Biomedical subjects
J G Quirk
Publications and source records attributed to J G 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.
Human papillomavirus is more prevalent in first trimester spontaneously aborted products of conception compared to elective specimens.
In this study the possible role of human papillomaviruses (HPV) in spontaneous abortions is addressed by assaying for HPV DNA in first trimester spontaneous and electively aborted products of conception materials enriched for chorionic villi. The presence of HPVs was measured by polymerase chain reaction (PCR) amplification and DNA dot blot hybridization using an internal probe. The "broad spectrum" HPV primers were directed to amplify E6/E7 junction sequences, while the probe was of an HPV-16 sequence with significant homology to HPV-6/11. The quantity and quality of isolated DNA was also analyzed and compared by observing the PCR amplification of a cellular sequence from the human beta-globin gene. Fifteen of the 25 spontaneous samples (60%) were found to be positive for HPV E6/E7 sequences. In comparison, only 3 of the 15 elective samples (20%) were positive. This is the first study of HPV in fetal materials to incorporate material from elective abortions as a control group. Although confounding contamination from the cervix and vagina can't be ruled out, these data are significant and strongly suggest that HPVs are elevated in spontaneously aborted products of conception. Furthermore, these results suggest the possibility that HPVs may be etiologic agents of at least some spontaneous abortions.
Multiple cellular proteins are recognized by the adeno-associated virus Rep78 major regulatory protein and the amino-half of Rep78 is required for many of these interactions.
Adeno-associated virus (AAV) encoded Rep78 is a multi-functional protein which is required for AAV DNA replication, is able to regulate both AAV and heterologous gene expression at the transcriptional level, and appears necessary for site specific integration of AAV DNA into human chromosome 19. By comparison with the analogous replication protein of the polyomaviruses, large T antigen, it seemed likely that Rep78 would interact with cellular proteins to carry out at least some its functions. This study demonstrates that Rep78 is able to interact with multiple cellular proteins, from cellular extracts as measured by West(far)-western, coimmunoprecipitation, and Rep78-affinity chromatography analysis. Eight cellular proteins of approximately 180, 140, 120, 95, 75, 55, 45, and 35 kDa (+/- 10%), were observed to bind Rep78 in all three assay systems. Two others, of 30 and 24 kDa, were observed in two of three assay systems. Furthermore, using truncated Rep78 proteins, it is demonstrated that the amino-terminus is required for most Rep78-cellular protein interactions. However, the extreme carboxy-terminus of Rep78 was found to be all that is required for binding to the 55 kDa cellular protein.
CA125 phosphorylation is associated with its secretion from the WISH human amnion cell line.
Evidence is presented suggesting that CA125 is a serine and/or threonine phosphoprotein and that its secretion from the human amnion WISH cell line is closely linked to its phosphorylation. It is also indicated that regulation of CA125 secretion requires protein(s) tyrosine phosphorylation. WISH cells treated with a tyrosine phosphatase inhibitor, vanadate/ H2O2, resulted in increased levels of CA125 secretion. Exposure of vanadate-treated cells to epidermal growth factor further enhanced this secretory activity. Immunohistochemistry of vanadate-treated cells resulted in a substantial increase in not only cytoplasmic tyrosine phosphoproteins but also in membrane-associated CA125 when stained with the PY20 anti-phosphotyrosine and M11 anti-CA125 monoclonal antibodies, respectively. M11 immunoprecipitation of CA125 from cells labelled with [32P]-orthophosphate was analyzed by SDS-PAGE and autoradiography. Immunoprecipitates from cell lysates demonstrated that a phosphoprotein of > 200 kD was isolated and immunoreacted with both the OC125 and M11 anti-CA125 monoclonal antibodies by Western blotting. CA125 immunoprecipitated from vanadate-treated cells showed a marked increase in cell-associated CA125 phosphorylation. Although CA125 could be immunoprecipitated from WISH cell media incubated with [32P]-orthophosphate in the presence or absence of vanadate as detected by Western blotting, autoradiographic analysis of the Western blots revealed no [32P]-labelled CA125 co-migrating with the 200-kD plus molecule detected by M11. When the PY20 anti-phosphotyrosine monoclonal antibody was used as the probe, no tyrosine-phosphorylated CA125 was detected in cell lysates.
Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno-associated virus DNA replication.
