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Objectives: In the Fluid and Catheter Treatment Trial (NCT00281268), adults with acute lung injury randomized to a conservative vs. liberal fluid management protocol had increased days alive and free of mechanical ventilator support (ventilator-free days). Recruiting sufficient children with acute lung injury into a pediatric trial is challenging. A Bayesian statistical approach relies on the adult trial for the a priori effect estimate, requiring fewer patients. Preparing for a Bayesian pediatric trial mirroring the Fluid and Catheter Treatment Trial, we aimed to: 1) identify an inverse association between fluid balance and ventilator-free days; and 2) determine if fluid balance over time is more similar to adults in the Fluid and Catheter Treatment Trial liberal or conservative arms.Design: Multicentered retrospective cohort study.Setting: Five pediatric intensive care units.Patients: Mechanically ventilated children (age≥1 month to <18 yrs) with acute lung injury admitted in 2007-2010.Interventions: None.Measurements and Main Results: Fluid intake, output, and net fluid balance were collected on days 1-7 in 168 children with acute lung injury (median age 3 yrs, median PaO2/FIO2 138) and weight-adjusted (mL/kg). Using multivariable linear regression to adjust for age, gender, race, admission day illness severity, PaO2/FIO2, and vasopressor use, increasing cumulative fluid balance (mL/kg) on day 3 was associated with fewer ventilator-free days (p=.02). Adjusted for weight, daily fluid balance on days 1-3 and cumulative fluid balance on days 1-7 were higher in these children compared to adults in the Fluid and Catheter Treatment Trial conservative arm (p<.001, each day) and was similar to adults in the liberal arm.Conclusions: Increasing fluid balance on day 3 in children with acute lung injury at these centers is independently associated with fewer ventilator-free days. Our findings and the similarity of fluid balance patterns in our cohort to adults in the Fluid and Catheter Treatment Trial liberal arm demonstrate the need to determine whether a conservative fluid management strategy improves clinical outcomes in children with acute lung injury and support a Bayesian trial mirroring the Fluid and Catheter Treatment Trial. [ABSTRACT FROM AUTHOR]

Objectives: To provide an updated review of the literature on nonpulmonary treatments for pediatric acute respiratory distress syndrome (PARDS) from the Second Pediatric Acute Lung Injury Consensus Conference., Data Sources: MEDLINE (Ovid), Embase (Elsevier), and CINAHL Complete (EBSCOhost)., Study Selection: Searches were limited to children with PARDS or hypoxic respiratory failure focused on nonpulmonary adjunctive therapies (sedation, delirium management, neuromuscular blockade, nutrition, fluid management, transfusion, sleep management, and rehabilitation)., Data Extraction: Title/abstract review, full-text review, and data extraction using a standardized data collection form., Data Synthesis: The Grading of Recommendations Assessment, Development, and Evaluation approach was used to identify and summarize evidence and develop recommendations. Twenty-five studies were identified for full-text extraction. Five clinical practice recommendations were generated, related to neuromuscular blockade, nutrition, fluid management, and transfusion. Thirteen good practice statements were generated on the use of sedation, iatrogenic withdrawal syndrome, delirium, sleep management, rehabilitation, and additional information on neuromuscular blockade and nutrition. Three research statements were generated to promote further investigation in nonpulmonary therapies for PARDS., Conclusions: These recommendations and statements about nonpulmonary treatments in PARDS are intended to promote optimization and consistency of care for patients with PARDS and identify areas of uncertainty requiring further investigation., Competing Interests: Dr. Morrow’s institution received funding from the National Research Foundation of Southern Africa, EuroQual Group, and Society of Critical Care Medicine/Pediatric Critical Care Medicine; she received funding from the University of Cape Town. Dr. Nadkarni is an elected member of the Executive Committee (Council) of the Society of Critical care Medicine. The views presented are those of the author and do not represent the views of the SCCM organization. Dr. Curley’s institution received funding from the National Heart, Lung, and Blood Institute and the National Institute for Child Health and Human Development. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2023 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

Critically ill children commonly receive coagulant products (plasma and/or platelet transfusions) to prevent or treat hemorrhage or correct coagulopathy. Unique aspects of pediatric developmental physiology, and the complex pathophysiology of critical illness must be considered and balanced against known transfusion risks. Transfusion practices vary greatly within and across institutions, and high-quality evidence is needed to support transfusion decision-making. We present recent recommendations and expert consensus statements to direct clinicians in the decision to transfuse or not to transfuse hemostatic blood products, including plasma, platelets, cryoprecipitate, and recombinant products to critically ill children., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 International Anesthesia Research Society.)

