J. Gerald Quirk

The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vectors.

Because of its ability to integrate chromosomally and its non‐pathogenic nature, adeno‐associated virus (AAV) has significant potential as a human gene therapy vector. Here we investigate the maximum amount of DNA which can be inserted into the AAV genome and still allow efficient packaging into an infectious virus particle. Altered wild‐type AAV genomes were constructed with inserts, which increased in size by 100 bp, ligated at map unit 96. These large wild‐type‐plus genomes were able to replicate and produce infectious virus, at levels slightly reduced but comparable to normal sized wild type, until the insert size reached 1 kb. These data indicate that the maximum effective packaging capacity of AAV is approximately 900 bp larger than wild type, or 119%. Furthermore, it is demonstrated that these large AAV genomes are able to latently infect cells by chromosomal integration as does wild‐type AAV. These data suggest that therapy vectors carrying a foreign gene of 900 bp or less can be generated from AAV, by ligation into non‐essential locations, and result in a recombinant AAV virus with a fully wild‐type phenotype. Such wild‐type‐plus AAV vectors will have both advantages and disadvantages over defective recombinant AAV virus — the most important advantages being the ease in which high titers of infectious virus can be generated and the ability to specifically integrate within chromosome 19. Once the concern subsides over the presence of wild‐type AAV in clinical applications, wild‐type AAV vectors may find specific application niches for use in human gene therapy.

Outcomes of newborns with gastroschisis: The effects of mode of delivery, site of delivery, and interval from birth to surgery

OBJECTIVEnOur purpose was to determine the impact of delivery site, delivery mode, and delivery-to-surgery interval on outcomes for neonates diagnosed with gastroschisis.nnnSTUDY DESIGNnData were obtained retrospectively by chart review on 56 newborns diagnosed with gastroschisis. Outcome measures examined included primary closure, days to enteral feeding, days in intensive care, total length of stay, and hospital charges.nnnRESULTSnInborn infants experienced fewer days to enteral feeding (p < 0.01)., shorter total lengths of hospital stay (p < 0.01), and lower hospital charges (p < 0.01). Newborns delivered by cesarean section tended to have longer lengths of stay (p = 0.07), greater hospital charges (p = 0.06), and significantly longer lengths of stay in intensive care (p = 0.05). Shorter intervals from delivery to surgery were observed for inborn neonates (p < 0.01) and for those delivered by cesarean section (p < 0.05). No relationships between hours from delivery to surgery and neonatal outcomes were observed.nnnCONCLUSIONSnDelivery at a regional center is associated with improved outcomes, whereas cesarean deliveries were associated with worse outcomes. We observed no salutary effect related to the interval between delivery and initial surgical repair.

Antenatal Betamethasone Compared With Dexamethasone (betacode Trial): A Randomized Controlled Trial

OBJECTIVE: To compare betamethasone with dexamethasone in terms of effectiveness in reducing perinatal morbidities and mortality among preterm infants. METHODS: We enrolled 299 women at risk for preterm delivery in a double-blind, placebo-controlled, randomized trial of antenatal betamethasone compared with dexamethasone at Stony Brook University Hospital from August 2002 through July 2004. We excluded women with clinical chorioamnionitis, fetal structural and chromosomal abnormalities, prior antenatal steroid exposure, and steroid use for other indications. Statistical analysis was performed in accordance of the intention-to-treat principle. RESULTS: There were no significant differences between the groups with regard to baseline characteristics. The rate of respiratory distress syndrome, need for vasopressor therapy, necrotizing enterocolitis, retinopathy of prematurity, patent ductus arteriosus, neonatal sepsis, and neonatal mortality were not significant different between the groups. However, the rates of intraventricular hemorrhage (6 of 105 [5.7%] compared with 17 of 100 [17.0%], relative risk [RR] 2.97, 95% confidence interval [CI] 1.22–7.24, P=.02) and any brain lesion (7 of 105 [6.7%] compared with 18 of 100 [18.0%], RR 2.7, 95% CI 1.18–6.19, P=.02) were significantly lower in neonates exposed to dexamethasone compared with betamethasone. The absolute risk reduction in the rate of intraventricular hemorrhage was 11.3 % ( 95% CI 2.7–11.9%), and the number needed to treat was 9 (95% CI 5–37) in favor of dexamethasone. CONCLUSION: Betamethasone and dexamethasone are comparable in reducing the rate of most major neonatal morbidities and mortality in preterm neonates. However, dexamethasone seems to be more effective in reducing the rate of intraventricular hemorrhage compared with betamethasone. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00418353 LEVEL OF EVIDENCE: I