The adeno-associated virus (AAV) terminal repeats (TR) are cis required, and the AAV encoded Rep78 protein is trans required, for AAV DNA replication. The Rep78 protein recognizes and interacts with at least three regions within the TR DNA. The major binding site, with the highest affinity for Rep78 binding, is within the TR stem (nt 36-16) and includes the 'core' GAGC trimer (GAGC3, nt 33-22; Fig. 2) sequence. In this study mutations were made within the GAGC trimer and these mutants assayed for their ability to allow for AAV double stranded (ds DNA, prepackaging DNA replication), and single stranded DNA (ss DNA, due to virion packaging) replication. Here, it is shown that when the two inside GAGC motifs are mutated, with only motif no. 1 left intact (see Fig. 2), the resulting AAV (mutA) genome was significantly defective for both ds DNA (17% of wild type) and ss DNA (9%). If the TRs contained only the two outside motifs intact (mutB), motifs no. 1 and 2, the AAV genome had a significant but reduced level of both ds (50%) and ss (34%) DNA replication. Finally, if only the middle motif no. 2 was mutated, with motifs no. 1 and 3 left intact (mutC), the resulting DNA replication for both ds and ss forms was essentially wild type (80% that of wild type). These data suggest that the GAGC trimer plays a role in AAV DNA replication, and that GAGC motif no. 3 is the most important of the three motifs for both ds and ss DNA replication.
Outcomes of newborns with gastroschisis: the effects of mode of delivery, site of delivery, and interval from birth to surgery.
OBJECTIVE: Our purpose was to determine the impact of delivery site, delivery mode, and delivery-to-surgery interval on outcomes for neonates diagnosed with gastroschisis. STUDY DESIGN: Data 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. RESULTS: Inborn 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. CONCLUSIONS: Delivery 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.
Duplication and deletion of chromosome band 2(p21p22) resulting from a familial interstitial insertion (2;11)(p21;p15).
Routine amniocentesis for advanced maternal age led to the prenatal diagnosis of a fetus with a karyotype of a 46,XX,del(2)(p21p22). At delivery the baby had holoprosencephaly as the major clinical finding, which has been associated with a deletion of band 2p21 in several other case reports. Chromosome studies of the parents showed a normal 46,XY karyotype in the father, and a balanced interstitial insertion 46,XX dir ins (11;2)(p15.1;p21p22) in the mother. Subsequent chromosome studies of other relatives documented a 23-year-old half-brother of the proposita with a partial trisomy for the segment deleted in the proposita. The half-brother showed the derivative chromosome 11 from the mother, resulting in a 46,XY,der(11)dup(2)(p21p22) karyotype. Major clinical findings include short stature, mild developmental delay, and behavior abnormalities. A half-sister of the proposita is also a balanced carrier of the dir ins (11;2) (p15.1;p21p22.2). The association of the deletion chromosome band 2p21 and the clinical finding of holoprosencephaly is further supported by the findings in this family.
Ultrasound evaluation of abdominal pregnancy.
The ultrasound diagnosis of abdominal pregnancy continues to be difficult in spite of recent advances in ultrasound technology. The principal impediment is a low suspicion for the diagnosis because of its relatively asymptomatic nature and the lack of specificity of symptoms when present. In the early days of sonography when static B-mode scanning was used, a "gestalt" diagnosis was suggested, based on one's ability to have an overall perspective of the intrauterine contents. With conversion to exclusive real time imaging and the inherent limitation of the field of view, this "gestalt perception" is no longer possible. It is therefore necessary to reassess our criteria based on this change and determine those that remain reliable for diagnosis.
Characterization of CA 125 synthesized by the human epithelial amnion WISH cell line.
CA 125 has been established as an important tumor marker for monitoring patients diagnosed with nonmucinous ovarian cystadenocarcinoma although it has also been shown to be expressed by other carcinomas, normal epithelial tissues, and fetal tissues. Current evidence implicates a role for CA 125 during early fetal development. The human epithelial amnion WISH cell line is a known secretor of CA 125. WISH cells have been investigated as a model system to characterize the structure of cell-associated and secreted CA 125 of fetal origin. CA 125 secretion was maximal in confluent monolayers of WISH cells where it averaged 2,081 units/ml/24 h. Secretion ranges from 600 to 900 units/10(6) cells/24 h. [35S]-Methionine-labelled CA 125 can be detected by 4 h and reached maximal levels of radioactive incorporation in tissue culture medium by 12 h when analyzed by immunoprecipitation with the M11 anti-CA 125 monoclonal antibody and SDS-PAGE, followed by autoradiography. Both cycloheximide and actinomycin D inhibited CA 125 synthesis. CA 125 was demonstrated to incorporate [3H]-galactose but the level of radioactive incorporation was greatly reduced when WISH cells were incubated in the presence of phenyl N-acetyl-alpha-D-galactosaminide (an inhibitor of O-linked glycosylation) or monensin (an inhibitor of intracellular protein transport within the Golgi complex). Treatment of WISH cells with tunicamycin (an inhibitor of N-linked glycosylation) only slightly decreased label incorporation.
