Kristin Greathouse

Innate Immune Function and Mortality in Critically Ill Children With Influenza: A Multicenter Study*

Objective:To prospectively evaluate relationships among serum cytokine levels, innate immune responsiveness, and mortality in a multicenter cohort of critically ill children with influenza infection. Design:Prospective, multicenter, observational study. Setting:Fifteen pediatric ICUs among members of the Pediatric Acute Lung Injury and Sepsis Investigators network. Patients:Patients ⩽18 yrs old admitted to a PICU with community-acquired influenza infection. A control group of outpatient children was also evaluated. Interventions:ICU patients underwent sampling within 72 hrs of ICU admission for measurement of a panel of 31 serum cytokine levels and quantification of whole blood ex vivo lipopolysaccharide-stimulated tumor necrosis factor-&agr; production capacity using a standardized stimulation protocol. Outpatient control subjects also underwent measurement of tumor necrosis factor-&agr; production capacity. Measurements and Main Results:Fifty-two patients (44 survivors, eight deaths) were sampled. High levels of serum cytokines (granulocyte macrophage colony-stimulating factor, interleukin-6, interleukin-8, interferon-inducible protein-10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1&agr;) were associated with mortality (p < 0.0016 for each comparison) as was the presence of secondary infection with Staphylococcus aureus (p = 0.007), particularly methicillin-resistant S. aureus (p < 0.0001). Nonsurvivors were immunosuppressed with leukopenia and markedly reduced tumor necrosis factor-&agr; production capacity compared with outpatient control subjects (n = 21, p < 0.0001) and to ICU survivors (p < 0.0001). This association remained after controlling for multiple covariables. A tumor necrosis factor-&agr; response <250 pg/mL was highly predictive of death and longer duration of ICU stay (p < 0.0001). Patients with S. aureus coinfection demonstrated the greatest degree of immunosuppression (p < 0.0001). Conclusions:High serum levels of cytokines can coexist with marked innate immune suppression in children with critical influenza. Severe, early innate immune suppression is highly associated with both S. aureus coinfection and mortality in this population. Multicenter innate immune function testing is feasible and can identify these high-risk children.

Immunosuppressive effects of red blood cells on monocytes are related to both storage time and storage solution

BACKGROUND: Reduced monocyte function is associated with adverse outcomes from critical illness. Red blood cells (RBCs) are thought to impair monocyte function but relationships between RBC storage solution and monocyte suppression are unknown. This study was designed to test the hypothesis that immunosuppressive effects of RBCs on monocytes are related to both storage time and preservative solution.

Innate immune function predicts the development of nosocomial infection in critically injured children.

ABSTRACT Background: Critical injury has been associated with reduction in innate immune function in adults, with infection risk being related to degree of immune suppression. This relationship has not been reported in critically injured children. Hypothesis: Innate immune function will be reduced in critically injured children, and the degree of reduction will predict the subsequent development of nosocomial infection. Methods: Children (⩽18 years old) were enrolled in this longitudinal, prospective, observational, single-center study after admission to the pediatric intensive care unit following critical injury, along with a cohort of outpatient controls. Serial blood sampling was performed to evaluate plasma cytokine levels and innate immune function as measured by ex vivo lipopolysaccharide-induced tumor necrosis factor &agr; (TNF-&agr;) production capacity. Results: Seventy-six critically injured children (and 21 outpatient controls) were enrolled. Sixteen critically injured subjects developed nosocomial infection. Those subjects had higher plasma interleukin 6 and interleukin 10 levels on posttrauma days 1–2 compared with those who recovered without infection and outpatient controls. Ex vivo lipopolysaccharide-induced TNF-&agr; production capacity was lower on posttrauma days 1–2 (P = 0.006) and over the first week following injury (P = 0.04) in those who went on to develop infection. A TNF-&agr; response of less than 520 pg/mL at any time in the first week after injury was highly associated with infection risk by univariate and multivariate analysis. Among transfused children, longer red blood cell storage age, not transfusion volume, was associated with lower innate immune function (P < 0.0001). Trauma-induced innate immune suppression was reversible ex vivo via coculture of whole blood with granulocyte-macrophage colony-stimulating factor. Conclusions: Trauma-induced innate immune suppression is common in critically injured children and is associated with increased risks for the development of nosocomial infection. Potential exacerbating factors, including red blood cell transfusion, and potential therapies for pediatric trauma-induced innate immune suppression are deserving of further study.

