Background Intravascular red cell hemolysis impairs NO-redox homeostasis, producing endothelial dysfunction, platelet activation and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or “microparticles” and hemolysis, which we hypothesized could impair vascular function and contribute to the putative “storage lesion” of banked blood. Methods and Results We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell free and microparticle-encapsulated hemoglobin which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator nitric oxide (NO). Using stopped-flow spectroscopy and laser triggered NO release from a caged NO compound we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies we show that hemoglobin, even at concentrations below 10 μM (in heme), produces potent vasoconstriction when infused into the rat circulation, while controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage related hemolysis. Conclusions The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.

Background: Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic environmental pollutants generated during incomplete combustion. After exposure and during metabolism, PAHs can form reactive epoxides that can covalently bind to DNA. These PAH–DNA adducts are established markers of cancer risk. PAH exposure has been associated with epigenetic alterations, including genomic cytosine methylation. Both global hypomethylation and hypermethylation of specific genes have been associated with cancer and other diseases in humans. Experimental evidence suggests that PAH–DNA adduct formation may preferentially target methylated genomic regions. Early embryonic development may be a particularly susceptible period for PAH exposure, resulting in both increased PAH–DNA adducts and altered DNA methylation.Objective: We explored whether prenatal exposure to PAHs is associated with genomic DNA methylation in cord blood and whether methylation levels are associated with the presence of detectable PAH–DNA adducts.Methods: In a longitudinal cohort study of nonsmoking women in New York City, we measured PAH exposure during pregnancy using personal air monitors, assessed PAH internal dose using prenatal urinary metabolites (in a subset), and quantified benzo[a]pyrene–DNA adducts and genomic DNA methylation in cord blood DNA among 164 participants.Results: Prenatal PAH exposure was associated with lower global methylation in umbilical cord white blood cells (p = 0.05), but global methylation levels were positively associated with the presence of detectable adducts in cord blood (p = 0.01).Conclusions: These observations suggest that PAH exposure was adequate to alter global methylation in our study population. Additional epidemiologic studies that can measure site-specific cytosine methylation and adduct formation will improve our ability to understand this complex molecular pathway in vivo.

A quantitative competitive PCR (QC-PCR) assay for Epstein-Barr virus (EBV) has been developed to provide accurate measurement of EBV genome load in pediatric transplant recipients at risk for developing posttransplant lymphoproliferative disorder (PTLD). The assay quantifies between 8 and 5,000 copies of the EBV genome in 10(5) lymphocytes after a 30-cycle amplification reaction. For 14 pediatric patients diagnosed with PTLD, the median EBV genome load was 4,000, and 13 of the 14 patients had values of >500 copies per 10(5) lymphocytes. Only 3 of 12 control transplant recipients not diagnosed with PTLD had detectable viral genome loads (median value, 40). This median was calculated by using the highest value obtained by PCR testing on each of these patients posttransplantation. PCR values of >500 copies per 10(5) lymphocytes appear to correlate with a diagnosis of PTLD. By a modified protocol, the EBV genome copy number in latently infected adults was estimated to be <0.1 copy per 10(5) lymphocytes.

Inner-city, minority populations are high-risk groups for adverse birth outcomes and also are more likely to be exposed to environmental contaminants, including environmental tobacco smoke (ETS), benzo[a]pyrene (BaP), and other polycyclic aromatic hydrocarbons (PAHs) found in urban air. In a sample of nonsmoking African-American and Dominican women, we evaluated the effects on birth outcomes of prenatal exposure to ETS, using questionnaire data and plasma cotinine as a biomarker of exposure, and environmental PAHs using BaP-DNA adducts as a molecular dosimeter. We previously reported that among African Americans, high prenatal exposure to PAHs estimated by prenatal personal air monitoring was associated with lower birth weight (p = 0.003) and smaller head circumference (p = 0.01) after adjusting for potential confounders. In the present analysis, self-reported ETS was associated with decreased head circumference (p = 0.04). BaP-DNA adducts were not correlated with ETS or dietary PAHs. There was no main effect of BaP-DNA adducts on birth outcomes. However, there was a significant interaction between the two pollutants such that the combined exposure to high ETS and high adducts had a significant multiplicative effect on birth weight (p = 0.04) and head circumference (p = 0.01) after adjusting for ethnicity, sex of newborns, maternal body mass index, dietary PAHs, and gestational age. This study provides evidence that combined exposure to environmental pollutants at levels currently encountered in New York City adversely affects fetal development.

