Dai Kimura

The effects of CO2 on cytokine concentrations in endotoxin-stimulated human whole blood.

Objectives:Hypercapnia is known to modulate inflammation in lungs. However, the effect of hypocapnia and hypercapnia on blood cytokine production during sepsis is not well understood. We hypothesized that CO2 modulates ex vivo inflammatory cytokine production during endotoxin stimulation. To test this hypothesis, we measured the production of pro- and anti-inflammatory cytokines in endotoxin-stimulated human whole blood cultures under hypercapnic, normocapnic, and hypocapnic conditions. Design:Prospective randomized study. Setting:Basic research laboratory. Subjects:Ten male and 10 female volunteers. Interventions:Venous blood samples, taken from volunteers were cultured at 37°C, under hypocapnic (2% CO2), normocapnic (5% CO2), and hypercapnic (7% CO2) conditions, with and without endotoxin stimulation. After 24 hrs of incubation, each cultures supernatant was analyzed for tumor necrosis factor-α, interleukin-1β, interleukin-6, interleukin-10, and interferon-γ concentrations by enzyme-linked immunosorbent assay. Data were analyzed using nonparametric repeated measures of analysis of variance followed by Dunns multiple comparisons test. Analysis of variance with Bonferroni correction was used to compare gender differences in cytokine concentrations. The Pearson test was used to estimate correlation between hydrogen ion and individual cytokine concentrations. Measurements and Main Results:Concentrations of the proinflammatory cytokines tumor necrosis factor-α, interleukin-1β and of the anti-inflammatory cytokine interleukin-10 under hypercapnic condition were significantly decreased (p < 0.05, 0.01, and 0.001, respectively) for both genders when compared with either normocapnic or hypocapnic conditions. Concentrations of tumor necrosis factor-α and interleukin-1β were significantly higher in men. In women, concentrations of interleukin-6 were significantly decreased under hypercapnic condition when compared with hypocapnic condition. An inverse relationship was found between hydrogen ion concentration and concentrations of tumor necrosis factor-α and interleukin-10. Conclusions:Our results are consistent with the hypothesis that CO2 can affect the production of pro- and anti-inflammatory cytokines after ex vivo stimulation with endotoxin.

Double-blind, placebo-controlled pilot randomized trial of methylprednisolone infusion in pediatric acute respiratory distress syndrome.

Objective: Low-dose methylprednisolone therapy in adults with early acute respiratory distress syndrome reduces systemic inflammation, duration of mechanical ventilation, and ICU length of stay. We report a pilot randomized trial of glucocorticoid treatment in early pediatric acute respiratory distress syndrome. Design: Double-blind, placebo-controlled randomized clinical trial. Setting: Le Bonheur Children’s Hospital, Memphis, TN. Patients: Children (0–18 yr) with acute respiratory distress syndrome undergoing mechanical ventilation. Interventions: Patients were randomly assigned to steroid or placebo groups within 72 hours of intubation. IV methylprednisolone administered as loading dose (2 mg/kg) and continuous infusions (1 mg/kg/d) on days 1–7 and then tapered over days 8–14. Both groups were ventilated according to the Acute Respiratory Distress Syndrome Network protocol modified for children. Daily surveillance was performed for adverse effects. Measurements and Main Results: Thirty-five patients were randomized to the steroid (n = 17, no death) and placebo groups (n = 18, two deaths). No differences occurred in length of mechanical ventilation, ICU stay, hospital stay, or mortality between the two groups. At baseline, higher plateau pressures (p = 0.006) and lower Pediatric Logistic Organ Dysfunction scores (p = 0.04) occurred in the steroid group; other characteristics were similar. Despite higher plateau pressures on days 1 (p = 0.006) and 2 (p = 0.025) due to poorer lung compliance in the steroid group, they had lower PaCO2 values on days 2 (p = 0.009) and 3 (p = 0.014), higher pH values on day 2 (p = 0.018), and higher PaO2/FIO2 ratios on days 8 (p = 0.047) and 9 (p = 0.002) compared with the placebo group. Fewer patients in the steroid group required treatment for postextubation stridor (p = 0.04) or supplemental oxygen at ICU transfer (p = 0.012). Steroid therapy was not associated with detectable adverse effects. Conclusion: This study demonstrates the feasibility of administering low-dose glucocorticoid therapy and measuring clinically relevant outcomes in pediatric acute respiratory distress syndrome. Changes in oxygenation and/or ventilation are consistent with early acute respiratory distress syndrome pathophysiology and results of similar clinical trials in adults. We propose and design a larger randomized trial to define the role of glucocorticoid therapy in pediatric acute respiratory distress syndrome.

