A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

CD209a expression on dendritic cells is critical for the development of pathogenic Th17 cell responses in murine schistosomiasis.

In murine schistosomiasis, immunopathology and cytokine production in response to parasite eggs are uneven and strain dependent. CBA/J (CBA) mice develop severe hepatic granulomatous inflammation associated with prominent Th17 cell responses driven by dendritic cell (DC)-derived IL-1ß and IL-23. Such Th17 cells fail to develop in low-pathology C57BL/6 (BL/6) mice, and the reasons for these strain-specific differences in APC reactivity to eggs remain unclear. We show by gene profiling that CBA DCs display an 18-fold higher expression of the C-type lectin receptor CD209a, a murine homolog of human DC-specific ICAM-3-grabbing nonintegrin, compared with BL/6 DCs. Higher CD209a expression was observed in CBA splenic and granuloma APC subpopulations, but only DCs induced Th17 cell differentiation in response to schistosome eggs. Gene silencing in CBA DCs and overexpression in BL/6 DCs demonstrated that CD209a is essential for egg-elicited IL-1ß and IL-23 production and subsequent Th17 cell development, which is associated with SRC, RAF-1, and ERK1/2 activation. These findings reveal a novel mechanism controlling the development of Th17 cell-mediated severe immunopathology in helminthic disease.

Use of a ventricular assist device as a bridge to transplantation in a patient with single ventricle physiology and total cavopulmonary anastomosis.

Mechanical circulatory support can be used to manage acute and chronic cardiac failure in both adult and pediatric patients. Traditionally, extracorporeal membrane oxygenation (ECMO) has been the most common form of mechanical circulatory support in children. However, more recently, in cases of pure ventricular dysfunction, ventricular assist devices (VADs) have offered specific advantages over ECMO, including better ventricular recovery, reduced anticoagulation requirements, decreased use of blood products and decreased cost. We present the use of a VAD in an adolescent with single-ventricle physiology, who could not be weaned from cardiopulmonary bypass (CPB) after undergoing a revision of a modified Fontan operation. Gas exchange was provided by the patient's lungs while the centrifugal VAD was used successfully to support the circulation as a bridge, first to a totally implantable pulsatile VAD and subsequently to heart transplantation.

CD209a Synergizes with Dectin-2 and Mincle to Drive Severe Th17 Cell-Mediated Schistosome Egg-Induced Immunopathology.

The immunopathology caused by schistosome helminths varies greatly in humans and among mouse strains. A severe form of parasite egg-induced hepatic granulomatous inflammation, seen in CBA mice, is driven by Th17 cells stimulated by IL-1ß and IL-23 produced by dendritic cells that express CD209a (SIGNR5), a C-type lectin receptor (CLR) related to human DC-SIGN. Here, we show that CD209a-deficient CBA mice display decreased Th17 responses and are protected from severe immunopathology. In vitro, CD209a augments the egg-induced IL-1ß and IL-23 production initiated by the related CLRs Dectin-2 and Mincle. While Dectin-2 and Mincle trigger an FcRγ-dependent signaling cascade that involves the tyrosine kinase Syk and the trimolecular Card9-Bcl10-Malt1 complex, CD209a promotes the sustained activation of Raf-1. Our findings demonstrate that CD209a drives severe Th17 cell-mediated immunopathology in a helminthic disease based on synergy between DC-SIGN- and Dectin-2-related CLRs.

Heparin-induced thrombocytopenia in the pediatric intensive care unit population.

