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Preterm labor (PTL) is frequently associated with inflammation. We hypothesized that biomarkers during pregnancy can identify pregnancies most at risk for development of PTL. An inflammation-induced mouse model of PTL was used. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to analyze and compare the plasma protein (PP) profile between CD-1 mice injected intrauterine with either lipopolysaccharide (LPS) or PBS on d 14.5 of gestation. The median differences of normalized PP peaks between the two groups were determined using the Mann-Whitney U test and the false discovery rate. In a second series of experiments, both groups of mice were injected with a lower dose of LPS. A total of 1665 peaks were detected. Thirty peaks were highly differentially expressed (p < 0.0001) between the groups. Two 11 kDa protein peaks were identified by MALDI-TOF/TOF-MS and confirmed to be mouse serum amyloid A (SAA) 1 and 2. Plasma SAA2 levels were increased in LPS-treated animals compared with controls and in LPS-treated animals that delivered preterm vs. those that delivered at term. SAA2 has the potential to be a plasma biomarker that can identify pregnancies at risk for development of PTL.

Preterm infants are at risk of developing sepsis, necrotizing enterocolitis (NEC), chronic lung disease (CLD), and retinopathy of prematurity (ROP). We used high-throughput mass spectrometry to investigate whether cord blood proteins can be used to predict development of these morbidities. Cord blood plasma from 44 infants with a birth weight of1500 g was analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). Six infants developed ROPor=stage II, 10 CLD, three sepsis, and one NEC. We detected 814 protein signals representing 330 distinct protein species. Nineteen biomarkers were associated with development ofor=stage II ROP [false-discovery rate (FDR)5%] and none with CLD. Several proteins with molecular weight (Mr) 15-16 kD and pI 4-5 were detected with increased abundance in infants with ROP, while similar Mr proteins with pI 7-9 were less abundant in these patients. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sequence analysis identified these proteins as alpha-, beta-, and gamma-globin chains. Partial deamidation of Asn139 in beta-globin chains was observed only in the pI 4-5 proteins. We conclude that there are several promising biomarkers for the risk of ROP. Deamidation of globin chains is especially promising and may indicate underlying prenatal pathologic mechanisms in ROP. Validation studies will be undertaken to determine their clinical utility.

Extracellular glutathione peroxidase (E-GPx) is a selenoenzyme that reduces hydrogen peroxide and organic peroxides. All plasma glutathione peroxidase (GPx) activity in humans is attributable to E-GPx. The gastrointestinal (GI) tract also synthesizes and secretes E-GPx into the extracellular milieu. Endogenously generated oxidants have been implicated in inflammatory bowel disease (IBD). We evaluated E-GPx levels in a mouse model of IBD using dextran sodium sulfate (DSS). Histologic lesions of the lower GI tract consisted of multifocal areas of mucosal erosion denuded of epithelial cells, reduction in goblet cells, dilated crypts, crypt collapse, submucosal edema, and transmural distribution of mixed inflammatory infiltrates. On d 7, plasma GPx activity in the DSS group increased by 61% compared with the control group (p < 0.05). Western blot analysis demonstrated a 64% increase in E-GPx protein in the plasma of the DSS group after 7 d of treatment (p < 0.01). As the major source of plasma GPx is the kidney, we determined whether the increase in plasma GPx activity and protein was caused by a change in E-GPx synthesis by the kidney. After 3 and 7 d of DSS treatment, E-GPx mRNA levels, relative to glyceraldehyde-3-phosphate dehydrogenase, increased in the kidney (p < 0.05) without a concomitant increase in cellular GPx mRNA on d 7. These results suggest that the inflammatory injury in the intestine elicits an increase in E-GPx in the plasma that is associated with an increase in E-GPx mRNA in the kidney. This implies that renal production of E-GPx may be sensitive to insults distal to the kidney.