Objectives: Critically ill children frequently receive plasma and platelet transfusions. We sought to determine evidence-based recommendations, and when evidence was insufficient, we developed expert-based consensus statements about decision-making for plasma and platelet transfusions in critically ill pediatric patients., Design: Systematic review and consensus conference series involving multidisciplinary international experts in hemostasis, and plasma/platelet transfusion in critically ill infants and children (Transfusion and Anemia EXpertise Initiative-Control/Avoidance of Bleeding [TAXI-CAB])., Setting: Not applicable., Patients: Children admitted to a PICU at risk of bleeding and receipt of plasma and/or platelet transfusions., Interventions: None., Measurements and Main Results: A panel of 29 experts in methodology, transfusion, and implementation science from five countries and nine pediatric subspecialties completed a systematic review and participated in a virtual consensus conference series to develop recommendations. The search included MEDLINE, EMBASE, and Cochrane Library databases, from inception to December 2020, using a combination of subject heading terms and text words for concepts of plasma and platelet transfusion in critically ill children. Four graded recommendations and 49 consensus expert statements were developed using modified Research and Development/UCLA and Grading of Recommendations, Assessment, Development, and Evaluation methodology. We focused on eight subpopulations of critical illness (1, severe trauma, intracranial hemorrhage, or traumatic brain injury; 2, cardiopulmonary bypass surgery; 3, extracorporeal membrane oxygenation; 4, oncologic diagnosis or hematopoietic stem cell transplantation; 5, acute liver failure or liver transplantation; 6, noncardiac surgery; 7, invasive procedures outside the operating room; 8, sepsis and/or disseminated intravascular coagulation) as well as laboratory assays and selection/processing of plasma and platelet components. In total, we came to consensus on four recommendations, five good practice statements, and 44 consensus-based statements. These results were further developed into consensus-based clinical decision trees for plasma and platelet transfusion in critically ill pediatric patients., Conclusions: The TAXI-CAB program provides expert-based consensus for pediatric intensivists for the administration of plasma and/or platelet transfusions in critically ill pediatric patients. There is a pressing need for primary research to provide more evidence to guide practitioners., Competing Interests: Drs. Nellis, Bateman, Bembea, and Russell received support for article research from the National Institutes of Health. Dr. Bembea’s institution received funding from the National Institute of Neurologic Disorders and Stroke (R01NS106292), the National Institute of Child Health and Human Development, and Grifols Investigator Sponsored Research Grant. Dr. Nishijima received funding from Bristol Myers Squibb. Dr. Emani received funding from Cheisi Pharmaceuticals. Dr. Haas received funding from Octapharma. Dr. Goel received funding from the National Heart, Lung, and Blood Institute and Rigel Pharmaceuticals. Dr. Crighton disclosed that she is employed by Royal Children’s Hospital of Melbourne, Australia and that she was the Australian and New Zealand Society of Blood Transfusion President. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

Objectives: To present the consensus statements with supporting literature for plasma and platelet transfusions in critically ill neonates and children with malignancy, acute liver disease and/or following liver transplantation, and sepsis and/or disseminated intravascular coagulation from the Transfusion and Anemia EXpertise Initiative-Control/Avoidance of Bleeding., Design: Systematic review and consensus conference of international, multidisciplinary experts in platelet and plasma transfusion management of critically ill children., Setting: Not applicable., Patients: Critically ill neonates and children with malignancy, acute liver disease and/or following liver transplantation, and sepsis and/or disseminated intravascular coagulation., Interventions: None., Measurements and Main Results: A panel of 13 experts developed evidence-based and, when evidence was insufficient, expert-based statements for plasma and platelet transfusions in critically ill neonates and children with malignancy, acute liver disease and/or following liver transplantation, and sepsis and/or disseminated intravascular coagulation. These statements were reviewed and ratified by the 29 Transfusion and Anemia EXpertise Initiative-Control/Avoidance of Bleeding experts. A systematic review was conducted using MEDLINE, EMBASE, and Cochrane Library databases, from inception to December 2020. Consensus was obtained using the Research and Development/University of California, Los Angeles Appropriateness Method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method. We developed 12 expert consensus statements., Conclusions: In the Transfusion and Anemia EXpertise Initiative-Control/Avoidance of Bleeding program, the current absence of evidence for use of plasma and/or platelet transfusion in critically ill children with malignancy, acute liver disease and/or following liver transplantation, and sepsis means that only expert consensus statements are possible for these areas of practice., Competing Interests: Dr. Crighton disclosed that she is employed by the Royal Children’s Hospital of Melbourne, Australia, and she was the Australian and New Zealand Society of Blood Transfusion President. Dr. Nellis received support for article research from the National Institutes of Health. Dr. Steiner received funding from Pumps for Kids, Infants, and Neonates (PumpKIN) Data Safety and Monitoring Board and HealthCore. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

Objectives: To present a list of high-priority research initiatives for the study of plasma and platelet transfusions in critically ill children from the Transfusion and Anemia EXpertise Initiative-Control/Avoidance of Bleeding., Design: Systematic review and consensus conference of international, multidisciplinary experts in platelet and plasma transfusion management of critically ill children., Setting: Not applicable., Patients: Critically ill pediatric patients at risk of bleeding and receiving plasma and/or platelet transfusions., Interventions: None., Measurements and Main Results: A panel of 13 experts developed research priorities for the study of plasma and platelet transfusions in critically ill children which were reviewed and ratified by the 29 Transfusion and Anemia EXpertise Initiative-Control/Avoidance of Bleeding experts. The specific priorities focused on the following subpopulations: severe trauma, traumatic brain injury, intracranial hemorrhage, cardiopulmonary bypass surgery, extracorporeal membrane oxygenation, oncologic diagnosis or stem cell transplantation, acute liver failure and/or liver transplantation, noncardiac surgery, invasive procedures outside of the operating room, and sepsis and/or disseminated intravascular coagulation. In addition, tests to guide plasma and platelet transfusion, as well as component selection and processing, were addressed. We developed four general overarching themes and 14 specific research priorities using modified Research and Development/University of California, Los Angeles methodology., Conclusions: Studies are needed to focus on the efficacy/harm, dosing, timing, and outcomes of critically ill children who receive plasma and/or platelet transfusions. The completion of these studies will facilitate the development of evidence-based recommendations., Competing Interests: Dr. Bembea’s institution received funding from the National Institute of Neurological Disorders and Stroke (R01NS106292), the National Institute of Child Health and Human Development, and Grifols Investigator Sponsored Research Grant. Drs. Bembea and Russell received support for article research from the National Institutes of Health. Dr. Steiner received funding from Pumps for Kids, Infants, and Neonates (PumpKIN) Data Safety and Monitoring Board and HealthCore. Dr. Crighton disclosed that she is employed by the Royal Children’s Hospital of Melbourne, Australia and was the Australian and New Zealand Society of Blood Transfusion President. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

