| Abstract |
BACKGROUND: Sepsis remains a critical problem that intensively employs the medical world due to high mortality. Although the rate has decreased over the last decade, thanks due to early detection, intervention and supportive care, the mortality rate of severe sepsis /septic shock remains high. The pathophysiology of sepsis remains a puzzle that’s still has missing pieces. Sepsis is an inflammatory process during which various mediators such as cytokines play an important role among microorganisms and the immune system of the host. It’s known that stimulation of cells by pathogens has as result the initiation of the cascade of pro-inflammatory and anti-inflammatory mediators such as cytokines, lipid markers and reactive oxygen species that together cause vasodilatation and increased permeability resulting in capillary leak, output of plasma and activation of leukocytes into tissues and organs.
Additionally, activated mediators stimulate the coagulation system causing disseminated intravascular coagulation. This sequence of tissue events results in hypoperfusion and hypoxia, which is the cause of the organic dysfunction, and which frequently represents the lethal phase of sepsis. An overwhelming immune response in infectious (S,SS) or non-infectious systemic inflammatory response syndrome (SIRS), as mediated by the uncontrolled release of pro-inflammatory mediators, can lead to shock, multiple organ system failure (MOSF), and even death. Identification of the high-risk patients and early treatment of severe sepsis remain a research challenge. Different biomarkers have been studied and proposed as indicators of severity and mortality in sepsis (bacterial agents, acute phase proteins (protein C, procalcitonin, LBP-LPS-binding protein), factors of coagulation (fibrin degrading products, antithrombin III, dimer D) cell membrane markers (HLA-DR, CD64, E-selectin), hormones (cortisol, ACTH) soluble receptors (sCD14, sTNFRI, sTRFRII) and cytokines (TNF, IL-6, IL-8, IL-10). Recent studies have concentrated on heat shock proteins as indicators of the severity of sepsis based on the fact that the HSPs induce the production of interleukins and stimulate immune cells during systemic inflammatory response. Contrasting to animal studies showing an HSP72 protective effect in experimental sepsis, human in vivo or in vitro studies are not conclusive, showing a possible relation to either protection or mortality and morbidity. Although increased circulation of stress proteins signals danger to inflammatory cells and aid in immune surveillance, they have also been liked to deleterious role in some diseases. Thus, increased extracellular HSP72 levels were detected in the plasma of children admitted to the PICU with septic shock and correlated with worse outcome.
Another major molecular chaperone, 90-kDa family of HSP (inducible HSP90α), has been shown to interact mainly with about 200 client proteins involved in transcription regulation and signaling-transduction pathways of glucocorticoid receptors, ribonucleoproteins, chromatin remodeling factors, transcription factors, and protein kinases. The guarding role of this abundant “chaperone” is to launch a rapid response to various insults such as heat, hypoxia, reactive oxygen species and injury-released growth factors. Following the contrasting HSP72 human studies, it has been recently shown that activation of the HSP90/Akt pathway induces caspase-3 activation provoking apoptosis in septic mice. Robust correlations, however, between extracellular HSP90α levels with clinically relevant outcome variables during the acute phase of SS or SIRS or with pro- or anti-inflammatory interleukins (IL), nCD64 or metabolic derangements are missing. Similarly, no studies in children have been conducted comparing HSP72, an HSP90α-machinery complement, among different PICU sub cohorts, including SIRS, or relating their levels to the acute phase nCD64 expression or early metabolic patterns, various interleukins, and MOSF.
OBJECTIVE: Extracellular heat-shock-proteins (HSP) act as inducers of interleukins (IL) and stimulants for immune cells during systemic inflammatory response syndrome (SIRS). Little is known about the alarming roles of extracellular HSP72 and HSP90α in acute phase of sepsis (S) or severe sepsis (SS).We hypothesized that increased HSP90α levels could be detected in the serum of critically ill children, especially those with severe sepsis and/or septic shock and in SIRS compared to healthy controls (H), and that extracellular HSP90α might better than the HSP72, also increased in SS, SIRS and MOSF, correlate with MOSF, early acute inflammatory and metabolic stress, severity of illness scores and outcome end-points.
METHODS: Critically ill children with S (n=20), SS (n=22) or SIRS (n=23) and H (n=25) were enrolled in the study. Immediately after PICU admission, Blood and urine cultures, pharyngeal and deep tissue swabs, and bronchoalveolar lavage (BAL) were examined and blood samples were taken for study of indicators of inflammation. We determined serum HSP90α, HSP72, IL-6, IL-8, IL-10, TNF-α and neutrophil CD64 (nCD64) expression in children with S or SS compared to SIRS (trauma) or healthy children (H).ELISA was used to evaluate HSPs, chemiluminescence to measure IL, and flow-cytometry to evaluate nCD64 expression. The severity of the illness in studied children was assessed with PRISM (Pediatric Risk of Mortality Score), TISS (Therapeutic Intervention Scoring System) modified for children and PELOAD score (Pediatric Logistic Organ Dysfunction Score) and MOSF. Length of stay (LOS), days on mechanical ventilation (DOMV) and PICU-mortality were the outcome endpoints recorded. Sepsis, severe sepsis, septic shock, and SIRS due to trauma were defined according to the International Pediatric Sepsis Consensus Conference definitions. The MOSF syndrome was defined using the criteria by Wilkinson.
RESULTS: Patients in both septic groups had elevated HSP90α, HSP72, IL-6, IL-8 and IL-10 levels compared to H, where as SS had increased HSP72, IL6 and TNF-α compared to SIRS (TBI).SIRS patients presented increased HSP90α, IL-6 and IL-8 compared to H. HSP72 related negatively to metabolic indices and positively to TNF-α and nCD64 only the relation to HDL reached a statistical significance (p<0.001). HSP90 related positively (p<0.001) to nCD64, IL-8, IL-10, CRP, PRISM, PELOD, TISS, and LOS and negatively to HDL (p<0.001) and LDL (p<0.02).HSP90α has a positive relation to various interleukins and acute phase proteins. Both HSPs were dramatically increased among non-survivors. HSP90α, along with HSP72, were dramatically increased among MOSF patients. The HSP90α and HSP72 increases in the MOSF group paralleled: a) significant increases of the inflammatory biomarkers nCD64, IL-6, CRP, and PCT; b) significant decreases of nutritional indices cholesterol, HDL, and LDL, but not glucose.
CONCLUSION: Extracellular HSHP72 and HSP90α are alarmingly elevated in critically ill children, especially in severe sepsis. For the first time we report that extracellular HSP90α is significantly elevated in critically ill children with SIRS, especially in pediatric patients with SS. More importantly, we showed the extracellular “chaperokine” HSP90α is associated with MOSF, inflammatory stress, severity of illness, organ dysfunction, length of stay, days on ventilator and predicted mortality. The HSP90α and HSP72 inverse relations to the low-LDL/low-HDL early septic metabolic derangement might well imply a coordinator role for their increased extracellular expressions at the onset of the host response in severe sepsis. Since both HSP are inversely related to the low-LDL / low-HDL septic metabolic pattern, extracellular HSP72 and HSP90α may also play a coordinator role in the host response during severe sepsis. Future studies will be required to further advance our understanding of mechanisms that regulate the immune response during severe sepsis and SIRS related MOSF that may lead to the identification of new diagnostic, prognostic or treatment targets.
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