CA 125 in tissues and amniotic fluid during pregnancy

CA 125 was assayed in amniotic fluid and tissue extracts by immunoradiometric assay, and immunohistochemical studies were performed on paraffin-embedded sections of endometrium, decidua, and fetal membranes with the monoclonal antibody OC 125 used as primary antibody. The concentration of CA 125 in amniotic fluid changes during pregnancy so that levels of 800 to 1000 U/ml are found before 12 weeks. Thereafter, levels of 4000 to 10,000 U/ml are detected routinely. As term approaches, amniotic fluid CA 125 concentrations fall to a range of 1000 to 2000 U/ml. Levels of CA 125 in tissue extracts of secretory endometrium and decidua were 65,000 and 29,500 U/gm of tissue, respectively. CA 125 was readily detected on the apical surfaces of glandular epithelium and in the secretions of endometrial glands obtained throughout the menstrual cycle. It was also detected in the lumina of decidualized glands throughout pregnancy. No antigen was detectable within glandular epithelial cells. We have previously reported high concentrations of CA 125 in chorionic tissue extracts (42,000 U/gm) and low concentrations in amniotic tissue extracts (275 U/gm). In contrast to those findings, immunohistochemical techniques detected CA 125 within the intercellular canaliculi that surround amniotic epithelial cells but not in chorion. We conclude that the likely source of amniotic fluid CA 125 is the decidua and that it gains access to the amniotic fluid via the intercellular canalicular system that traverses the amniotic epithelium.

Distribution of CA 125 in embryonic tissues and adult derivatives of the fetal periderm

New murine monoclonal antibodies to a partially purified CA 125 antigen were developed and identified as M 2 and M 11. With immunohistochemical techniques, these new antibodies and OC 125 antibody were used to search for CA 125 in embryonic tissues and adult apocrine sweat glands and mammary glands. The embryonic skin, the periderm, expressed CA 125 antigen and its adult derivatives, the mammary glands and apocrine sweat glands, expressed CA 125 while in the active state of secretion. In a 6-week-old formalin-fixed and paraffin-embedded ectopic embryo specimen, antibodies M 2 and M 11 recognized CA 125 in the periderm, the notochord, the myocardium, the pericardium, the gastroenteric tract, enteric duct remnants in the umbilical cord (vitelline and allantoic ducts), the mesonephric duct, and the amnion. OC 125 staining of these formalin-fixed specimens was either very faint or absent. In a formalin-fixed and paraffin-embedded specimen of apocrine sweat glands from the axilla, antibodies M 2 and M 11 detected CA 125 antigen intracellularly in the secretory cells. Again no staining was observed with OC 125 antibody. In a frozen and acetone-fixed specimen of lactating mammary glands, antibodies M 2 and OC 125 detected CA 125 antigen intraductally. Colostrum and milk collected from 25 mothers at various stages post partum had mean CA 125 levels of 34,213 U/ml in colostrum, 1469 U/ml at 3 to 7 days, and 105 U/ml at 5 to 26 weeks.

Aortic stenosis, cesarean delivery, and epidural anesthesia.

A 23-year-old female was referred to the University of Arkansas for Medical Sciences at 32 weeks gestation with a history of aortic stenosis following aortic valve replacement. Evaluation by echocardiography showed an approximately 90 mmHg transvalvular pressure gradient. Pregnancy progressed to 36 weeks gestation without problem, at which time the patient underwent cesarean section with lumbar epidural anesthesia. Invasive hemodynamic monitors were used to assess cardiac performance and as a guide for anesthetic management. The impact of aortic stenosis on pregnancy is discussed, as are management aspects of lumbar epidural anesthesia in such patients.

A rapid and specific enzymatic method for the quantification of phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol in amniotic fluid

An enzymatic procedure for the quantification of phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol in amniotic fluid is described. By use of this method, choline and glycerol are released enzymatically from phosphatidylcholine and phosphatidylglycerol, respectively, in reactions catalyzed by phospholipase D. The hydrogen peroxide generated from choline (by the action of choline oxidase) and from glycerol (by the combined action of glycerokinase and glycerol-3-phosphate oxidase) is quantified spectrophotometrically after the addition of horseradish peroxidase, aminoantipyrine, and phenol. The phosphatidylcholine concentration in amniotic fluid was found to be approximately 10 to 30 nmol/ml between the twenty-third and thirty-sixth week of gestation and increased sevenfold to eightfold between the thirty-seventh week and term. The procedure can be modified for the quantification of disaturated phosphatidylcholine. The concentration of phosphatidylglycerol was approximately 2 nmol/ml between the twenty-third and thirty-sixth week and increased to 10 to 20 nmol/ml between the thirty-seventh and forty-first week of pregnancy. Since contamination of amniotic fluid with bile pigments does not interfere with either assay, the phosphatidylcholine and phosphatidylglycerol concentrations in amniotic fluid can be determined in samples that are contaminated with meconium.