In this study of Epstein-Barr virus (EBV) latency, the polymerase chain reaction was used in modified form for amplification and detection of viral mRNA sequences in peripheral blood lymphocytes from healthy seropositive adults. Six known promoters for latent gene expression and eight known gene products were identified in in vitro-immortalized lymphocytes and in the cell lines established spontaneously from seropositive adults. We examined whether mRNA expression in uncultured B cells from four seropositive adults was the same as that which occurred in spontaneously established EBV-positive B-cell lines from the same individuals. A minimum of 17 polymerase chain reaction targets was required to circumscribe the known latent mRNA structures. Expression of the C promoter for the EBNA genes was detected in B-cell RNA from three of the four subjects. Transcripts initiated from the alternative W promoter for EBNA expression were not detected. The spliced transcripts detected in the B cells contained only the C2-to-Wl alternative splice, which was nonproductive for EBNA4 gene expression. None of the other EBNA open reading frames were detected spliced onto the 3' ends of the C promoter-initiated RNAs. Spliced RNA from the TP gene was detected in all four subjects. Expression of the TP gene was restricted to TP1 promoter-initiated RNAs, as no TP2 promoterinitiated transcripts were detected. Expression of RNA from the LMP gene was not detected. The F promoter which is active in the restricted expression latency that occurs in Burkitt's lymphoma cells was not detected being expressed in peripheral blood B cells. This pattern of latent gene expression is unique to uncultured B cells, indicating that there are profound differences between viral lptent states in vitro and in situ and suggesting a central role for the TP gene in the latency of EBV.

BackgroundCoal burning provides 70% of the energy for China’s industry and power, but releases large quantities of polycyclic aromatic hydrocarbons (PAHs) and other pollutants. PAHs are reproductive and developmental toxicants, mutagens, and carcinogens.ObjectiveWe evaluated the benefit to neurobehavioral development from the closure of a coal-fired power plant that was the major local source of ambient PAHs.MethodsThe research was conducted in Tongliang, Chongqing, China, where a coal-fired power plant operated seasonally before it was shut down in May 2004. Two identical prospective cohort studies enrolled nonsmoking women and their newborns in 2002 (before shutdown) and 2005 (after shutdown). Prenatal PAH exposure was measured by PAH–DNA adducts (benzo[a]pyrene–DNA) in umbilical cord blood. Child development was assessed by the Gesell Developmental Schedules at 2 years of age. Prenatal exposure to other neurotoxicants and potential confounders (including lead, mercury, and environmental tobacco smoke) was measured. We compared the cohorts regarding the association between PAH–DNA adduct levels and neurodevelopmental outcomes.ResultsSignificant associations previously seen in 2002 between elevated adducts and decreased motor area developmental quotient (DQ) (p = 0.043) and average DQ (p = 0.047) were not observed in the 2005 cohort (p = 0.546 and p = 0.146). However, the direction of the relationship did not change.ConclusionThe findings indicate that neurobehavioral development in Tongliang children benefited by elimination of PAH exposure from the coal-burning plant, consistent with the significant reduction in PAH–DNA adducts in cord blood of children in the 2005 cohort. The results have implications for children’s environmental health in China and elsewhere.

The predictive negative value of persistently low or nondetectable EBV viral loads was 100% in this study. Patients with nondetectable or low viral loads for the first 6 months after ITx did not develop PTLD regardless of their pretransplant EBV serological status. The frequency of viral load monitoring can be safely decreased for patients whose viral loads remain low for the first 6 months ITx.

Pediatric solid organ transplant recipients are at risk for Epstein-Barr virus (EBV)-driven lymphoproliferative disease. The expression of 5 sentinel EBV genes (EBNA1, EBNA2a, LMP1, LMP2a, and ZEBRA) was examined in solid organ transplant recipients who developed persistent virus loads in their peripheral blood lymphocytes after transplantation. Two distinct groups were identified. LMP2a gene expression alone was detected among 12 of 14 patients carrying EBV loads < or =100 copies/10(5) lymphocytes. The other 2 low-load carriers made LMP2a RNA but also expressed LMP1 RNA. In contrast, LMP2a and LMP1 gene expression was detected among 11 of 13 patients carrying a virus load >100 copies/10(5) lymphocytes. Two high-load carriers made LMP1 RNA but not the RNA for LMP2a or any of the other viral genes. Therefore, persistent low-load carriers appear to maintain an apparently normal state of latent viral infection, whereas high-load carriers display a unique LMP1:LMP2a pattern of viral gene expression that has not been previously described.