A multicenter evaluation of viral bloodstream detections in children presenting to the Emergency Department with suspected systemic infection.

BACKGROUND: Fever is a common symptom in children presenting to the Emergency Department (ED). We aimed to describe the epidemiology of systemic viral infections and their predictive values for excluding serious bacterial infections (SBIs), including bacteremia, meningitis and urinary tract infections (UTIs) in children presenting to the ED with suspected systemic infections. METHODS: We enrolled children who presented to the ED with suspected systemic infections who had blood cultures obtained at seven healthcare facilities. Whole blood specimens were analyzed by an experimental multiplexed PCR test for 7 viruses. Demographic and laboratory results were abstracted. RESULTS: Of the 1114 subjects enrolled, 245 viruses were detected in 224 (20.1%) subjects. Bacteremia, meningitis and UTI frequency in viral bloodstream-positive patients was 1.3, 0 and 10.1% compared to 2.9, 1.3 and 9.7% in viral bloodstream-negative patients respectively. Although viral bloodstream detections had a high negative predictive value for bacteremia or meningitis (NPV = 98.7%), the frequency of UTIs among these subjects remained appreciable (9/89, 10.1%) (NPV = 89.9%). Screening urinalyses were positive for leukocyte esterase in 8/9 (88.9%) of these subjects, improving the ability to distinguish UTI. CONCLUSIONS: Viral bloodstream detections were common in children presenting to the ED with suspected systemic infections. Although overall frequencies of SBIs among subjects with and without viral bloodstream detections did not differ significantly, combining whole blood viral testing with urinalysis provided high NPV for excluding SBI.

Doxorubicin induces cell senescence preferentially over apoptosis in the FU-SY-1 synovial sarcoma cell line.

Surgical resection coupled with adjuvant radiotherapy and/or doxorubicin based chemotherapy are the mainstays of synovial sarcoma (SS) treatment. Although effective as a SS adjuvant, the proposed mechanism of action of doxorubicin remains controversial. Current opinion supports DNA damage-induced apoptosis. This in vitro study used cDNA gene expression profiling to investigate whether apoptosis, alone or in combination with cell senescence, is induced by doxorubicin in SS cells. Cell cultures of the FU-SY-1 SS, the pleomorphic SW982 sarcoma, and a primary dermal fibroblast (NHDF), were exposed to 500 nM doxorubicin, and then processed for cDNA microarray analysis. The one class response option of SAM (Significance Analysis of Microarrays) was used to test for significant overexpression of 15 apoptosis-related genes and nine senescence-related genes. Drug-induced cell senescence was quantified by measuring beta-galactosidase activity. None of 15 apoptosis-related genes and only two of nine senescence-related genes were identified by SAM as significantly overexpressed in doxorubicin-treated cultures. Drug-induced senescence as reflected by beta-galactosidase activity was significantly increased (p < 0.05) only in FU-SY-1 SS cultures. Apoptosis does not appear to be a major determinant of doxorubicin-induced mortality in FU-SY-1 SS or NHDF cultures, but may impact SW982 cells via the overexpression of BAX relative to Bcl-2. Doxorubicin-induced cell senescence was prominent in FU-SY-1 SS cultures, but negligible in SW982 and NHDF cultures. Likely, both apoptosis and cell senescence contribute to doxorubicin-induced cell death in this synovial sarcoma cell line.

G3139 antisense oligonucleotide directed against antiapoptotic Bcl-2 enhances doxorubicin cytotoxicity in the FU-SY-1 synovial sarcoma cell line.

