable ages as the healthy volunteers) and old patients.
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conclusions as to whether there is a causative relation between nDNA levels, inflammation, shock, and organ damage. However, in a recent murine study, administration of DNAses (to remove free nucleic acids from the circulation) resulted in decreased coagulation and inflammation, suppression of organ damage, and improved outcome in a cecal ligation and puncture sepsis model 22. These findings suggest a detrimental role of nucleic acids in sepsis.
Third and finally, although statistically significant, the correlations between nDNA and markers of inflammation, shock, kidney, and liver damage in septic patients were relatively weak. Therefore caution should be taken when interpreting these results. The well-known high heterogeneity of septic shock patients might contribute to this.
In conclusion, plasma levels of nucleic acids mtDNA and especially nDNA are increased in septic shock patients and healthy volunteers during experimental human endotoxemia, and nDNA levels are related with inflammatory cytokines. This suggests that nucleic acids, particularly nDNA, are both actively and passively released; as a result of inflammation and cell damage, respectively. Furthermore, nDNA levels correlate with markers of shock and organ damage in septic shock patients. It remains to be determined whether nDNA is merely a marker or directly involved in the pathophysiology of septic shock.
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