OBJECTIVES: To report the occurrence of heparin-induced thrombocytopenia (HIT), discuss its pathophysiology, and outline an approach to management in the pediatric intensive care unit (ICU) patient. DESIGN: Retrospective case reports. SETTING: Pediatric ICU in a tertiary-care center. PATIENTS AND RESULTS: Two pediatric ICU patients (2 and 6 mos of age) who developed HIT in the pediatric ICU. One was receiving heparin as a flush solution through a central line and the other had full heparinization during cardiopulmonary bypass. Both had received heparin during their neonatal course and developed thrombocytopenia; however, HIT was not considered as a possible diagnosis. HIT was diagnosed using a heparin-induced platelet aggregation study. The thrombocytopenia resolved with the cessation of heparin administration. One of the patients developed a deep vein thrombosis around a femoral venous catheter. CONCLUSION: Although well described in the adult literature, there have been a limited number of reports of HIT in pediatric-aged patients. Given its potential for morbidity, HIT should be considered in the differential diagnosis of thrombocytopenia in the pediatric ICU patient.

The intracellular cargo receptor ERGIC-53 is required for the production of infectious arenavirus, coronavirus, and filovirus particles.

Arenaviruses and hantaviruses cause severe human disease. Little is known regarding host proteins required for their propagation. We identified human proteins that interact with the glycoproteins (GPs) of a prototypic arenavirus and hantavirus and show that the lectin endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), a cargo receptor required for glycoprotein trafficking within the early exocytic pathway, associates with arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus GPs. ERGIC-53 binds to arenavirus GPs through a lectin-independent mechanism, traffics to arenavirus budding sites, and is incorporated into virions. ERGIC-53 is required for arenavirus, coronavirus, and filovirus propagation; in its absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identified a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the propagation of several highly pathogenic RNA virus families.

Changes in near infrared spectroscopy during deep hypothermic circulatory arrest.

Monitoring cerebral oxygenation with near infrared spectroscopy may identify periods of cerebral desaturation and thereby the patients at risk for perioperative neurocognitive issues. Data regarding the performance of near infrared spectroscopy monitoring during deep hypothermic circulatory arrest are limited. The current study presents data regarding use of a commercially available near infrared spectroscopy monitor during deep hypothermic circulatory arrest in paediatric patients undergoing surgery for congenital heart disease. The cohort included 8 patients, 2 weeks to 6 months of age, who required deep hypothermic circulatory arrest for repair of congenital heart disease. The baseline cerebral oxygenation was 63 +/- 11% and increased to 88 +/- 7% after 15 min of cooling to a nasopharyngeal temperature of 17-18 degrees C on cardiopulmonary bypass. In 5 of 8 patients, the cerebral oxygenation value had achieved its peak value (either >or=90% or no change during the last 2-3 min of cooling on cardiopulmonary bypass). In the remaining 3 patients, additional time on cardiopulmonary bypass was required to achieve a maximum cerebral oxygenation value. The duration of deep hypothermic circulatory arrest varied from 36 to 61 min (43.4 +/- 8 min). After the onset of deep hypothermic circulatory arrest, there was an incremental decrease in cerebral oxygenation to a low value of 53 +/- 11%. The greatest decrease occurred during the initial 5 min of deep hypothermic circulatory arrest (9 +/- 3%). Over the entire period of deep hypothermic circulatory arrest, there was an average decrease in the cerebral oxygenation value of 0.9% per min (range of 0.5 to 1.6% decline per minute). During cardiopulmonary bypass, cooling and deep hypothermic circulatory arrest, near infrared spectroscopy monitoring followed the clinically expected parameters. Such monitoring may be useful to identify patients who have not achieved the highest possible cerebral oxygenation value despite 15 min of cooling on cardiopulmonary bypass. Future studies are needed to define the cerebral oxygenation value at which neurological damage occurs and if interventions to correct the decreased cerebral oxygenation will improve perioperative outcomes.

Variation in the ligand binding domains of the CD94/NKG2 family of receptors in the squirrel monkey.