Clinical Outcomes of Children Receiving Intensive Cardiopulmonary Support During Hematopoietic Stem Cell Transplant

Objective: We investigated the short-term and 1-year clinical outcomes of 129 children who received intensive cardiopulmonary support during hematopoietic stem cell transplant. Intensive cardiopulmonary support was defined as receiving at least one of the following interventions: continuous positive pressure ventilation, dopamine infusion greater than or equal to 10 mcg/kg/minute, or the use of any other vasoactive infusion. Duration of intensive cardiopulmonary support, survival to hospital discharge, and predictors of these outcome variables were compared with 387 hematopoietic stem cell transplant patients who did not receive intensive support during the same period. We also report the 1-year survival; presence of chronic graft-versus-host disease; and renal, cardiac, and pulmonary function for all patients. Design: A multicenter retrospective cohort study. Setting: The ICU and hematopoietic stem cell transplant unit of nine pediatric tertiary care centers. Patients: Children undergoing hematopoietic stem cell transplant who required intensive cardiopulmonary support. Interventions: None. Results: Predictors of the need for intensive support included unrelated donor allogeneic transplant, glomerular filtration rate less than 85 mL/minute/1.73 m2, and nonmalignant disease as the indication for transplant. The survival to discontinuation of intensive support for all patients was 62% and 58% for patients who received invasive mechanical ventilatory support. The duration of mechanical ventilation was not predictive of survival. Predictors of intensive support mortality included macroscopic bleeding, engraftment, and pediatric logistic organ dysfunction score greater than one in two domains. Survival to hospital discharge was 50% for the intensive support group and 99% for the nonintensive support group. Overall 1-year survival was 40% in the intensive support population and 65% in the nonintensive support group. There were no significant differences in the survival, rates of chronic graft-versus-host disease, creatinine, forced expiratory volume in 1-minute, cardiac shortening fraction, or performance status in intensive and nonintensive support patients who survived to hospital discharge. Conclusion: Intensive cardiopulmonary support plays an important and potentially life-saving role in the care of pediatric stem cell transplant patients. Survivors of intensive support do not have compromised 1-year survival or organ function compared with children who did not receive intensive support.

Gene transfer to the gastrointestinal tract after peroral administration of recombinant adeno-associated virus type 2 vectors.

Objectives: The transfer of exogenous genetic material to cells within the gastrointestinal (GI) tract has many potential therapeutic applications. An attractive feature of the GI tract for gene transfer is its accessibility through the orogastric route. In this study, we evaluated the stability of recombinant adeno-associated virus type 2 (rAAV2) vectors within the GI tract and whether rAAV2-mediated gene transfer could be increased through manipulation of the intraluminal environment. Methods: The stability of rAAV2 vectors carrying &bgr;-galactosidase and enhanced green fluorescence protein transgenes was determined in the presence of hydrochloric acid, pepsin, trypsin, chymotrypsin gastric fluid and intestinal fluid and after in vivo administration. For in vivo experiments, the rAAV2 vector carrying the &bgr;-galactosidase transgene was administered perorally to FVB/NJ mice. Groups of mice received the vector alone or in combination with sodium bicarbonate and aprotinin. Gene transfer to the stomach and small intestine was evaluated by polymerase chain reaction and histochemical assays. Results: The stability of rAAV2 was reduced by hydrochloric acid, trypsin, chymotrypsin, gastric fluid and intestinal fluid. The vector was not stable within the lumen of the GI tract. Gastric acid neutralization with sodium bicarbonate and protease inhibition with aprotinin increased the in vivo stability of the vector and the level of gene transfer to the stomach and all regions of the small bowel. In both groups of mice (vector alone and vector plus sodium bicarbonate and aprotinin), transgene-derived protein expression (&bgr;-galactosidase) was below the level of detection of the histochemical assay. Conclusions: Recombinant AAV2 are adversely affected by physiological conditions within the proximal GI tract. Gastric acid neutralization and inhibition of intestinal protease activity improved rAAV2 stability and increased the level of gene transfer within the GI tract. Despite these changes, transduction of the GI tract after peroral rAAV2 administration remained low.

