Efrossini Briassouli

Glutamine Randomized Studies in Early Life: The Unsolved Riddle of Experimental and Clinical Studies

Glutamine may have benefits during immaturity or critical illness in early life but its effects on outcome end hardpoints are controversial. Our aim was to review randomized studies on glutamine supplementation in pups, infants, and children examining whether glutamine affects outcome. Experimental work has proposed various mechanisms of glutamine action but none of the randomized studies in early life showed any effect on mortality and only a few showed some effect on inflammatory response, organ function, and a trend for infection control. Although apparently safe in animal models (pups), premature infants, and critically ill children, glutamine supplementation does not reduce mortality or late onset sepsis, and its routine use cannot be recommended in these sensitive populations. Large prospectively stratified trials are needed to better define the crucial interrelations of “glutamine-heat shock proteins-stress response” in critical illness and to identify the specific subgroups of premature neonates and critically ill infants or children who may have a greater need for glutamine and who may eventually benefit from its supplementation. The methodological problems noted in the reviewed randomized experimental and clinical trials should be seriously considered in any future well-designed large blinded randomized controlled trial involving glutamine supplementation in critical illness.

Influence of different ventilator modes on Vo2 and Vco2 measurements using a compact metabolic monitor.

OBJECTIVE We assessed the influence of different ventilator modes on carbon dioxide elimination (Vco(2)) and oxygen uptake (Vo(2)) using a new compact modular metabolic monitor (E-COVX) and its impact on calculated respiratory quotient (RQ) and resting energy expenditure (REE) in critically ill children. METHODS Sequential 30-min ventilation by pressure-regulated volume controlled ventilation (PRVC), synchronized intermittent mandatory ventilation (SIMV), and biphasic intermittent positive airway pressure/airway pressure release ventilation (BiVent) in mechanically ventilated critically-ill children was assessed. To determine within- or between-day variations, 30-min Vo(2) and Vco(2) measurements were repeated at four separate occasions. RESULTS A total of 3960pulmonary 1-min gas exchange measurements were recorded in the 44 sessions for the three ventilator modes. Vo(2), Vco(2), and REE did not differ significantly among the PRVC, SIMV, and BiVent sequence of measurements. RQ (0.86+/-0.1) in the SIMV and Vco(2) (113+/-55mL/min) in the BiVent mode had a higher trend compared with PRVC (0.82+/-0.01, P<0.05, and 103+/-49mL/min, P<0.2, respectively). All three modes displayed good agreement and there were no significant differences between the first and second same-day or between the first- and second-day measurements or sequentially changed ventilator modes. Bland-Altman plots comparing the means of sequential REE, Vo(2), Vco(2), and RQ during the PRVC, SIMV, and BiVent modes of ventilation indicated that the average paired differences were <-5.5%. CONCLUSION The influence of different ventilator modes on Vo(2) and Vco(2) measurements in adequately sedated critically ill children is not significant. The E-COVX metabolic module is suitable for repeated measurements in well-sedated mechanically ventilated children with stable respiratory patterns using the PRVC, SIMV, or BiVent modes of ventilation.

Heat Shock Protein 72 Expressing Stress in Sepsis: Unbridgeable Gap between Animal and Human Studies—A Hypothetical “Comparative” Study

Heat shock protein 72 (Hsp72) exhibits a protective role during times of increased risk of pathogenic challenge and/or tissue damage. The aim of the study was to ascertain Hsp72 protective effect differences between animal and human studies in sepsis using a hypothetical “comparative study” model. Forty-one in vivo (56.1%), in vitro (17.1%), or combined (26.8%) animal and 14 in vivo (2) or in vitro (12) human Hsp72 studies (P < 0.0001) were enrolled in the analysis. Of the 14 human studies, 50% showed a protective Hsp72 effect compared to 95.8% protection shown in septic animal studies (P < 0.0001). Only human studies reported Hsp72-associated mortality (21.4%) or infection (7.1%) or reported results (14.3%) to be nonprotective (P < 0.001). In animal models, any Hsp72 induction method tried increased intracellular Hsp72 (100%), compared to 57.1% of human studies (P < 0.02), reduced proinflammatory cytokines (28/29), and enhanced survival (18/18). Animal studies show a clear Hsp72 protective effect in sepsis. Human studies are inconclusive, showing either protection or a possible relation to mortality and infections. This might be due to the fact that using evermore purified target cell populations in animal models, a lot of clinical information regarding the net response that occurs in sepsis is missing.