Synovial sarcoma (SS) is a highly aggressive, periarticular soft tissue sarcoma that causes death in more than half of affected children, adolescents, and young adults. Five- and 10-year survival rates are as low as 36 and 20%, respectively. Bcl-2, a negative regulator of apoptosis, is overexpressed in up to 90% of SS. Increased Bcl-2 expression not only leads to the development of cancer, but also to resistance of many anticancer chemotherapeutic agents. We hypothesized reducing Bcl-2 expression in SS should enhance doxorubicin cytotoxicity. Cell cultures representing two human sarcomas (FU-SY-1 SS and the pleomorphic SW982) and a primary human dermal fibroblast comparator (NHDF) were exposed in vitro to doxorubicin, or to doxorubicin preceded by Bcl-2 (G3139) antisense oligonucleotides, and assayed for cell survival, apoptosis, and modulations in Bcl-2 and Bcl-xL mRNA and protein content. SW982 sarcoma cells proved most susceptible to doxorubicin, while NHDF mesenchymal cells were least sensitive to doxorubicin. Treatment of FU-SY-1 SS with G3139 reduced Bcl-2 mRNA and protein levels, which enhanced doxorubicin-induced cell killing. There was a concurrent reduction in Bcl-xL mRNA following G3139 application in FU-SY-1 and NHDF cultures, but not in SW982. G3139 anti-Bcl-2 intervention sensitized the FU-SY-1 SS to doxorubicin, due to increased apoptosis. G3139 intervention was ineffective in the two non-SS cell lines.

Discriminate gene lists derived from cDNA microarray profiles of limited samples permit distinguishing mesenchymal neoplasia ex vivo.

BACKGROUND: Mesenchymal neoplasia comprises a heterogeneous group of tumors with over 200 benign neoplasms and 100 sarcomas. Currently, tumors are classified using histologic and immunocytologic characteristics, with diagnostic error rates reported as high as 40% of cases. As a feasibility study, our goal was to generate a preliminary discriminatory gene list for selected mesenchymal tumors, including sarcomas. This technique may enable an eventual molecular classification schema based on expression profiles that can complement current clinical and pathologic diagnostic procedures in mesenchymal tumors. METHODS: cDNA microarray analyses were preformed on connective tissue tumors obtained at time of surgical resection or biopsy. Messenger RNA (mRNA) from four general tumor classes was competitively hybridized against a human dermal fibroblast cell line comparator and the resulting gene expression profiles processed by ANOVA and linear discriminate analysis. RESULTS: The tissue classification involved 18 patients with malignant peripheral nerve sheath tumors, giant cell containing tumors, benign spindle cell lesions, or Ewing's family of tumors. Lymph nodes from two patients served comparative purposes. Twenty-five differentially regulated genes considered most variable among the five tissue classes were identified. The tissues were segregated into five classes by linear discriminate analysis. CONCLUSIONS: Linear discriminate analysis of cDNA gene expression profiles partitioned mesenchymal tumor classes, even when constrained by limited sample sizes.

Transit tumor retrieval preserves RNA fidelity and obviates snap-freezing.

UNLABELLED: Genetic expression profiling is enabling investigators to discover new diagnostic and possibly therapeutic pathways in sarcoma biology. To draw substantial conclusions from these molecular analyses, adequate tissue samples must be accrued. Beyond cohort size, the most variable and limiting aspect of doing gene expression analyses on fresh human tissue is the preservation of labile ribonucleic acids extracted from clinical specimens. We have developed a novel retrieval protocol that is readily amenable to the clinical constraints placed on surgeons and pathologists that minimizes variables that can corrupt ribonucleic acid fidelity. We evaluate critically genomic message integrity of mesenchymal tumors derived from transcontinental inter-institutional collaboration. Intact total ribonucleic acid was isolated and assessed for quality and quantity. Ribosomal RNA integrity was quantified using a bioanalyzer. Ribonucleic acid from 42 mesenchymal tumors was isolated and quantified, with selected samples amplified. The mean ribosomal ratios for collaborative institutions ranged from 1.0 to 1.18. Samples remained at 4 degrees C before processing from 1 to 17 days. Tumors stabilized using this protocol retained total ribonucleic acid integrity suitable for amplification and genomic expression analysis regardless of the institutional source or preprocessing duration, enabling a potential consortium of investigators to collaborate in the expression profiling of sarcomas. LEVEL OF EVIDENCE: Diagnostic study, Level III-3 (no consistently applied gold standard). See the Guidelines for Authors for a complete description of levels of evidence.