Objectives: Pediatric acute respiratory distress syndrome is heterogeneous, with a paucity of risk stratification tools to assist with trial design. We aimed to develop and validate mortality prediction models for patients with pediatric acute respiratory distress syndrome., Design: Leveraging additional data collection from a preplanned ancillary study (Version 1) of the multinational Pediatric Acute Respiratory Distress syndrome Incidence and Epidemiology study, we identified predictors of mortality. Separate models were built for the entire Version 1 cohort, for the cohort excluding neurologic deaths, for intubated subjects, and for intubated subjects excluding neurologic deaths. Models were externally validated in a cohort of intubated pediatric acute respiratory distress syndrome patients from the Children's Hospital of Philadelphia., Setting: The derivation cohort represented 100 centers worldwide; the validation cohort was from Children's Hospital of Philadelphia., Patients: There were 624 and 640 subjects in the derivation and validation cohorts, respectively., Interventions: None., Measurements and Main Results: The model for the full cohort included immunocompromised status, Pediatric Logistic Organ Dysfunction 2 score, day 0 vasopressor-inotrope score and fluid balance, and PaO2/FIO2 6 hours after pediatric acute respiratory distress syndrome onset. This model had good discrimination (area under the receiver operating characteristic curve 0.82), calibration, and internal validation. Models excluding neurologic deaths, for intubated subjects, and for intubated subjects excluding neurologic deaths also demonstrated good discrimination (all area under the receiver operating characteristic curve ≥ 0.84) and calibration. In the validation cohort, models for intubated pediatric acute respiratory distress syndrome (including and excluding neurologic deaths) had excellent discrimination (both area under the receiver operating characteristic curve ≥ 0.85), but poor calibration. After revision, the model for all intubated subjects remained miscalibrated, whereas the model excluding neurologic deaths showed perfect calibration. Mortality models also stratified ventilator-free days at 28 days in both derivation and validation cohorts., Conclusions: We describe predictive models for mortality in pediatric acute respiratory distress syndrome using readily available variables from day 0 of pediatric acute respiratory distress syndrome which outperform severity of illness scores and which demonstrate utility for composite outcomes such as ventilator-free days. Models can assist with risk stratification for clinical trials.

Objectives: Observational studies have shown that fluid overload is independently associated with increased morbidity in critically ill children, especially with respiratory pathology. It is unknown if recent evidence has influenced clinical practice. We sought to describe current IV fluid management in pediatric acute respiratory distress syndrome., Design: Multinational, cross-sectional electronic survey., Setting: Pediatric Acute Lung Injury and Sepsis Investigators Network., Subjects: Pediatric intensivists., Interventions: None., Measurements and Main Results: One-hundred fifty-four respondents (43% response rate) had a median 10 years of experience (Q1-Q3, 4-17.8), in ICUs with a median 24 beds (18-36), where 86% provided extracorporeal membrane oxygenation. For maintenance IV fluid, 96% used the "4-2-1" rule to determine rate, and 59% used dextrose with normal saline for content. For fluid resuscitation, 77% use normal saline in 10 milliliters per kilogram aliquots (42%) or as fluid challenges (37%). Less than 20% of respondents reported resuscitating with 20 mL/kg boluses. Documented intake over output is the favored vital sign to assess (75% vs 57%) and guide fluid management (97% vs 14%) over central venous pressure. The majority of respondents chose 10% fluid overload as the threshold to act in children with pediatric acute respiratory distress syndrome. The majority (77%) agreed that fluid accumulation contributes to worse outcomes in pediatric acute respiratory distress syndrome and should be treated. Ninety-one percent reported conservative fluid management in pediatric acute respiratory distress syndrome is likely to be beneficial or protective., Conclusions: Pediatric intensivists agree that acting on 10% fluid overload in children with pediatric acute respiratory distress syndrome is important. Decisions are being made largely using intake and output documentation, not central venous pressure. These findings are important for future pediatric acute respiratory distress syndrome interventional trials.

Objectives: This article describes the methodology used for Pediatric Critical Care Transfusion and Anemia Expertise Initiative Consensus Conference., Design: Consensus conference of international experts in pediatric critical care and transfusion medicine, following standards set by the Institute of Medicine, using the Research and Development/UCLA Appropriateness Method, modeled after the Pediatric Acute Lung Injury Consensus Conference. Topics related to RBC transfusion in children with or at risk for critical illness were divided into nine subgroups with a systematic review of the literature., Methods: The panel of 38 content and four methodology experts met three times over the course of 2 years and collaborated to develop evidence-based and, when evidence was lacking, expert-based clinical recommendations as well as research priorities for RBC transfusions in critically ill children or those at risk for critical illness. Electronic searches were conducted using PubMed, Embase, and Cochrane Library databases from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA Appropriateness Method. We used a standardized data extraction form to construct evidence tables and graded the evidence using the Grading of Recommendations Assessment, Development, and Evaluation system., Main Results: The consensus conference resulted in 102 recommendation statements, of which 57 were clinical (20 evidence based and 37 based on expert consensus) and 45 detailed recommendations for future research. Dissemination was done via decision tree, a primary publication listing all statements, and separate publications for each subtopic that include supporting arguments for each recommendation., Conclusions: A consensus conference of experts from around the world developed recommendations for RBC transfusions in critically ill children or children at risk for critical illness, the identification of current research gaps, and future research priorities.

Objectives: To provide context for the implementation of the Pediatric Critical Care Transfusion and Anemia Expertise Initiative recommendations for RBC transfusions including a review of prior research related to implementation of transfusion guidelines, efforts to facilitate implementation through Transfusion and Anemia Expertise Initiative, and to provide a framework for recommendation implementation., Design: Review of existing clinical literature and description of a comprehensive approach to implementation based on Implementation Science principles., Results: The Transfusion and Anemia Expertise Initiative recommendations on RBC transfusions are based on clinical evidence and aim to limit unnecessary and potentially harmful transfusions. Prior efforts to use transfusion guidelines include use of provider education, local guidelines, visual aids, prospective and retrospective audit and feedback as well as computerized decision support tools; however, no single approach has been identified as optimal for implementation in pediatric critical care settings. Evidence around provider beliefs and transfusion decision-making point to the need for additional provider education, emphasizing the importance of limiting transfusions, and the development of recommendations, such as the Transfusion and Anemia Expertise Initiative guidelines, that can be applied to specific clinical conditions., Conclusions: The Transfusion and Anemia Expertise Initiative guidelines will be broadly disseminated; however, coordinated implementation efforts will be required to impact practice. An approach that encourages involvement of a wide range of multiprofessional stakeholders, formal agreement on the implemented guidelines, selection of strategies that are practical and feasible, and active monitoring of clinical practice and outcomes throughout implementation is recommended. A formal second stage Transfusion and Anemia Expertise Initiative - Continuous Assessment of Blood-use is proposed to enhance implementation of the recommendations, follow uptake and impact on practice and patient outcomes, and ensure integration of new clinical evidence into the existing guideline as it is developed.