Regulation of inflammatory biomarkers by intravenous methylprednisolone in pediatric ARDS patients: Results from a double-blind, placebo-controlled randomized pilot trial.

OBJECTIVE A double-blind, randomized controlled trial showed that low-dose glucocorticoid therapy in pediatric ARDS patients is feasible and may improve both ventilation and oxygenation indices in these patients. However, the molecular mechanisms underlying potential changes in outcomes remain unclear. Based on these clinical findings, this study was designed to examine the effects of intravenous methylprednisolone on circulating inflammatory biomarkers in pediatric ARDS patients. DESIGN Double-blind, placebo-controlled randomized trial with blood collection on study entry and day 7. SETTING Tertiary care childrens hospital. PATIENTS Children (0-18years) with ARDS undergoing mechanical ventilation. INTERVENTIONS 35 children were randomized within 72h of mechanical ventilation. The glucocorticoid group received methylprednisolone 2mg/kg loading dose followed by 1mg/kg/day continuous infusion from days 1 to 7. Both groups were ventilated following the ARDSnet recommendations. WBC and differential cell counts, plasma cytokines and CRP levels, and coagulation parameters were analyzed on days 0 and 7. RESULTS At study entry, the placebo group had higher IL-15 and basophil levels. On day 7, in comparison to study entry, the placebo group had lower IL-1α, IFN-γ and IL-10 levels. The glucocorticoid group had lower INF-α, IL-6, IL-10, MCP-1, G-CSF and GM-CSF levels, and higher IL-17α levels on day 7 in comparison to study entry. Total and differential cell counts remained unchanged within the placebo group between days 0 and 7, whereas in the glucocorticoid group total WBC and platelets counts were increased on day 7. Pearsons correlation studies within the placebo and glucocorticoid groups revealed positive and negative correlations between cytokine levels, cell counts, coagulation parameters and relevant clinical parameters of disease severity identified in our previous study. Multiple regression models identified several cytokines as predictors for alterations in clinical parameters of disease severity. CONCLUSION This pilot study shows the feasibility of simultaneously measuring multiple inflammatory cytokines, cell counts and coagulation parameters in pediatric ARDS patients. We report statistical models that may be useful for future, larger trials to predict ARDS severity and outcomes.

Immunological Response to (1,4)‐α‐d‐Glucan in the Lung and Spleen of Endotoxin‐Stimulated Juvenile Rats

We investigated the effects of (1,4)-alpha-D-glucan (alpha-DG), a novel immune stimulatory drug from Tinospora cordifolia, on the concentration of pro- and anti-inflammatory cytokines (interleukin [IL]-1beta, IL-6, tumour necrosis factor-alpha [TNF-alpha], gamma-interferon [IFN-gamma] and IL-10) in the lung and spleen of endotoxin-stimulated juvenile rats. Experimental groups (n = 16/group) included controls with an intraperitoneal injection of saline, endotoxaemic rats with a non-lethal dose of 10 mg/kg Escherichia coli endotoxin, and endotoxaemic rats treated with two doses of 10 mg/kg alpha-DG, intraperitoneally, 2 and 4 hr after endotoxin injection. At 24 hr of treatment, rats were euthanized and lungs and spleen were removed for cytokines determination and lung injury. Endotoxaemia increased IL-1beta concentration by fivefold in both organs, while creating a moderate pulmonary hypercellularity (demonstrated by about 11% increase in the alveolar-septal thickening and 11% decrease in the alveolar-interstitial space ratio). In the lung, alpha-DG treatment reduced concentrations of IL-1beta by 30% (p > 0.05), IL-6 by 43% (p < 0.01), IFN-gamma by 46% (p < 0.01) and the anti-inflammatory cytokine, IL-10, by 31% (p > 0.05) compared to endotoxaemia. In the spleen, alpha-DG treatment decreased the ratio of IL-1beta to IL-10 by 55% (p < 0.05), demonstrating an anti-inflammatory trend. These data suggest that alpha-DG differentially modulates cytokine response in the lung and spleen and modifies the pro- and anti-inflammatory balance during an early period of endotoxaemia in juvenile rats.

Admission and treatment factors associated with the duration of acidosis in children with diabetic ketoacidosis.