Perinatal lethal conditions: the effect of diagnosis on decision making.
OBJECTIVE: To identify factors influencing pregnancy management decisions following identification of a perinatal lethal condition. METHODS: One hundred thirty pregnancies with perinatal lethal conditions diagnosed before 24 weeks' gestation were examined. Information collected included demographic data, estimated gestational age at presentation, referral indication, nature of the defect, and performance of autopsy. RESULTS: Eighty-seven families elected to abort affected pregnancies and 43 elected to continue. Demographic factors did not influence decision making, nor did gestational age at diagnosis or referral indication. When comparing the diagnosis of one lethal condition with diagnoses of all other lethal conditions, pregnancies with a central nervous system defect or severe urinary tract defect were more often aborted; those with unexplained severe oligohydramnios and twin pregnancies in which at least one twin was affected were more often continued. Autopsy was obtained much more often in pregnancies that were aborted than in those that were continued. CONCLUSION: The type of defect correlates well with the pregnancy management decision. It is important to consider the type of malformation, certainty of the diagnosis, and level of medical understanding when counseling patients after the diagnosis of a lethal fetal defect. Because many patients will continue pregnancies diagnosed with a perinatal lethal condition, the physician should convey understanding and acceptance of a decision not to abort such a pregnancy. The importance of follow-up testing, including autopsy when appropriate, should be stated clearly.
MR imaging in high-risk obstetric patients: a valuable complement to US.
The accuracy of prenatal diagnosis has become increasingly critical in the field of high-risk obstetrics. Although ultrasound (US) provides adequate information in most cases and continues to be the initial prenatal examination of choice, there are instances in which the results of the US study may be equivocal. The role of magnetic resonance (MR) imaging was explored in 27 selected patients with various indications to determine its effectiveness as a complement to US. MR imaging was most helpful in the diagnosis of extrauterine gestation, evaluation of placental position, determination of extent or nature of masses associated with pregnancy, and differentiation between diaphragmatic hernia and a thoracic mass. Although MR imaging did not add information that affected the accuracy of the diagnosis of oligohydramnios, in all other cases it provided an extra dimension in diagnosis by showing clearer anatomic relationships in the pelvis. It has proved to be a valuable complement to an equivocal US study.
Alteration in age-specific risks for chromosomal trisomy by maternal serum alpha-fetoprotein and human chorionic gonadotropin screening.
Maternal serum screening for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG) increases the detection rate of Down's syndrome (DS) pregnancies. To estimate the risk of a DS pregnancy for a particular woman, Wald et al. combine a trivariate function of AFP, hCG, and unconjugated oestriol with age-specific risk. Calculation of independent likelihood ratios (LRs) for AFP and hCG has allowed us to examine the predictive value of each test alone and the combination. AFP and hCG were measured in stored serum samples from 672 normal, 8 trisomy 21 (DS), 9 trisomy 18, and 2 trisomy 13 pregnancies. AFP and hCG multiples of the median (MOM) were calculated for each sample. The LRs for AFP MOM and hCG MOM were calculated and combined with age-specific risk. Of eight DS pregnancies, six had increased risk based on age and AFP. Addition of hCG detected two additional DS pregnancies. Of nine trisomy 18 pregnancies, four (44 per cent) had hCG MOM under 0.25. Three out of nine would have been classified as high risk by AFP, but none by combined AFP and hCG. Amniocentesis would have been recommended in 74 per cent of aneuploid pregnancies if both age and serum screening were used. Abandonment of amniocentesis based on age alone would have excluded two abnormal pregnancies from detection. Screening programmes should note that combined risk figures are specific for DS and do not include other trisomies. Detection of other trisomies requires inclusion of low hCG level as a discriminator and continuation of age-based testing.
New monoclonal antibodies identify the glycoprotein carrying the CA 125 epitope.