Natural killer cells are regulated, in part, by cell surface expression of the inhibitory CD94/NKG2A heterodimer and the activating CD94/NKG2C heterodimer. In the present study, we characterize the CD94/NKG2 family in the squirrel monkey, a New World monkey species. Full-length CD94, NKG2A, and NKG2CE complementary deoxyribonucleic acid molecules were identified in three unrelated squirrel monkeys. Three alternatively spliced forms of CD94 were detected in which part of intron 4 was included in the mature transcript, suggesting evolutionary pressure for changes in the corresponding loop 3 region of the lectin domain in squirrel monkeys. Squirrel monkey NKG2A contains a three-nucleotide indel that results in an additional amino acid in the predicted NKG2A protein compared to NKG2A in other species. This NKG2A insertion tracks to loop five of the lectin domain, as is seen with the recently described marmoset NKG2CE indel. Transmembrane-deleted forms of CD94 and NKG2CE were also expressed in the squirrel monkey. Analysis of full-length squirrel monkey and additional primate CD94/NKG2 sequences demonstrated statistically significant increases in the Ka/Ks ratio in the putative major histocompatibility complex E (MHC-E) binding domain compared to the non-binding domain. Furthermore, positive selection was detected in the MHC-E binding domain of primate NKG2 family members, and purifying selection was detected in the primate CD94 binding domain. Purifying selection was also detected in the nonbinding domains of primate CD94 and NKG2 molecules.

Noninvasive monitoring of carbon dioxide in infants and children with congenital heart disease: end-tidal versus transcutaneous techniques.

End-tidal CO2 (ET(CO2)) monitoring and transcutaneous (TC) CO2 monitoring were prospectively compared in 53 patients, 1 month to 16 years of age, with congenital heart disease (CHD). There were 32 patients with cyanotic CHD and 21 with acyanotic CHD. The TC-Pa(CO2) difference was 2 +/- 1 mm Hg and the ET-Pa(CO2) difference was 5 +/- 3 mm Hg (P < .0001). The TC-Pa(CO2) difference was < or = 2 mm Hg in 30 of 53 patients and < or = 5 mm Hg in 53 of 53 patients. The ET-Pa(CO2) difference was < or = 2 mm Hg in 9 of 53 patients and < or = 5 mm Hg in 30 of 53 patients (P < .001). No variation in the TC-Pa(CO2) difference was noted based on the type of CHD (acyanotic vs cyanotic) or age. The ET-Pa(CO2) difference was greater in patients with cyanotic versus acyanotic CHD (7 +/- 3 mm Hg vs 4 +/- 2 mm Hg, P < .0001) and in patients < or = 1 year of age versus patients > or = 1 year of age (6 +/- 3 mm Hg vs 4 +/- 2, P = .008). In infants and children with CHD, TC monitoring provides a more accurate estimation of Pa(CO2) than ET monitoring.

Dexmedetomidine in the treatment of withdrawal syndromes in cardiothoracic surgery patients.

Dexmedetomidine (Precedex, Abbott Laboratories, Abbott Park, IL) is an alpha 2 adrenergic agonist that possesses a high ratio of specificity for the alpha 2 versus the alpha 1 receptor. It is currently approved for the provision of sedation during mechanical ventilation in adults. Given previous experience with clonidine for the treatment of substance withdrawal and the preliminary anecdotal experience with dexmedetomidine, it appears that dexmedetomidine may be a useful agent for treatment of substance withdrawal in the intensive care setting. The authors present their experience with the use of dexmedetomidine to control withdrawal behavior in 3 patients following cardiothoracic surgery. Previous reports regarding the use of dexmedetomidine to treat withdrawal and its potential application in this clinical arena are reviewed.

Argatroban for anticoagulation during cardiopulmonary bypass in an infant.

Heparin induced thrombocytopenia (HIT) is a rare, but potentially life-threatening complication of heparin therapy. In patients with HIT, alternative means of anticoagulation are necessary. The authors present an infant with HIT who required anticoagulation during cardiopulmonary bypass for tricuspid valve excision in the treatment of bacterial endocarditis. The direct thrombin inhibitor, argatroban, was successfully used. Previous reports regarding the use of argatroban and other nonheparin anticoagulants for anticoagulation are reviewed and suggestions regarding argatroban dosing in infants are presented.