Objectives: To present the recommendations and supporting literature for RBC transfusions in critically ill children with acquired and congenital heart disease developed by the Pediatric Critical Care Transfusion and Anemia Expertise Initiative., Design: Consensus conference series of 38 international, multidisciplinary experts in RBC transfusion management of critically ill children., Methods: Experts developed evidence-based and, when evidence was lacking, expert-based clinical recommendations and research priorities for RBC transfusions in critically ill children. The cardiac disease subgroup included three experts. Electronic searches were conducted using PubMed, EMBASE, and Cochrane Library databases from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA appropriateness method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method., Results: Twenty-one recommendations were developed and reached agreement. For children with myocardial dysfunction and/or pulmonary hypertension, there is no evidence that transfusion greater than hemoglobin of 10 g/dL is beneficial. For children with uncorrected heart disease, we recommended maintaining hemoglobin greater than 7-9.0 g/dL depending upon their cardiopulmonary reserve. For stable children undergoing biventricular repairs, we recommend not transfusing if the hemoglobin is greater than 7.0 g/dL. For infants undergoing staged palliative procedures with stable hemodynamics, we recommend avoiding transfusions solely based upon hemoglobin, if hemoglobin is greater than 9.0 g/dL. We recommend intraoperative and postoperative blood conservation measures. There are insufficient data supporting shorter storage duration RBCs. The risks and benefits of RBC transfusions in children with cardiac disease requires further study., Conclusions: We present RBC transfusion management recommendations for the critically ill child with cardiac disease. Clinical recommendations emphasize relevant hemoglobin thresholds, and research recommendations emphasize need for further understanding of physiologic and hemoglobin thresholds and alternatives to RBC transfusion in subpopulations lacking pediatric literature.

Objectives: To present the recommendations and supporting literature for RBC transfusions in critically ill children with bleeding developed by the Pediatric Critical Care Transfusion and Anemia Expertise Initiative., Design: Consensus conference series of international, multidisciplinary experts in RBC transfusion management of critically ill children., Methods: The panel of 38 experts developed evidence-based and, when evidence was lacking, expert-based clinical recommendations as well as research priorities for RBC transfusions in critically ill children. The respiratory subgroup included six experts. Electronic searches were conducted using PubMed, EMBASE, and Cochrane Library databases from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA Appropriateness Method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method., Results: Transfusion and Anemia Expertise Initiative experts developed seven recommendations focused on children with acute respiratory failure. All recommendations reached agreement (> 80%). Transfusion of RBCs in children with respiratory failure with an hemoglobin level less than 5 g/dL was strongly recommended. It was strongly recommended that RBCs not be systematically administered to children with respiratory failure who are hemodynamically stable and who have a hemoglobin level greater than or equal to 7 g/dL. Experts could not make a recommendation for children with hemodynamic instability, with severe hypoxemia and/or with an hemoglobin level between 5 and 7 g/dL. Specific RBC transfusion strategies using physiologic-based metrics and biomarkers could not be elaborated., Conclusions: The Transfusion and Anemia Expertise Initiative Consensus Conference developed specific recommendations regarding RBC transfusion management in critically ill children with respiratory failure, as well as recommendations to guide future research. Clinical recommendations emphasize relevant hemoglobin thresholds. Research recommendations emphasize the need to identify appropriate physiologic thresholds, suggest a better understanding of alternatives to RBC transfusion, and identify the need for better evidence on hemoglobin thresholds that might be used in specific subpopulations of critically ill children.

Objectives: To present the recommendations and supporting literature for RBC transfusions in critically ill children with nonhemorrhagic shock developed by the Pediatric Critical Care Transfusion and Anemia Expertise Initiative., Design: Consensus conference series of international, multidisciplinary experts in RBC transfusion management of critically ill children., Methods: The panel of 38 experts developed evidence-based, and when evidence was lacking, expert-based clinical recommendations as well as research priorities for RBC transfusions in critically ill children. The nonhemorrhagic shock subgroup included five experts. Electronic searches were conducted using PubMed, EMBASE, and Cochrane Library databases from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA Appropriateness Method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method., Results: Transfusion and Anemia Expertise Initiative Consensus Conference experts developed and voted on a total of four clinical and four research recommendations focused on RBC transfusion in the critically ill child with nonhemorrhagic shock. All recommendations reached agreement (> 80%). Of the four clinical recommendations, three were based on consensus panel expertise, whereas one was based on weak pediatric evidence. In hemodynamically stabilized critically ill children with a diagnosis of severe sepsis or septic shock, we recommend not administering a RBC transfusion if the hemoglobin concentration is greater than or equal to 7 g/dL. Future studies are needed to determine optimum transfusion thresholds for critically ill children with nonhemorrhagic shock undergoing acute resuscitation., Conclusions: The Transfusion and Anemia Expertise Initiative Consensus Conference developed pediatric-specific clinical and research recommendations regarding RBC transfusion in the critically ill child with nonhemorrhagic shock. Although agreement among experts was strong, available pediatric evidence was scant-revealing significant gaps in the existing literature.