Immunoparalysis in Pediatric Critical Care

Although many forms of critical illness are initiated by a proinflammatory stimulus, a compensatory anti-inflammatory response can occur with systemic inflammation. Immunoparalysis, an important form of acquired immunodeficiency, affects the innate and adaptive arms of the immune system. Immunoparalysis has been associated with increased risks for nosocomial infection and death in a variety of pediatric critical illnesses. Evidence suggests that immunoparalysis is reversible with immunostimulants. Highly standardized, prospective immune monitoring regimens are needed to better understand the immunologic effects of critical care treatment regimens and to enrich clinical trials with subjects most likely to benefit from immunostimulatory therapies.

246. Comparison of rAAV2-Mediated Expression of Human and Murine |[beta]|-Glucuronidase in Adult MPS VII Mice

Top of pageAbstract Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease (LSD) caused by a deficiency of -glucuronidase. This enzyme deficiency results in the accumulation of glycosaminoglycans in most organs including the brain. We previously demonstrated that intrahepatic administration of a recombinant adeno-associated virus type 2 (rAAV2) vector carrying the murine -glucuronidase cDNA leads to improvement in both the peripheral and central manifestations of disease in adult MPS VII mice. The objective of this study was to compare the effects of rAAV2-mediated expression of human to murine -glucuronidase in this disease model. Methods: rAAV2 vectors carrying the murine -glucuronidase (Gusb) cDNA and human -glucuronidase (GUSB) cDNA under the transcriptional direction of the human elongation factor-1 promoter were administered to 7-week old MPS VII mice (n=3, each vector). Mice received 1.31011 DNase resistant particles by intrahepatic injection and were sacrificed 6 months post-vector administration. Tissues and sera were evaluated as indicated for vector genomes (quantitative PCR assay), -glucuronidase activity (fluorometric assay), and -galactosidase activity (fluorometric assay). Results: In the livers of the rAAV2-hGUSB (human)-injected group, the mean level of vector genomes was 2.6-fold lower than in the group receiving rAAV2-mGusb (murine) (P=0.03). There was no difference in hepatic -glucuronidase activity between the rAAV2-hGUSB-injected (11267% of normal, wild-type levels; meanS.E.M.) and rAAV2-mGusb-injected (16242%) groups. However, hepatic gene transfer was more variable in the group receiving rAAV2-hGUSB; one mouse had 4% of wild-type -glucuronidase activity and 5-fold less vector genomes than the other mice in this group. -Glucuronidase activity within the serum of individual mice in the rAAV2-hGUSB group was 0, 86, and 135% and in the rAAV2-mGusb group 8, 11, and 26% of wild-type. The -glucuronidase serum-to-liver ratio was 0.00, 0.37, and 1.31 in the rAAV2-hGUSB group and 0.08, 0.07, and 0.10 in the rAAV2-mGusb group. In the brain, vector genomes were detected at <1 copy per 1000 cell genome equivalents and -glucuronidase activity was no different from control MPS VII mice in both groups of mice. -Galactosidase levels (secondarily elevated by the storage abnormality) were reduced in the brains of the rAAV2-hGUSB (16425% of wild-type) and rAAV2-mGusb (1618% of wild type) groups as compared to control MPS VII (24836% of wild-type) mice (rAAV2-hGUSB vs. control, P<0.02; rAAV2-mGusb vs. control, P<0.01). Summary: These data demonstrate that the human and murine -glucuronidase transgenes delivered by an rAAV2 vector have different properties in MPS VII mice. In this study, the vector carrying the human transgene resulted in a lower and more variable levels of transduction than the vector carrying the murine transgene. Also, the human -glucuronidase transgene resulted in higher serum enzyme levels than those observed with the murine transgene. Despite the differences, biochemical improvement of disease within the brain was observed for both transgenes.