The effects of endotracheal suctioning on the accuracy of oxygen consumption and carbon dioxide production measurements and pulmonary mechanics calculated by a compact metabolic monitor.

BACKGROUND:Open endotracheal suctioning (ETS), which is performed regularly in mechanically ventilated patients to remove obstructive secretions, can cause an immediate decrease in dynamic compliance and expired tidal volume and result in inadequate or inaccurate sidestream respiratory monitoring, necessitating prolonged periods of stabilization of connected metabolic monitors. We investigated the immediate effect of open ETS on the accuracy of oxygen consumption (VO2) and carbon dioxide production (VCO2) measurements and calculated lung mechanics, respiratory quotient, and resting energy expenditure in mechanically ventilated children without severe lung pathology, when using a compact modular metabolic monitor (E-COVX) continuously recording patient spirometry and gas exchange measurements. METHODS:Open ETS was performed when clinically indicated in 11 children mechanically ventilated for sepsis or head injury. A total of 2800 pulmonary 1-min gas exchange measurements were recorded in 28 ETS instances for 50 consecutive minutes before and 50 min after the standardized procedure. RESULTS:Pulmonary mechanics and indirect calorimetry did not differ between pre- and postsuction sets of measurements. Pre- and postsuction VO2, VCO2, dynamic airway resistance, dynamic compliance, and expiratory minute ventilation remained stable from 5 to 55 min after tracheal suctioning and did not differ among different ventilatory modes. Average paired differences of sequential pre- and postsuction VO2, VCO2, respiratory quotient, and resting energy expenditure were −0.6%, −1%, −0.1%, and −0.3%. Ratio differences between the first and the second periods of measurements (1–25 vs 26–50 sets of 1-min measurements) did not differ in the two groups. CONCLUSIONS:Pulmonary mechanics and indirect calorimetry measurements are not influenced after uneventful open ETS in well-sedated patients. The E-COVX is able to reliably record spirometry and metabolic indices as early as 5 min after suctioning at different ventilator modes.

Early Response Roles for Prolactin Cortisol and Circulating and Cellular Levels of Heat Shock Proteins 72 and 90α in Severe Sepsis and SIRS

Objective. To evaluate the early heat shock protein (HSP) and hormonal stress response of intensive care unit (ICU) patients with severe sepsis/septic shock (SS) or systemic inflammatory response syndrome (SIRS) compared to healthy subjects (H). Methods. Patients with early (first 48 hrs) SS (n = 29) or SIRS (n = 29) admitted to a university ICU and 16 H were enrolled in the study. Serum prolactin, cortisol, and plasma ACTH were determined using immunoassay analyzers. ELISA was used to evaluate extracellular HSPs (eHSP90α, eHSP72) and interleukins. Mean fluorescence intensity (MFI) values for intracellular HSPs (iHSP72, iHSP90α) were measured using 4-colour flow-cytometry. Results. Prolactin, cortisol, and eHSP90α levels were significantly increased in SS patients compared to SIRS and H (P < 0.003). ACTH and eHSP72 were significantly higher in SS and SIRS compared to H (P < 0.005). SS monocytes expressed lower iHSP72 MFI levels compared to H (P = 0.03). Prolactin was related with SAPS III and APACHE II scores and cortisol with eHSP90α, IL-6, and lactate (P < 0.05). In SS and SIRS eHSP90α was related with eHSP72, IL-6, and IL-10. Conclusion. Prolactin, apart from cortisol, may have a role in the acute stress response in severe sepsis. In this early-onset inflammatory process, cortisol relates to eHSP90α, monocytes suppress iHSP72, and plasma eHSP72 increases.