Objectives: To present the recommendations and supporting literature for selection and processing of RBC products in critically ill children developed by the Pediatric Critical Care Transfusion and Anemia Expertise Initiative., Design: Consensus conference series of international, multidisciplinary experts in RBC transfusion management of critically ill children METHODS:: The panel of 38 experts developed evidence-based, and when evidence was lacking, expert-based clinical recommendations as well as research priorities for RBC transfusions in critically ill children. The RBC processing subgroup included five experts. Electronic searches were conducted using PubMed, EMBASE, and Cochrane Library databases from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA Appropriateness Method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method., Results: Five recommendations reached agreement (> 80%). Irradiated cellular products are recommended for children at risk of transfusion-associated graft versus host disease due to severe congenital or acquired causes of immune deficiency or when the blood donor is a blood relative. Washed cellular blood components and avoidance of other plasma-containing products are recommended for critically ill children with history of severe allergic reactions or anaphylaxis to blood transfusions, although patient factors appear to be important in the pathogenesis of reactions. For children with history of severe allergic transfusion reactions, evaluation for allergic stigmata prior to transfusion is recommended. In children with severe immunoglobulin A deficiency with evidence of antiimmunoglobulin A antibodies and/or a history of a severe transfusion reaction, immunoglobulin A-deficient blood components obtained either from an immunoglobulin A-deficient donor and/or washed cellular components is recommended., Conclusions: The Transfusion and Anemia Expertise Initiative consensus conference developed recommendations for selection and processing of RBC units for critically ill children. Recommendations in this area are largely based on pediatric and adult case report data.

Objectives: To present the recommendations and supporting evidence for RBC transfusions in critically ill children with hematologic and oncologic disease from the Pediatric Critical Care Transfusion and Anemia Expertise Initiative., Design: Consensus conference series of international, multidisciplinary experts in RBC transfusion management of critically ill children., Methods: The panel of 38 experts developed evidence-based and, when evidence was lacking, expert-based clinical recommendations and research priorities for RBC transfusions in critically ill children. The hematologic/oncologic subgroup included seven experts. Electronic searches were conducted using PubMed, EMBASE, and Cochrane Library databases from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA Appropriateness Method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method., Results: The hematologic/oncologic subgroup developed 14 recommendations (seven clinical, seven research); all achieved greater than 80% agreement. In patients with sickle cell disease, Transfusion and Anemia Expertise Initiative recommends: 1) RBC transfusion to achieve a target hemoglobin concentration of 10 g/dL rather than hemoglobin of less than 30% prior to surgical procedures requiring general anesthesia and 2) exchange transfusion over simple (nonexchange) transfusion if the child's condition is deteriorating (based on clinical judgment), otherwise a simple, nonexchange RBC transfusion is recommended. There is insufficient evidence to make recommendations on transfusion thresholds for patients with sickle cell disease prior to minor procedures, with acute stroke or with pulmonary hypertension. For patients with oncologic disease or undergoing hematopoietic stem cell transplant, a hemoglobin concentration of 7-8 g/dL is recommended. Due to lack of evidence, research is needed to clarify the appropriate transfusion thresholds in these patients., Conclusions: Transfusion and Anemia Expertise Initiative developed specific pediatric recommendations regarding RBC transfusion management in critically ill children with sickle cell disease, oncologic disease, and hematopoietic stem cell transplant and recommendations to help guide future research priorities.

Objectives: To present the recommendations and supporting literature for RBC transfusions in critically ill children with bleeding developed by the Pediatric Critical Care Transfusion and Anemia Expertise Initiative., Design: Consensus conference series of international, multidisciplinary experts in RBC transfusion management of critically ill children., Methods: The panel of 38 experts developed evidence-based and, when evidence was lacking, expert-based clinical recommendations as well as research priorities for RBC transfusions in critically ill children. The bleeding subgroup included five experts. Electronic searches were conducted using PubMed, EMBASE, and Cochrane Library databases from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA Appropriateness Method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method., Results: Transfusion and Anemia Expertise Initiative Consensus Conference experts developed a total of six recommendations focused on transfusion in the critically ill child with acute bleeding. In critically ill children with nonlife-threatening bleeding, we recommend giving a RBC transfusion for a hemoglobin concentration less than 5 g/dL, and be considered for a hemoglobin concentration between 5 and 7 g/dL. In critically ill children with hemorrhagic shock, we suggest that RBCs, plasma and platelets transfusion ratio between 2:1:1 to 1:1:1 until the bleeding is no longer life-threatening. We recommend future studies to develop physiologic and laboratory measures to indicate the need for RBC transfusions, and to determine if goal directed hemostatic resuscitation improves survival. Finally, we recommend future studies to determine if low titer group O whole blood is more efficacious and safe compared with reconstituted whole blood in children with hemorrhagic shock., Conclusions: The Transfusion and Anemia Expertise Initiative Consensus Conference developed pediatric specific recommendations regarding RBC transfusion management in the critically ill child with acute bleeding, as well as recommendations to help guide future research priorities.

Objectives: To present the consensus recommendations and supporting literature for RBC transfusions in general critically ill children from the Pediatric Critical Care Transfusion and Anemia Expertise Initiative., Design: Consensus conference series of international, multidisciplinary experts in RBC transfusion management of critically ill children., Methods: The panel of 38 experts developed evidence-based and, when evidence was lacking, expert-based recommendations and research priorities regarding RBC transfusions in critically ill children. The subgroup on RBC transfusion in general critically ill children included six experts. Electronic searches were conducted using PubMed, EMBASE, and Cochrane Library databases from 1980 to May 30, 2017, using a combination of keywords to define concepts of RBC transfusion and critically ill children. Recommendation consensus was obtained using the Research and Development/UCLA Appropriateness Method. The results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method., Results: Three adjudicators reviewed 4,399 abstracts; 71 papers were read, and 17 were retained. Three papers were added manually. The general Transfusion and Anemia Expertise Initiative subgroup developed, and all Transfusion and Anemia Expertise Initiative members voted on two good practice statements, six recommendations, and 11 research questions; in all instances, agreement was reached (> 80%). The good practice statements suggest a framework for RBC transfusion in PICU patients. The good practice statements and recommendations focus on hemoglobin as a threshold and/or target. The research questions focus on hemoglobin and physiologic thresholds for RBC transfusion, alternatives, and risk/benefit ratio of transfusion., Conclusions: Transfusion and Anemia Expertise Initiative developed pediatric-specific good practice statements and recommendations regarding RBC transfusion management in the general PICU population, as well as recommendations to guide future research priorities. Clinical recommendations emphasized relevant hemoglobin thresholds, and research recommendations emphasized a need for further understanding of physiologic thresholds, alternatives to RBC transfusion, and hemoglobin thresholds in populations with limited pediatric literature.