Increase in the plasma levels of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during normal pregnancy*

Abstract The influence of pregnancy on the circulating concentrations of atrial natriuretic factor, the 28 amino acid carboxy (C)-terminal end of the 126 amino acid atrial natriuretic factor prohormone, and the amino (N)-terminus of the prohormone was studied with three specific radioimmunoassays recognizing: (1) atrial natriuretic factor (i.e., amino acids 99 through 126), (2) the 98 amino acid N-terminus, and (3) amino acids 31 through 67 from the midportion of the N-terminus of the prohormone. Plasma atrial natriuretic factor in normal pregnant women gradually increased as pregnancy progressed, with the mean -t SEM being 58 ± 4 pg/ml in the first trimester, 74 ± 5 pg/ml in the second trimester, and 89 ± 7 pg/ml in the third trimester. Likewise, proatrial natriuretic factor 31 through 67 increased from 1421 ± 76 pg/ml (first trimester) to 1509 ± 84 pg/ml (second trimester) to 1758 ± 83 pg/ml in the third trimester, whereas the whole N-terminus of the prohormone increased from 1804 ± 98 pg/ml (first trimester) to 1909 ± 111 pg/ml (second trimester) to 2160 ± 79 pg/ml in the third trimester. These results suggest that release of the N-terminus of the prohormone, as well as atrial natriuretic factor, increases with the rise in blood volume associated with a normal pregnancy. The circulating concentrations of both the C-terminus and N-terminus of the atrial natriuretic factor prohormone increased further in the 48 hours after delivery. Because both the C-terminus and N-terminus of the atria[ natriuretic factor prohormone contain diuresis-producing peptides, these results suggest that postpartum diuresis may be mediated by these peptides.

Epidermal Growth Factor Enhances Secretion of the Ovarian Tumor-Associated Cancer Antigen CAI25 From the Human Amnion WISH Cell Line

OBJECTIVE : We studied the relation between epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) and CA125 production in WISH cells. METHODS: We investigated quantitatively and immunohistochemically EGF-stimulated CA125 release from WISH cells and the effect of EGF on CA125 phosphorylation. RESULTS: Immunohistochemical staining demonstrated that CA125 and EGFR expression on the plasma membrane of the WISH cells was closely correlated with cell density. The WISH cell monolayers (day 4) stained for CA125 in both the cytoplasm and plasma membrane. By day 8, cells began to form clumps in the surrounding monolayer that were positive for membrane- associated CA125 and EGFR, while the monolayer was almost negative for both molecules. Four-day and 8-day cells exposed to EGF demonstrated a loss of both CA125 and EGFR staining. Epidermal growth factor increased the secreted CA125 levels by 50% in day-4 cells but had no effect on day-8 cells. CA125 from WISH cells was phosphorylated, and EGF further enhanced this phosphorylation. CONCLUSION: Release of CA125 from the plasma membrane is enhanced by EGF and may be regulated by phosphorylation. (J Soc Gynecol Invest 1994;1:89-96)

Second-trimester ultrasonography: determinants of visualization of fetal anatomic structures.

Little information has been published regarding rates of visualization of fetal anatomic structures or factors affecting visualization in unselected patients. We prospectively studied these points by scoring visualization of intracranial, spinal, cardiac, ventral wall, umbilical cord, stomach, bladder, and genital anatomy in 573 consecutive midtrimester scans in which no fetal anomalies were seen. Each feature was scored 0, 1, or 2, corresponding to poor, adequate, or optimal visualization, and these eight numbers were summed for an overall visualization score. Overall visualization fell abruptly beyond maternal weight 165 pounds (p less than 0.001) and rose with advancing gestational age (p less than 0.001). Placental location did not significantly influence visualization. Overall, an adequate screening scan, defined as adequate visualization of central nervous system, heart, stomach, ventral wall, bladder, and cord was obtained in 51% of scans, and optimal visualization of each of these in only 9% of cases. Factors that might improve anatomic visualization and the implications of these results for the use of ultrasonography in screening for congenital anomalies are discussed.