Glutamine May Repress the Weak LPS and Enhance the Strong Heat Shock Induction of Monocyte and Lymphocyte HSP72 Proteins but May Not Modulate the HSP72 mRNA in Patients with Sepsis or Trauma

Objective. We assessed the lipopolysaccharide (LPS) or heat shock (HS) induction of heat shock protein-72 (HSP72) in peripheral blood mononuclear cells (PBMCs) of patients with severe sepsis (SS) or trauma-related systemic inflammatory response syndrome (SIRS), compared to healthy individuals (H); we also investigated any pre- or posttreatment modulating glutamine (Gln) effect. Methods. SS (11), SIRS (10), and H (19) PBMCs were incubated with 1 μg/mL LPS or 43°HS. Gln 10 mM was either added 1 h before or 1 h after induction or was not added at all. We measured monocyte (m), lymphocyte (l), mRNA HSP72, HSP72 polymorphisms, interleukins (ILs), monocyte chemoattractant protein-1 (MCP-1), and cortisol levels. Results. Baseline lHSP72 was higher in SS (p < 0.03), and mHSP72 in SIRS (p < 0.02), compared to H. Only HS induced l/mHSP72/mRNA HSP72; LPS induced IL-6, IL-8, IL-10, and MCP-1. Induced mRNA was related to l/mHSP72, and was related negatively to cytokines. Intracellular l/mHSP72/HSP72 mRNA was related to serum ILs, not being influenced by cortisol, illness severity, and HSP72 polymorphisms. Gln did not induce mRNA in any group but modified l/mHSP72 after LPS/HS induction unpredictably. Conclusions. HSP72 mRNA and l/mHSP72 are higher among critically ill patients, further induced by HS, not by LPS. HSP72 proteins and HSP72 mRNA are related to serum ILs and are negatively related to supernatant cytokines, not being influenced by HSP72 polymorphisms, cortisol, or illness severity. Gln may depress l/mHSP72 after LPS exposure and enhance them after HS induction, but it may not affect early induced HSP72 mRNA.

Immunoparalysis: Clinical and immunological associations in SIRS and severe sepsis patients.

Introduction: This study was designed to identify changes in the monocytic membrane marker HLA‐DR and heat shock proteins (HSPs) in relation to T‐regulatory cells (T‐regs) and other immunological marker changes in patients with systemic inflammatory response syndrome (SIRS) or sepsis/septic shock. Methods: Healthy volunteers, intensive care unit (ICU) patients with SIRS due to head injury and ICU patients with severe sepsis/septic shock were enrolled in the current study. Determination of CD14+/HLA‐DR+ cells, intracellular heat‐shock proteins and other immunological parameters were performed by flow cytometry and RT‐PCR techniques as appropriate. Univariate and multivariate analysis examined associations of CD14/HLA‐DR, HSPs, T‐regs and suppressor of cytokine signalling (SOCS) proteins with SIRS, sepsis and outcome. Results: Fifty patients (37 with severe sepsis and 13 with SIRS) were enrolled, together with 20 healthy volunteers used as a control group. Compared to healthy individuals, patients with SIRS and severe sepsis showed progressive decline of their CD14/HLA‐DR expression (0% to 7.7% to 50% within each study subpopulation, p < 0.001). Mean fluorescent intensity (MFI) levels of HSP70 and HSP90 on monocytes and polymorphonuclear cells were significantly higher in SIRS patients compared to controls and fell significantly in severe sepsis/septic shock patients (p < 0.05 for all comparisons). There was no statistically significant difference between subgroups for levels of T‐regulatory cells or relative copies of Suppressor of Cytokine Signalling 3 (SOCS3) proteins. In univariate models percent of CD14/HLA‐DR was associated with mortality (OR: 1.8 95%CI 1.02–3.2, p = 0.05), while in multivariate models after adjusting for CD14/HLA‐DR only younger age and lower Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were associated with increased chances of survival (beta −0.05, OR 0.9, 95% CI 0.9–0.99, p = 0.038 for age and beta −0.11, OR 0.89, 95% CI 0.8–0.99, p = 0.037 for APACHE II score). Conclusions: Significant associations with SIRS and sepsis were found for CD14/HLA‐DR expression and monocyte and polymorphonuclear cell levels of HSP70 and 90. The role of these biomarkers in assessing the prognosis of sepsis needs to be further explored and validated in prospective studies.