Objectives: To present recommendations and supporting literature for RBC transfusions in critically ill children supported with extracorporeal membrane oxygenation, ventricular assist devices, or renal replacement therapy., Design: Consensus conference series of international, multidisciplinary experts in RBC transfusion management of critically ill children., Methods: The panel of 38 experts developed evidence-based, and when evidence was lacking, expert-based clinical recommendations as well as research priorities for RBC transfusions in critically ill children. The extracorporeal membrane oxygenation/ventricular assist device/renal replacement therapy subgroup included six experts. We conducted electronic searches of the PubMed, EMBASE, and Cochrane Library databases from 1980 to May 2017, using medical subject heading terms and text words to define concepts of RBC transfusion, extracorporeal membrane oxygenation, ventricular assist device, and renal replacement therapy. We used a standardized data extraction form to construct evidence tables and graded the evidence using the Grading of Recommendations Assessment, Development, and Evaluation system. Recommendations developed and supporting literature were reviewed and scored by all panel members. Agreement was obtained using the Research and Development/UCLA Appropriateness Method., Results: For inpatients requiring extracorporeal membrane oxygenation, ventricular assist device, or renal replacement therapy support, there was expert agreement (> 80%) on five good practice statements aimed to improve accuracy and uniform reporting of RBC transfusion data in pediatric extracorporeal membrane oxygenation, ventricular assist device, and renal replacement therapy studies and quality improvement projects; four clinical recommendations of physiologic metrics and biomarkers of oxygen delivery, in addition to hemoglobin concentration, to guide RBC transfusion, acknowledging insufficient evidence to recommend specific RBC transfusion strategies; and eight research recommendations., Conclusions: Further research surrounding indications, risks, benefits, and alternatives to RBC transfusion in children on extracorporeal devices is clearly needed. Using a structured literature review and grading process, the Transfusion and Anemia Expertise Initiative panel concluded that there is currently insufficient evidence to recommend specific RBC transfusion variables in children requiring extracorporeal membrane oxygenation, ventricular assist device, or renal replacement therapy support.

Objectives: To date, there are no published guidelines to direct RBC transfusion decision-making specifically for critically ill children. We present the recommendations from the Pediatric Critical Care Transfusion and Anemia Expertise Initiative., Design: Consensus conference series of multidisciplinary, international experts in RBC transfusion management of critically ill children., Setting: Not applicable., Intervention: None., Subjects: Children with, or children at risk for, critical illness who receive or are at risk for receiving a RBC transfusion., Methods: A panel of 38 content and four methodology experts met over the course of 2 years to develop evidence-based, and when evidence lacking, expert consensus-based recommendations regarding decision-making for RBC transfusion management and research priorities for transfusion in critically ill children. The experts focused on nine specific populations of critically ill children: general, respiratory failure, nonhemorrhagic shock, nonlife-threatening bleeding or hemorrhagic shock, acute brain injury, acquired/congenital heart disease, sickle cell/oncology/transplant, extracorporeal membrane oxygenation/ventricular assist/ renal replacement support, and alternative processing. Data to formulate evidence-based and expert consensus recommendations were selected based on searches of PubMed, EMBASE, and Cochrane Library from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA Appropriateness Method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method., Measurements and Results: The Transfusion and Anemia Expertise Initiative consensus conference developed and reached consensus on a total of 102 recommendations (57 clinical [20 evidence based, 37 expert consensus], 45 research recommendations). All final recommendations met agreement, defined a priori as greater than 80%. A decision tree to aid clinicians was created based on the clinical recommendations., Conclusions: The Transfusion and Anemia Expertise Initiative recommendations provide important clinical guidance and applicable tools to avoid unnecessary RBC transfusions. Research recommendations identify areas of focus for future investigation to improve outcomes and safety for RBC transfusion.

There are regular, and therefore predictable, environmental changes on Earth due to the rotation of the planet on its axis and its orbit around the sun. Thus organisms have adapted their metabolism, physiology and behaviour to minimise stresses caused by unfavourable conditions and maximise efficiency of growth. Additionally, most organisms are able to anticipate these changes and accordingly maximise metabolic efficiency and growth, because they have a complex biological time-keeping system commonly referred to as the circadian clock. Multiple pathways in plants are organised in a temporal manner through circadian clock-regulation of gene transcription and post-translational modifications. What is becoming more apparent is the bidirectional nature of interactions between the clock and stress response pathways. Until recently, the focus of many studies had been on the unidirectional, hierarchical control of biological processes by the circadian clock, and impacts on the clock in response to environmental stress had been largely ignored. Studies of interactions of the circadian clock with the environment have primarily been to understand mechanisms of entrainment. We review the evidence and implications of the reciprocal interactions between the clock and the environment.