Background Our goal was to determine the factors associated with prolonged acidosis in children with diabetic ketoacidosis. Method The records of all children (109 admissions, 86 patients) admitted to the pediatric intensive care unit (PICU) during a 3-year period with the diagnosis of diabetic ketoacidosis were analyzed. Results The charts were reviewed after institutional review board approval was obtained. Demographic and serial laboratory data, time to correction of acidosis, as well as the first 24-hour chloride load, total fluid administered, fluid balance, and PICU and hospital lengths of stay were recorded. The anion gap (AG = Na − Cl − HCO3) and the delta gap (DG = AG − 12 − [24 − HCO3]) were calculated. Prolonged acidosis (HCO3 < 15 mEq/L at 24 hours) was analyzed against various independent factors on admission and during therapy. Low Na (128 vs 133 mEq/L), HCO3 (4.7 vs 9.5 mEq/L), DG (−6.3 vs −2.8 mEq/L), pH (6.97 vs 7.16), PaCO2 (15 vs 23 mm Hg), and base excess (−26 vs −18) as well as high chloride load (17 vs 11 mEq/kg per 24 hours) were associated with prolonged acidosis (t test, P < 0.05). Stepwise logistic regression eliminated all except base excess and DG in the model. Children with prolonged acidosis had longer PICU (45 vs 34 hours) and hospital stays (5.5 vs 2.5 days) (P < 0.05). The AG was normal in all cases at 24 hours. There were no deaths. Conclusions Nongap acidosis was present in many children with prolonged metabolic acidosis. We suggest that a continuous acetate or bicarbonate therapy via maintenance fluid might be beneficial in this subgroup of patients.

Corticosteroids in pediatric ARDS: all cards on the table

Intensive Care Med DOI 10.1007/s00134-015-4027-3 Andreas Schwingshackl Gianfranco Umberto Meduri Dai Kimura Stephania A. Cormier Kanwaljeet J. S. Anand Corticosteroids in pediatric ARDS: all cards on the table Accepted: 8 August 2015 O Springer-Verlag Berlin Heidelberg and ESICM 2015 Dear Editor, Hardly any topic in modern critical care medicine remains as controver- sial as steroid administration in acute respiratory distress syndrome (ARDS), despite multiple adult ran- domized controlled trials (RCTs) and recent pediatric data. The article by Yehya et al. [1] and the editorial commentary by Peters et al. [2] are vital, since few, if any, therapeutic approaches are simultaneously asso- ciated with such profound potential benefits and risks as steroid therapy in critically ill patients. Marked contradiction, however, exists between the Yehya et al. data and the findings of well-designed and protocol-driven RCTs in adult ARDS patients. These studies consistently reported significant improvements in markers of systemic inflammation, ventilator-free days, ICU-free days, no changes or actually improved survival, and either no increase or decreases in infection rate [3, 4]. The findings of Yehya et al. cannot be interpreted because the specific indi- cations for corticosteroid use were not reported. To imply that any type of steroid, at any concentration, and CO RRESPONDENCE used for more than 24 h represents a protocol-driven treatment for pedi- atric ARDS (PARDS) is simply not justifiable. Further, grouping short-term (less than 24 h) and non-corticosteroid exposed patients together is an improper control for evaluating ster- oid therapy. Corticosteroids can exert important, non-genomic effects within minutes, including decreased cell adhesion, phosphokinase activa- tion, MCP-1 and H 2 O 2 release, CD63 translocation, TNF-a and IL-6 expression. Possible corticosteroid effects cannot be assessed unless exposed and non-exposed patients are categorically separated. Thus, proposing that this single-center, observational study ‘‘has relevance for clinical practice’’, a conclusion unsupported by data, will likely mis- lead and confound many bedside physicians. Undoubtedly, the most likely explanation for Yehya et al.’s findings are (1) selection of steroid therapy for the sickest patients (con- founding by indication) and (2) rebound effects resulting from abrupt discontinuation of corticosteroids, as is well documented by worsening PaO 2 /FiO 2 ratios and increasing CRP levels. Owing to the wide-ranging impli- cations and inherent responsibility of publishing patient data, it is impera- tive that we treat this topic with the utmost equipoise until clear evidence for or against steroid use in ARDS/ PARDS is gained. Whether compar- ative effectiveness research (CER) can provide such evidence is ques- tionable, since the US Food and Drug Administration, European Medicines Agency, or other labeling agencies do not consider this research methodol- ogy Level 1 evidence. CER studies can ‘‘adjust’’ the outcomes for measured confounders, and boot- strapping techniques like propensity scoring can reduce the margin of inferential errors, but only large, well- designed RCTs can control for unmeasured and non-measurable confounders. We recommend caution in drawing conclusions from a data set with multiple confounding vari- ables and improper controls. Statistical approaches such as propensity score matching can only take into account measured con- founding factors, whereas randomized trials allow for control- ling of both measured and unmeasured confounders. The bene- ficial or no-harm results reported in adult RCTs cannot be disregarded unless systematically investigated in pediatric patients. We recently pub- lished a feasibility RCT investigating prolonged low-dose methylpred- nisolone in pediatric ARDS [5] and, undoubtedly, there is an urgent need to conduct a large-scale, well-de- signed RCT in PARDS. References 1. Yehya N, Servaes S, Thomas NJ, Nadkarni VM, Srinivasan V (2015) Corticosteroid exposure in pediatric acute respiratory distress syndrome. Intensive Care Med. doi: 10.1007/s00134-015-3953-4 2. Peters MJ, Ray S, Kneyber M (2015) Corticosteroids for paediatric ARDS: unjustified—even unjustifiable? Intensive Care Med. doi: 10.1007/s00134-015-3963-2 3. Confalonieri M, Urbino R, Potena A, Piattella M, Parigi P, Puccio G, Della Porta R, Giorgio C, Blasi F, Umberger R, Meduri GU (2005) Hydrocortisone infusion for severe community-acquired pneumonia: a preliminary randomized study. Am J Respir Crit Care Med