Extracellular glutathione peroxidase (E-GPx) is a selenium-dependent enzyme that can reduce hydrogen peroxide and phospholipid hydroperoxides. E-GPx is found in plasma and extracellular fluids such as bronchoalveolar lavage fluid. Because lung is one of the tissues that is capable of synthesizing and secreting E-GPx, the effect of exposure to hyperoxia on E-GPx in plasma and lung were studied in an injury model of hyperoxia exposure in adult mice. Exposure to 100% oxygen for 72 h resulted in an increase of 55% in plasma GPx activity and an increase of 50% in the amount of E-GPx protein in the plasma. Exposure to hyperoxia was also associated with an increase in the amount of E-GPx protein in lungs. The 7-fold increase in the amount of E-GPx protein in lungs was not due to plasma contamination of lungs from mice exposed to hyperoxia. E-GPx in the lung is calculated to account for 10% of lung GPx activity in control mice. However, E-GPx is calculated to account for 45% of lung GPx activity in the lungs of mice exposed to hyperoxia for 72 h. Further studies are needed to determine whether the increase in lung E-GPx is due to changes in translation or stability of E-GPx. The role of E-GPx in protecting the lung from oxidative damage warrants further study.

Hypoxic Induction of Vascular Endothelial Growth Factor (VEGF) and HIF-1 Protein in Retinal Pigment Epithelial Cells

Retinopathy of prematurity (ROP) is a neovascularizing disease that causes blindness in premature infants. ROP is initiated by an arrest of normal retinal blood vessel development. Subsequently, the non-perfused peripheral retina becomes hypoxic leading to retinal neovascularization. Vascular endothelial growth factor (VEGF) has been implicated in playing a role in the pathogenesis of ROP. HIF-1 is a heterodimeric DNA binding protein consisting of an α and β subunit. HIF-1 protein expression and DNA binding increases in response to hypoxia. HIF-1 binding sites are present in several hypoxia inducible genes and HIF-1 has been implicated in the activation of VEGF transcription in response to hypoxia. It is therefore possible that HIF-1 plays a role in the pathogenesis of ROP.

Erythropoietin (Epo) is a glycoprotein hormone that is the primary regulator of erythropoiesis. Transcription of the Epo gene increases in response to hypoxia or anemia. Epo is synthesized in the liver in fetal life and in the kidney later in gestation. Hypoxia inducible factor (HIF-1) is a DNA binding protein that binds to a hypoxia inducible enhancer in the 3′ region of the Epo gene. HIF-1 is a heterodimer consisting of a α andβ sub-unit. HIF-1 α and β mRNA is present in all tissues in adult mice. HIF-1β transcripts were detected in various tissues of 9.5 day fetal mice by in situ hybridization. Epo mRNA is present in only fetal mouse liver at day 14 and in kidney by day 19 of gestation. In the human fetus Epo mRNA is present in fetal kidney by 11 weeks gestation. In order to determine if there is a relationship between the expression of HIF-1 α and β sub-units with the shift in expression of the Epo gene from the liver to the kidney we analyzed the expression of these mRNA's in mouse and human fetuses. Total RNA was extracted from mouse brain, heart, kidney, liver and lung at day 15, 17 and 19 of gestation in the mouse and 12-22 weeks of gestation in the human fetus. RNA was analyzed by northern blot and dot blot hybridization using appropriate cDNA probes. HIF-1α and β were expressed in all tissues tested and at all stages of gestation in mouse and human. Expression of mouse HIF-1α in the heart and kidney was mildly in excess of other organs, and expression in the brain was minimal. Expression of human HIF-1α in the kidney and lung was several-fold higher than in other organs. In the human, increased expression of HIF-1β was present in the lung, and expression in the brain was minimal. Human Epo mRNA was expressed in the kidney and liver from 12-22 weeks of gestation. In summary, both HIF-1α and β sub-units are expressed ubiquitously at the various stages of gestation tested. Our studies indicate that either sub-unit of the transcription factor HIF-1 probably does not play a major role in either tissue-specific expression or in the switch of Epo gene expression from the fetal liver to the kidney. However, other unidentified members of this family of transcription factors may play a part in this mechanism.

Extracellular glutathione peroxidase (EGPx) is a member of a selenium-dependent antioxidant enzyme family. To better understand its potential functions in vivo, we examined the postimplantation expression of EGPx mRNA from E7.5 to E16.5 of mouse development. During this period, the message for EGPx shifted major sites of accumulation, but maintained high concentration at borders between the developing embryo and its environment. Thus, at E7.5 (neural plate stage) the highest EGPx expression was found in the maternally-derived deciduum, with minor accumulation in the visceral yolk sac endoderm. At E9.5, the major sites of expression were the yolk sac endoderm and developing heart musculuture. By E16.5, EGPx mRNA expression persisted in the yolk sac endoderm, but also accumulated significantly in a ventral to dorsal gradient in the developing skin. Expression in the heart and lungs was primarily restricted to atrially-derived myocytes. At E16.5, we also detected significant EGPx mRNA accumulation in the intestinal epithelium and minor accumulation in developing kidney, the principle site of EGPx synthesis in the adult. Since EGPx is expressed in the yolk sac endoderm, we explored its in vitro expression in F9 teratocarcinoma cells. EGPx mRNA is induced along with α-fetoprotein when F9 cells are differentiated toward visceral, but not parietal endoderm. Further studies of EGPx function and gene regulation can now be performed in this in vitro system.