Objectives: To determine whether weight extremes impact clinical outcomes in pediatric acute respiratory distress syndrome., Design: Post hoc analysis of a cohort created by combining five multicenter pediatric acute respiratory distress syndrome studies., Setting: Forty-three academic PICUs worldwide., Patients: A total of 711 subjects prospectively diagnosed with pediatric acute respiratory distress syndrome., Intervention: Subjects more than 2 years were included and categorized by Center for Disease Control and Prevention body mass index z score criteria: underweight (< -1.89), normal weight (-1.89 to +1.04), overweight (+1.05 to +1.64), and obese (≥ +1.65). Subjects were stratified by direct versus indirect lung injury leading to pediatric acute respiratory distress syndrome. The primary outcome was in-hospital mortality. In survivors, secondary analyses included duration of mechanical ventilation and ICU length of stay., Measurements and Main Results: A total of 331 patients met inclusion criteria; 12% were underweight, 50% normal weight, 11% overweight, and 27% obese. Overall mortality was 20%. By multivariate analysis, body mass index category was independently associated with mortality (p = 0.004). When stratified by lung injury type, there was no mortality difference between body mass index groups with direct lung injury; however, in the indirect lung injury group, the odds of mortality in the obese were significantly lower than normal weight subjects (odds ratio, 0.11; 95% CI, 0.02-0.84). Survivors with direct lung injury had no difference in the duration of mechanical ventilation or ICU length of stay; however, those with indirect lung injury, the overweight required longer duration of mechanical ventilation than other groups (p < 0.001)., Conclusions: These data support the obesity paradox in pediatric acute respiratory distress syndrome. Obese children with indirect lung injury pediatric acute respiratory distress syndrome have a lower risk of mortality. Importantly, among survivors, the overweight with indirect lung injury requires longer duration of mechanical ventilation. Our data require prospective validation to further elucidate the pathobiology of this phenomenon., Competing Interests: The remaining authors have disclosed that they do not have any potential conflicts of interest.

Objectives: To determine the frequency of low-tidal volume ventilation in pediatric acute respiratory distress syndrome and assess if any demographic or clinical factors improve low-tidal volume ventilation adherence., Design: Descriptive post hoc analysis of four multicenter pediatric acute respiratory distress syndrome studies., Setting: Twenty-six academic PICU., Patients: Three hundred fifteen pediatric acute respiratory distress syndrome patients., Measurements and Main Results: All patients who received conventional mechanical ventilation at hours 0 and 24 of pediatric acute respiratory distress syndrome who had data to calculate ideal body weight were included. Two cutoff points for low-tidal volume ventilation were assessed: less than or equal to 6.5 mL/kg of ideal body weight and less than or equal to 8 mL/kg of ideal body weight. Of 555 patients, we excluded 240 for other respiratory support modes or missing data. The remaining 315 patients had a median PaO2-to-FIO2 ratio of 140 (interquartile range, 90-201), and there were no differences in demographics between those who did and did not receive low-tidal volume ventilation. With tidal volume cutoff of less than or equal to 6.5 mL/kg of ideal body weight, the adherence rate was 32% at hour 0 and 33% at hour 24. A low-tidal volume ventilation cutoff of tidal volume less than or equal to 8 mL/kg of ideal body weight resulted in an adherence rate of 58% at hour 0 and 60% at hour 24. Low-tidal volume ventilation use was no different by severity of pediatric acute respiratory distress syndrome nor did adherence improve over time. At hour 0, overweight children were less likely to receive low-tidal volume ventilation less than or equal to 6.5 mL/kg ideal body weight (11% overweight vs 38% nonoverweight; p = 0.02); no difference was noted by hour 24. Furthermore, in the overweight group, using admission weight instead of ideal body weight resulted in misclassification of up to 14% of patients as receiving low-tidal volume ventilation when they actually were not., Conclusions: Low-tidal volume ventilation is underused in the first 24 hours of pediatric acute respiratory distress syndrome. Age, Pediatric Risk of Mortality-III, and pediatric acute respiratory distress syndrome severity were not associated with improved low-tidal volume ventilation adherence nor did adherence improve over time. Overweight children were less likely to receive low-tidal volume ventilation strategies in the first day of illness.

Objective: Delayed enteral nutrition, defined as enteral nutrition started 48 hours or more after admission to the PICU, is associated with an inability to achieve full enteral nutrition and worse outcomes in critically ill children. We reviewed nutritional practices in six medical-surgical PICUs and determined risk factors associated with delayed enteral nutrition in critically ill children., Design: Retrospective cross-sectional study using medical records as source of data., Setting: Six medical-surgical PICUs in northeastern United States., Patients: Children less than 21 years old admitted to the PICU for 72 hours or more excluding those awaiting or recovering from abdominal surgery., Measurements and Main Results: A total of 444 children with a median age of 4.0 years were included in the study. Enteral nutrition was started at a median time of 20 hours after admission to the PICU. There was no significant difference in time to start enteral nutrition among the PICUs. Of those included, 88 children (19.8%) had delayed enteral nutrition. Risk factors associated with delayed enteral nutrition were noninvasive (odds ratio, 3.37; 95% CI, 1.69-6.72) and invasive positive-pressure ventilation (odds ratio, 2.06; 95% CI, 1.15-3.69), severity of illness (odds ratio for every 0.1 increase in pediatric index of mortality 2 score, 1.39; 95% CI, 1.14-1.71), procedures (odds ratio, 3.33; 95% CI, 1.67-6.64), and gastrointestinal disturbances (odds ratio, 2.05; 95% CI, 1.14-3.68) within 48 hours after admission to the PICU. Delayed enteral nutrition was associated with failure to reach full enteral nutrition while in the PICU (odds ratio, 4.09; 95% CI, 1.97-8.53). Nutrition consults were obtained in less than half of the cases, and none of the PICUs used tools to assure the adequacy of energy and protein nutrition., Conclusions: Institutions in this study initiated enteral nutrition for a high percentage of patients by 48 hours of admission. Noninvasive positive-pressure ventilation was most strongly associated with delay enteral nutrition. A better understanding of these risk factors and assessments of nutritional requirements should be explored in future prospective studies.