Management of massive diffuse alveolar hemorrhage in a child with systemic lupus erythematosus

Diffuse alveolar hemorrhage (DAH) from systemic lupus erythematosus (SLE) is a rare but potentially life-threatening condition. We report the case of a 14-year-old female with SLE who developed hypoxia and shock secondary to severe alveolar hemorrhage. She was successfully managed by placement on extracorporeal membrane oxygenation (ECMO) followed by emergent pulmonary lobectomy and medical treatment including high-dose methylprednisolone, cyclophosphamide, intravenous immunoglobulin, and plasmapheresis.

Plasma Biomarker Analysis in Pediatric ARDS: Generating Future Framework from a Pilot Randomized Control Trial of Methylprednisolone: A Framework for Identifying Plasma Biomarkers Related to Clinical Outcomes in Pediatric ARDS

Objective Lung injury activates multiple pro-inflammatory pathways, including neutrophils, epithelial, and endothelial injury, and coagulation factors leading to acute respiratory distress syndrome (ARDS). Low-dose methylprednisolone therapy (MPT) improved oxygenation and ventilation in early pediatric ARDS without altering duration of mechanical ventilation or mortality. We evaluated the effects of MPT on biomarkers of endothelial [Ang-2 and soluble intercellular adhesion molecule-1 (sICAM-1)] or epithelial [soluble receptor for activated glycation end products (sRAGE)] injury, neutrophil activation [matrix metalloproteinase-8 (MMP-8)], and coagulation (plasminogen activator inhibitor-1). Design Double-blind, placebo-controlled randomized trial. Setting Tertiary-care pediatric intensive care unit (ICU). Patients Mechanically ventilated children (0–18 years) with early ARDS. Interventions Blood samples were collected on days 0 (before MPT), 7, and 14 during low-dose MPT (n = 17) vs. placebo (n = 18) therapy. The MPT group received a 2-mg/kg loading dose followed by 1 mg/kg/day continuous infusions from days 1 to 7, tapered off over 7 days; placebo group received equivalent amounts of 0.9% saline. We analyzed plasma samples using a multiplex assay for five biomarkers of ARDS. Multiple regression models were constructed to predict associations between changes in biomarkers and the clinical outcomes reported earlier, including P/F ratio on days 8 and 9, plateau pressure on days 1 and 2, PaCO2 on days 2 and 3, racemic epinephrine following extubation, and supplemental oxygen at ICU discharge. Results No differences occurred in biomarker concentrations between the groups on day 0. On day 7, reduction in MMP-8 levels (p = 0.0016) occurred in the MPT group, whereas increases in sICAM-1 levels (p = 0.0005) occurred in the placebo group (no increases in sICAM-1 in the MPT group). sRAGE levels decreased in both MPT and placebo groups (p < 0.0001) from day 0 to day 7. On day 7, sRAGE levels were positively correlated with MPT group PaO2/FiO2 ratios on day 8 (r = 0.93, p = 0.024). O2 requirements at ICU transfer positively correlated with day 7 MMP-8 (r = 0.85, p = 0.016) and Ang-2 levels (r = 0.79, p = 0.036) in the placebo group and inversely correlated with day 7 sICAM-1 levels (r = −0.91, p = 0.005) in the MPT group. Conclusion Biomarkers selected from endothelial, epithelial, or intravascular factors can be correlated with clinical endpoints in pediatric ARDS. For example, MPT could reduce neutrophil activation (⇓MMP-8), decrease endothelial injury (⇔sICAM-1), and allow epithelial recovery (⇓sRAGE). Large ARDS clinical trials should develop similar frameworks. Trial Registration https://clinicaltrials.gov, NCT01274260.