Extracellular glutathione peroxidase (EGPx) is a unique seleno-enzyme found in plasma and other extracellular fluids. Based on the disappearance of plasma EGPx activity in nephrectomized animals and the high level of EGPx mRNA in renal tissue, it was previously concluded that the kidney was the major source of plasma EGPx activity. We have previously shown that humans with renal failure receiving chronic dialysis treatment possess 42% plasma GPx activity. We examined the effect of nephrectomy and renal transplantation on plasma GPx activity. Nephrectomized patients have 22% plasma GPx activity, which increases to ≥ 100% of normal within 2 weeks following transplantation.In situ hybridization studies have shown that EGPx message is predominantly localized to the proximal convoluted tubules. We have determined that the EGPx mRNA is localized specifically to the S1 and S2 segments of the proximal tubules. We therefore examined the effect of nephrotoxic agents known to affect proximal tubule function. In rabbits, a nephrotoxic dose of cephaloglycin (300 mg/kg) caused a 52% decrease in plasma GPx activity within 24 hours. This effect was prevented by concomitant treatment with probenecid. Patients receiving ifosfamide chemotherapy were found to have lower levels of plasma GPx activity. Three patients with Fanconi syndrome associated with ifosfamide therapy had 50, 56, and 23% of control plasma GPx activity. After resolution of the Fanconi syndrome in one patient, plasma GPx activity returned to normal within one month. We speculate that plasma GPx activity may be a sensitive and early indicator of damage to renal proximal tubules.

The anti-leukemic activity of ASNase is thought to result from depletion of serum ASN. In studies of the pharmacologic effects of ASNase, investigators have reported dramatic prolonged reduction in the serum concentration of ASN after the administration of ASNase. Such measurements, however, have not accounted for the problem of hydrolysis of ASN by ASNase that is present in blood samples. We examined recovery of 14C ASN from blood samples with and without added ASNase at various concentrations. In the presence of ≥0.001 IU/ml of ASNase, the amount of 14C ASN recovered was 90% of control. Blood samples drawn from patients at 1 and 7 days following ASNase injection were collected directly into tubes containing 14C ASN +/- ASA. Recovery with ASA, was 60-110% and 100-110% of control (i.e., no ASNase) on days 1 and 7 respectively. We conclude that: 1) continued hydrolysis of ASN by ASNase can result in falsely low serum ASN measurements; 2) ASA is a potent inhibitor of E. Coli ASNase; 3) ASA present in collection tubes obviates the problem of continued ASNase activity. Thus, for accurate measurements of the rate and degree of ASN depletion by ASNase, ASA should be present in the blood collection system.

In order to determine the role of the glutathione cycle in granulocyte (PMN) oxidative metabolism, rats were made glutathione peroxidase (GSH-Px) deficient(def), by feeding them a selenium def diet. After 12-15 weeks def rat PMN GSH-Px was 23 U/mg protein compared to 155 U/mg protein for controls. We studied the effect of GSH-Px deficiency on phorbol myristate acetate (PMA) stimulated PMN hexose monophosphate shunt (HMPS) activity and O2− production. HMPS activity was similar for def and control rats for the first 5 min. of incubation with PMA. However, over 20 min., def PMN produce 480 cpm/2.5×106 cells and control 920 cpm/2.5×106 cells. When stimulated with L-amino acid oxidase and L-leucine HMPS was 220 in def and 570 cpm/20 min/2.5×106 cells in control PMN. Initial rates of PMA stimulated O2− production were the same in def and control (2.8, 2.78 nmol O2−/min/106 PMN). However, at 5, 10, and 20 min. the rates of O2− production in the def were 44%, 14%, and 3% of O2− produced at 2 min. This is compared to 52%, 36%, and 23% O2− produced at 2 min. in the control. Incubation of PMN with L-amino acid oxidase and L-leucine for 20 min. resulted in a loss of 85% of PMA stimulatable O2− production in def compared with 43% in controls. This effect was abolished with the concomittant addition of catalase. We conclude that H2O2 in the rat PMN is metabolized through the glutathione cycle and GSH-Px protects PMN against H2O2 mediated destruction of their O2− generating system.