Objective: To describe the recommendations from the Pediatric Acute Lung Injury Consensus Conference on nonpulmonary treatments in pediatric acute respiratory distress syndrome., Design: Consensus conference of experts in pediatric acute lung injury., Methods: A panel of 27 experts met over the course of 2 years to develop a taxonomy to define pediatric acute respiratory distress syndrome and to make recommendations regarding treatment and research priorities. The nonpulmonary subgroup comprised three experts. When published data were lacking, a modified Delphi approach emphasizing strong professional agreement was utilized., Results: The Pediatric Acute Lung Injury Consensus Conference experts developed and voted on a total of 151 recommendations addressing the topics related to pediatric acute respiratory distress syndrome, 30 of which related to nonpulmonary treatment. All 30 recommendations had strong agreement. Patients with pediatric acute respiratory distress syndrome should receive 1) minimal yet effective targeted sedation to facilitate mechanical ventilation; 2) neuromuscular blockade, if sedation alone is inadequate to achieve effective mechanical ventilation; 3) a nutrition plan to facilitate their recovery, maintain their growth, and meet their metabolic needs; 4) goal-directed fluid management to maintain adequate intravascular volume, end-organ perfusion, and optimal delivery of oxygen; and 5) goal-directed RBC transfusion to maintain adequate oxygen delivery. Future clinical trials in pediatric acute respiratory distress syndrome should report sedation, neuromuscular blockade, nutrition, fluid management, and transfusion exposures to allow comparison across studies., Conclusions: The Consensus Conference developed pediatric-specific definitions for pediatric acute respiratory distress syndrome and recommendations regarding treatment and future research priorities. These recommendations for nonpulmonary treatment in pediatric acute respiratory distress syndrome are intended to promote optimization and consistency of care for patients with pediatric acute respiratory distress syndrome and identify areas of uncertainty requiring further investigation.

Objective: Results of a large multicenter randomized clinical trial published in 2007 demonstrated no benefit in using a liberal versus conservative RBC transfusion threshold in stable critically ill children. Using the conservative threshold decreased the number of RBC transfusions without increasing adverse outcomes. We aimed to determine if wide dissemination of this evidence altered the hemoglobin threshold used for RBC transfusions in our pediatric medical-surgical ICU., Design: Before-after retrospective cohort study using multiple administrative databases and chart review., Setting: PICU serving medical and surgical patients., Patients: All potentially stable children receiving a RBC transfusion in the PICU in 2006 (prepublication) and in 2009-2010 (postpublication). Children were considered unstable and excluded if they were severely hypoxic, receiving renal replacement therapy, hemodynamically unstable, or bleeding., Interventions: Physician education on evidence supporting hemoglobin transfusion thresholds in teaching conferences, staff meetings, and via e-mail., Measurements and Main Results: In 2006, 14.6% of patients (n = 285/1,940) received a RBC transfusion. In 2009-2010, 12.1% of patients (n = 551/4,542) received a RBC transfusion. We evaluated patients transfused when they were potentially clinically stable, including 145 children in 2006 (191 transfusion days) and 266 children in 2009-2010 (369 transfusion days). We found no significant differences in age, sex, race, diagnoses, postoperative status, illness severity scores, mortality, or length of stay between these two groups. The median hemoglobin transfusion threshold decreased significantly from 8.0 g/dL (interquartile range 7.3, 8.6 g/dL) in 2006 to 7.5 g/dL (interquartile range 6.9, 8.1 g/dL) in 2009-2010 (p = 0.001). The percentage of transfusion days using a hemoglobin threshold more than 7 g/dL decreased from 81% (n = 154) in 2006 to 71% (n = 261) in 2009-2010., Conclusion: Although transfusion thresholds in potentially stable critically ill children in our PICU significantly decreased after dissemination of best available evidence, 71% of patients were transfused at a hemoglobin threshold more than 7 g/dL.

Cerebellar granule cells (CGC) deprived of serum or trophic factors develop sensitivity to kainate neurotoxicity that is mediated by the alpha-amino-3-hydroxy-5-methyl-isoxazole proprionic acid (AMPA) subtypes of glutamate receptors (GluR). The L-type voltage-gated calcium channel (L-type VGCC) blocker nifedipine increases the potency of kainate 50-fold. Thus, one goal of this laboratory is to determine the underlying protective mechanism triggered by calcium influx through this channel. The cell-permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine effected complete protection against kainate treatment in the presence of nifedipine, as did the iron chelator deferoxamine. The chelatable heavy metal pool decreased approximately 70% immediately following treatment with kainate, but did not change following kainate/nifedipine treatment. Tetramethylrhhodamine ethyl ester (TMRE) fluorescence, an indicator of mitochondrial membrane potential, decreased approximately 70% following kainate treatment but displayed a more modest decrease ( approximately 15%) when CGC were treated with kainate/nifedipine. Reactive oxygen species (ROS) formation decreased in CGC immediately following kainate treatment but was slightly elevated following kainate/nifedipine treatment. Electron microscopic examinations of the CGC indicated severe swelling and distortion of mitochondria immediately following kainate/nifedipine treatment and the appearance of mitochondrial herniations, whorls, and bridges 2 h later, features that were rarely observed following kainate treatment. These results support the hypothesis that calcium entry through L-type VGCCs protects CGC during kainate treatment by lowering the chelatable heavy metal pool and the mitochondrial membrane potential, thereby mitigating the formation of ROS., (Copyright 2001 Wiley-Liss, Inc.)

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels., (Copyright 1999 Elsevier Science B.V.)

Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate and glutamate. GCP II also hydrolyzes gamma-glutamyl bonds in folylpolyglutamate. The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose crystal structures have been determined. These aminopeptidases are cocatalytic zinc metallopeptidases belonging to the peptidase family M28. Specific zinc and substrate ligands have been proposed in GCP II based on the amino acid sequence alignment to these M28 family members. In the present study, site-directed mutagenesis has been used to test the assignment of these putative ligands in human GCP II. Substitutions to the five putative zinc ligands resulted in severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis. In addition, substitutions of amino acids near the putative zinc ligands have identified other specific residues important for enzyme structure and/or function. Substitutions to putative substrate ligands were less perturbing, and increases in Km were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu). The results from substitutions at the proposed zinc and substrate ligands are consistent with the assignment of these residues and suggest that GCP II has a three-dimensional structure similar to other members of the peptidase family M28.