230: PULMONARY HYPERTENSION IN INFANTS WITH RSV BRONCHIOLITIS IS NOT ASSOCIATED WITH VIRAL LOAD

Critical Care Medicine • Volume 46 • Number 1 (Supplement) www.ccmjournal.org Learning Objectives: Infants with severe respiratory syncytial virus (RSV) bronchiolitis can develop pulmonary hypertension (PHTN). Higher viral loads (VL) have been associated with mechanical ventilation (MV) and ICU admission; however, it is unknown whether high VL is associated with the development of PTHN. Methods: This prospective observational study of pediatric patients aged < 2 years, with RSV bronchiolitis, who required MV, was conducted in a tertiary children’s hospital from 2015–2016. Patients with congenital heart disease were excluded. Serial VL measurements were obtained from tracheal aspirates (TA) and nasal washings (NW) and compared to echocardiogram results obtained within the first 72 hrs. Oxygen saturation index (OSI) trends were used as a disease severity marker. Results: We recruited 33 patients age 106 ± 117.8 days old and gestational age 35.5 ± 3.1 weeks at birth were recruited. On average, the patients required MV for 12.6 ± 10.4 days. Ten subjects had evidence of mild-moderate PHTN, which was associated with lower birth weight (2090.3 g vs 2877.5 g, p = 0.005). There was no significant difference in viral load between patients with and without PTHN on day 1 (5.69 vs 6.29 NWPCR log (PFUe/ml), p = 0.17) through day 5 (5.20 vs 6.28 TAPCR log (PFUe/ml), p = 0.16). There was a trend in the subjects who had evidence of PHTN with prematurity (33.7 vs 36.22 weeks gestation, p = 0.08), higher OSI on day 3 (9.0 vs 6.85, p = 0.3), required high frequency oscillatory ventilator (HFOV) (40% vs 13%, p = 0.08), and longer ventilator duration (16.45 vs 10.93 days p = 0.27). Conclusions: In mechanically ventilated infants with severe RSV bronchiolitis, mild-moderate PHTN was identified in 30% of the subjects, and associated with lower birthweight. Higher viral loads were not associated with PTHN. Although not statistically significant, patients with PHTN tended to have longer MV, higher OSI and need for HFOV even with the same or lower viral loads. Further larger study is warranted.

New mouse model of pulmonary hypertension induced by respiratory syncytial virus bronchiolitis

Pulmonary hypertension (PH) has been observed in up to 75% of infants with moderate to severe respiratory syncytial virus (RSV) bronchiolitis and is associated with significant morbidity and mortality in infants with congenital heart disease. The purpose of the present study was to establish a mouse model of PH secondary to RSV bronchiolitis that mimics the disease etiology as it occurs in infants. Neonatal mice were infected with RSV at 5 days of age and then reinfected 4 wk later. Serum-free medium was administered to age-matched mice as a control. Echocardiography and right ventricular systolic pressure (RVSP) measurements via right jugular vein catheterization were conducted 5 and 6 days after the second infection, respectively. Peripheral capillary oxygen saturation monitoring did not indicate hypoxia at 2-4 days post-RSV infection, before reinfection, and at 2-7 days after reinfection. RSV-infected mice had significantly higher RVSP than control mice. Pulsed-wave Doppler recording of the pulmonary blood flow by echocardiogram demonstrated a significantly shortened pulmonary artery acceleration time and decreased pulmonary artery acceleration time-to-ejection time ratio in RSV-infected mice. Morphometry showed that RSV-infected mice exhibited a significantly higher pulmonary artery medial wall thickness and had an increased number of muscularized pulmonary arteries compared with control mice. These findings, confirmed by RVSP measurements, demonstrate the development of PH in the lungs of mice infected with RSV as neonates. This animal model can be used to study the pathogenesis of PH secondary to RSV bronchiolitis and to assess the effect of treatment interventions. NEW & NOTEWORTHY This is the first mouse model of respiratory syncytial virus-induced pulmonary hypertension, to our knowledge. This model will allow us to decipher molecular mechanisms responsible for the pathogenesis of pulmonary hypertension secondary to respiratory syncytial virus bronchiolitis with the use of knockout and/or transgenic animals and to monitor therapeutic effects with echocardiography.