Van Rij reported proximal muscle weakness in a case of selenium (Se) deficiency in New Zealand in 1979. Since then cardiomyopathy, but not proximal muscle weakness, has been reported in the USA. A 33 y.o. white female was on home parenteral nutrition for 4½ years due to a duodenocecostomy secondary to a traumatic jejunoilectomy. During the first year she noted proximal muscle weakness which did not improve over the next 3½ years. She noted weakness with arm lifting and inability to rise from a squatting position. During a study of Se function, glutathione peroxidase levels were found to be profoundly low in plasma and blood cells. Plasma Se level initially was 32.5 ng.ml (nl=60-120). After 3-4 weeks of treatment with Se (400 μg/d IV) as selenous acid, the patient's muscle strength testing markedly improved as the GSHPx activities in plasma, platelets, granulocytes, & mononuclear cells became normal. Red cell GSHPx activity approached normal levels much more slowly, and may not be a sensitive reflection of whole body selenium status.

PMN have Fc Receptors (FcR) for IgG when part of an IC. We employed homologous IC (tetanus toxin and human anti-tecanus toxin IgG) as a stimulus for human PMN responsiveness. IC, at equivalence, stimulated PMN to produce superoxide (O2−) in a dose-dependent manner. Neither anttgen nor antibody alone had PMN stimulatory activity. The lag time for activation was 40-50 seconds and was dose-dependent. The addition of IC to PMN resulted in apparent membrane potential depolarization within 20 seconds (prior to the onset of superoxide production). IC-stimulated PMN increased their intracellular calcium, aggregated and degranulated. The addition of fresh human serum to IC resulted in enhanced O2− production and degranulation at less than saturating amounts of IC. Human monoraeric IgG (3 mg/ml) did not inhibit IC mediated PMN O2− production. The FAB fragment of a monoclonal antibody (IV3) specific for a 40 kD FcR on human PMN selectively inhibited IC-mediated O2− generation by approximately 75%. The same observations were obtained when another monoclonal antibody (3G8) FAB fragment, specific for a 51-75 kC FcR on human PMN was used. Complete inhibition of IC-mediated PMN O2− production was achieved only when FAB fragments of both antibodies were utilized. These results show that human IC can stimulate human PMN, that these interactions can be enhanced, by the presence of complement components and that monoclonal antibodies directed against both FcR are necessary to completely inhibit functional activity induced by human IC.

To provide a means for the prenatal diagnosis of CGD, we combined a modified nitroblue tetrazolium (NBT) slide test with quinacrine staining for Y chromosome fluorescence. Blood samples (10 μ1) are applied to glass slides and the adherent cells incubated with NBT and phorbol myristate acetate, an activator of oxidative metabolism. Resultant metabolic activity produces microscopically observable formazan particles in 99-100% of adult and 95-100% of fetal granulocytes (PMN), in 0-1% of PMN from 4 CGD patients, and in 18-53% of PMN from 3 obligate CGD carriers. Since placental venipuncture samples are often contaminated with maternal blood, it is necessary to distinguish fetal and maternal cells in such mixtures. Quinacrine staining reveals a Y-body in 50-90% of male fetal PMN. Such identification of male PMN by Y-fluorescence allows detection and assessment of oxidative activity of CGD cells diluted 1:100 in maternal cells. Thus, the modified NBT slide test with quinacrine staining of blood obtained by placental venipuncture provides a method for the prenatal diagnosis of CGD in male fetuses at risk for the X-linked or autosomal recessive forms of the disease.

591 BACTERIAL KILLING AND OXIDATIVE METABOLISM IN CHLORPRDMAZINE (CPZ) TREATED POLYMORPHONUCLEAR LEUKOCYTES (PMN): A MODEL FOR CHRONIC GRANULOMATOUS DISEASE

Although annual influenza(flu) vaccination is recommended for patients(pts) in designated high-risk categories, little data exist to support these recommendations in pediatric pts. We prospectively followed a group of oncology pts (OP) on chemRx or off Rx for