CA 125 is an antigenic determinant located on the surface of ovarian carcinoma cells and elevated in the serum of greater than 90% of patients with carcinoma. The antigen, derived from the ovarian epithelium, has been described as a mucinlike glycoprotein greater than 200 kd. To date little is known of the metabolic regulation or expression of this antigen in either normal or neoplastic tissues. New monoclonal antibodies that we describe here recognize both unique and similar epitopes to OC 125. These reagents may allow for a more complete definition of the structure and expression of the CA 125 complex. These antibodies recognize high-molecular weight (greater than 200 kd) subspecies and a lower-molecular-weight (68 kd) subspecies of the antigen and identify it in the cytoplasm and the extracellular matrix of CA 125-producing cells.
The N-terminal and C-terminal portions of the atrial natriuretic factor prohormone increase during preeclampsia.
The influence of preeclampsia on the circulating concentrations of the 28-amino-acid carboxy terminus (C-terminus) (i.e., atrial natriuretic factor) and the amino terminus (N-terminus) of the 126-amino-acid atrial natriuretic factor prohormone (pro ANF) was studied in the third trimester with the use of three specific radioimmunoassays that recognize: (1) atrial natriuretic factor (i.e., amino acids 99 to 126), (2) the whole 98-amino-acid N-terminus, and (3) amino acids 31 to 67 from the midportion of the N-terminus of the prohormone. The C-terminus was significantly increased (p less than 0.001) in the third trimester in women with preeclampsia, the mean +/- SEM of 15 subjects was 150 +/- 7 pg/ml versus 89 +/- 7 pg/ml in the third trimester in 12 women during normal pregnancies and 65 +/- 2 pg/ml in 19 healthy nonpregnant women. The whole 98-amino-acid N-terminus, likewise, was significantly increased (p less than 0.001) in women with preeclampsia to 4706 +/- 629 pg/ml versus 2160 +/- 79 pg/ml in women in the third trimester of normal pregnancies and versus the circulating concentration of 1847 +/- 127 pg/ml in healthy nonpregnant women. ProANF 31 to 67 mean circulating concentration in preeclampsia was 4638 +/- 725 pg/ml, which was also significantly (p less than 0.001) increased compared with its mean circulating concentration in the third trimester of normal pregnancy of 1758 +/- 83 pg/ml or that in healthy nonpregnant women (1400 +/- 105 pg/ml). The circulating concentrations of both the N-terminus and C-terminus of the atrial natriuretic factor prohormone decreased within 24 hours after delivery in contrast to a normal pregnancy in which they both increase post partum. These results indicate a marked difference in the metabolism of both the N-terminus and the C-terminus of the atrial natriuretic factor prohormone in women with preeclampsia versus that in women with normal pregnancies or that in healthy nonpregnant women.
How do perinatologists manage preeclampsia?
The members of the Society of Perinatal Obstetricians were surveyed regarding management of preeclampsia, with focus on drug therapy, use of invasive monitors, and both general policies and treatment of hypothetical cases of preterm severe preeclampsia. There was agreement that magnesium sulfate should be given to all patients with preeclampsia during labor and postpartum and that blood pressure should be held to about 160/105 mmHg. The drugs of choice for control of blood pressure were hydralazine, alpha-methyldopamine, and cardioselective beta-blockers. Most perinatologists use invasive monitors only for specific indications, but a substantial minority use either arterial lines or central venous pressure monitors routinely in severe preeclampsia. There was no consensus with respect to management of preterm, severe preeclampsia, but even among the 49% of respondents who volunteered an unequivocal policy of "deliver regardless of gestational age," over three fourths would hospitalize and observe in selected cases meeting American College of Obstetrics and Gynecology criteria for severe preeclampsia.
Landry Guillain-Barré Strohl syndrome in pregnancy: report of three cases treated with plasmapheresis.
Landry Guillain-Barré Strohl syndrome complicating pregnancy is a rare occurrence, with only 31 cases previously reported in the obstetric literature. Cytomegalovirus as a potential etiologic agent has been documented in only two of these cases. In the past, symptomatic treatment of pregnant patients with severe and progressive symptoms has been associated with an unacceptably high rate of maternal morbidity and mortality. Recent studies have shown convincingly that plasmapheresis is the treatment of choice for nonpregnant patients acutely ill with Guillain-Barré syndrome, but data regarding its use in pregnancy are limited. We report three patients, two of whom had positive cytomegalovirus titers, in whom aggressive plasmapheresis prevented the need for ventilatory support and its inherent increase in maternal morbidity and mortality.
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.