Glutamine suppresses Hsp72 not Hsp90α and is not inducing Th1, Th2, or Th17 cytokine responses in human septic PBMCs.

OBJECTIVE L-Alanyl-glutamine (L-Ala-Gln) is a pharmaco-nutrient commonly used in nutrition regimens due to its immunomodulatory effects. In critically ill patients who are septic, L-Ala-Gln was associated with an increase in mortality. The aim of this study was to investigate whether L-Ala-Gln modulated heat shock protein (Hsp)-72, 90-α, T helper (Th)1, Th2, and Th17 cytokine expression in the peripheral blood mononuclear cells (PBMC) of patients with severe sepsis. METHODS Time-dose effects of L-Ala-Gln were compared with those of L-glutamine (L-Gln) and lipopolysaccharide (LPS) and to healthy controls. PBMCs were incubated with 1 or 10 μg/mL LPS, 5 or 10 mM L-Gln, and 5 or 10 mM L-Ala-Gln for different periods of time (0; 4; 24 h) when culture supernatants were harvested. RESULTS In both groups, basal Hsp72 increased over time (P < 0.02); Hsp90-α levels declined in controls (P < 0.02) but remained increased in septic patients (P < 0.02), not exhibiting any significant time-response trend. Both Glns suppressed Hsp72 in septic and controls at 10 mM by 4 h (P < 0.045) and Hsp90-α in the control group by 24 h (P < 0.045). LPS did not induce Hsps in either group. L-Ala-Gln did not induce any of the Th1, Th2, and Th17 cytokines in either group. CONCLUSION High doses of L-Gln or L-Ala-Gln do not induce any of the Th1, Th2, and Th17 cytokines in either healthy or septic human PBMCs. High Gln doses suppress Hsp72 in septic and control PBMCs. Hsp90-α time-series expression declines, contrasting the increasing trend of Hsp72 in healthy controls. Hsp90-α sustains increased levels in septic supernatants, showing a characteristic longitudinal behavior needed further elucidation.

Potential for Glutamine Supplementation in Critically Ill Children

Glutamine serves as a primary fuel for rapidly dividing cells, such as in the gut and immune system, and is used as a source of nitrogen to refill the citric acid cycle. During critical illness, the demand for glutamine may exceed that which can be mobilised from muscle stores. Clinical data over the past 20 years have provided evidence that glutamine supplementation may reduce mortality, the occurrence of infections and hospital length of stay in such patients. Experimental work has proposed various mechanisms of glutamine action but none of the randomised studies in early life could demonstrate any effect on mortality and only a few showed some effect in inflammatory response, organ function and a trend for infection control. However, the beneficial effect of glutamine, in adult and experimental models of sepsis, appears to be HSP70 dependent. The aim of this systematic literature review is to examine whether glutamine supplementation improves outcome in infants and children. Methodological problems in clinical trials and interrelations with stress-induced heat-shock protein and inflammatory response should be considered in future research involving glutamine supplementation in premature infants and critically ill children.

0104. Modulatory effects of heat shock with or without glutamine compared to LPS on peripheral blood mononuclear cells heat-shock-protein 90α expression in severe sepsis and trauma

Inflammatory stimuli cause posttranslational modifications of inducible 90α-kDa-heat-shock-protein (HSP90α) that are Hsp90-inhibitor sensitive and may be important to the pro-inflammatory actions of Hsp90α.