Envelope-dependent transactivation by the retroviral oncoprotein v-Rel is required for efficient malignant transformation of chicken spleen cells.

The retroviral oncoprotein v-Rel is a chimeric protein that has 11 helper virus-derived Envelope (Env) amino acids (aa) at its N terminus. Within these N-terminal Env aa of v-Rel there are three aa substitutions compared to the Rev-A helper virus Env. These aa substitutions have previously been shown to impart a number of unique properties onto v-Rel, including increased transforming and transactivating ability. In this study, we have analysed the sequence requirements for the Env aa to influence several properties of v-Rel. Phe residues at aa 3 and 9 are critical for an N-terminal transactivation function of v-Rel, and the analysis of several Env mutants demonstrates that transactivation ability parallels the transforming ability of v-Rel. Substitutions of conservative aa, such as leucine and tyrosine, for Phe 3 and 9 are tolerated for transactivation in chicken embryo fibroblasts and for transformation of chicken spleen cells. In contrast, the substitution of 10 Phe residues at the N terminus of v-Rel does not enable transactivation, indicating that a distinct structure surrounding Phe-3 and Phe-9 is essential for v-Rel function. We also show that the addition of the v-Rel Env aa to the N terminus of human c-Rel can enable it to activate transcription. Taken together, these results indicate that Phe residues at positions 3 and 9 have been selected for their ability to enhance the oncogenicity of v-Rel by increasing its ability to activate transcription.

Differential increases in AFP, hCG, and uE3 in twin pregnancies: impact on attempts to quantify Down syndrome screening calculations.

Since the advent of multiple marker screening (MMS) for Down syndrome (DS) risk calculations, limitations for twins have been apparent. Recent attempts have been made to extrapolate mathematically singleton risks to twins. Here we investigate the pattern of levels among AFP, hCG, and uE3 in twins. MMS screening data from 4,443 twin pregnancies were compared to those from 258,885 singletons from 14-21 weeks of gestational age during a 3-year period (1992-1994) in our laboratory. Medians were determined for singletons and twins, and the ratios of twins to singletons were derived. Median AFP levels for twins are approximately double those of singletons, but median increases for hCG and uE3 are less than double. The data were divided further by ethnic groups (white, African American, Asian, and Hispanic), among which there were significant variations in medians, but not in the ratios of twins to singletons. The increased serum levels of different markers in twins are not consistent across analytes, possibly reflecting independent development of different compartments. Such differences mean that a mere mathematical conversion of singleton DS risks would be imbalanced among the analytes and cannot be applied reasonably to twins. Ethnic-specific databases are as important in twins as they are in singletons.

Wide variation in maternal serum alpha-fetoprotein reports in one metropolitan area: concerns for the quality of prenatal testing.

The variation in reported laboratory values, medians, and confounding variables used in the interpretation of alpha-fetoprotein (AFP) values from 14 laboratories servicing the Detroit-Metropolitan area was assessed by reviewing the laboratory report forms. The wide variation found in reported values, medians, and use of correction factors makes interpretation of results difficult and inaccurate. The need for large data bases for continuous correction of medians, uniform use of correction factors, and clinical decision making is emphasized.

The inadequacy of the current correction for maternal weight in maternal serum alpha-fetoprotein interpretation.

The application of correction factors for maternal serum alpha-fetoprotein (MSAFP) is expected to increase the accuracy of this screening tool. Correction for maternal weight compensates for the dilution effect of a larger plasma volume in women of greater weight. We analyzed the effect of a previously published linear correction formula for weight on the frequency of abnormal MSAFP results. Serum samples from 8276 patients were studied for AFP and were grouped according to maternal weight in 50-lb increments. Abnormal results were defined as 0.4 or less or 2.5 or more multiples of the median for gestational age. Without correction, the highest rate (15%) of low MSAFP results was obtained in the obese population. Conversely, the rate of elevated MSAFP was highest (3.8%) in the lowest maternal weight group. Correction for weight up to 250 lb significantly increased the rate of abnormally high results in the obese group, indicating an overcorrection effect. The number of amniocenteses indicated for abnormal MSAFP was reduced by about 9% with weight correction. We suggest that linear correction of serum AFP results is adequate for maternal weights up to 200 lb. Results in women weighing more than this upper limit should be corrected as if the weight were 200 lb only. This balances the frequency of abnormal results in all weight groups and maintains the reduction in number of procedures performed.

Counseling for low maternal serum alpha-fetoprotein should emphasize all chromosome anomalies, not just Down syndrome.

In common usage, a low maternal serum alpha-fetoprotein (MSAFP) value is associated with an increased risk of Down syndrome. We have performed amniocenteses for the indication of age-adjusted low MSAFP in 1154 patients and found 13 chromosomally abnormal conceptions. Autosomal trisomies were detected in half the cases. Additional abnormalities included sex-chromosome aberrations, deletions, or triploidy in proportions consistent with those seen in an advanced-maternal-age population. Patients with low serum AFP should be counseled that not only Down syndrome, but other aneuploidies as well, may be diagnosed. The risk quoted should be that of all chromosomal abnormalities, which is about twice the risk calculated for Down syndrome.

Race-ethnicity-specific variation in multiple-marker biochemical screening: alpha-fetoprotein, hCG, and estriol.

OBJECTIVE: To identify any race-ethnicity-specific differences in serum alpha-fetoprotein (AFP), hCG, and unconjugated estriol (E3) levels in women between 14 and 21 weeks' gestation. METHODS: Data from the 3-year period 1992-1994 were analyzed from 208,257 women who had AFP screening, of whom 155,142 also had hCG and 62,121 also had E3 screened, between 14 and 21 weeks' gestation. Subjects were categorized into four groups: white, black, Asian, and Hispanic. RESULTS: There was a consistent pattern of analyte differences across gestational ages. Levels for AFP were generally higher in Asian and black women than in Hispanic and white women (median AFP at 16 weeks-31.2, 30.9, 27.4, 27.3, respectively), and levels of hCG and E3 were highest in Asians (hCG at 16 weeks-34.7, 30.3, 28.2, 26.8, respectively). Weight correction for AFP, hCG, and E3 levels did not compensate for the ethnic differences. CONCLUSIONS: Because hCG and E3 demonstrate the same general pattern of differences as AFP among ethnic groups, averaging values for all ethnic groups tends inappropriately to lower calculated Down syndrome risks for black and Asian women. Additionally, the slopes of the curves are not parallel, such that separate data bases are preferable to multiplicative correction factors. Separate data bases should be used in laboratories with volume sufficient to permit the establishment of race-ethnicity-specific regressions. Use of separate data bases should result in more accurate screening.

Similarity of insulin-dependent diabetics' and non-insulin-dependent diabetics' levels of beta-hCG and unconjugated estriol with controls: no need to adjust as with alpha-fetoprotein.

OBJECTIVE: To determine if the same systematic alteration of serum values seen for alpha-fetoprotein (AFP) in diabetic patients is also seen for beta-hCG and unconjugated estriol (uE3). METHODS: Serum AFP, beta-hCG, and uE3 results were obtained in 18,639 patients for whom complete follow-up information was obtained. Patients were divided into euglycemic (n = 18,088), insulin-requiring diabetics (n = 104), and non-insulin-requiring diabetics (n = 437), as well as by race. RESULTS: The 20% adjustment used for AFP appropriately corrects serum values. No such systematic variation is seen for either beta-hCG or uE3 by glycemic status or race. CONCLUSION: No adjustment for beta-hCG or uE3 is necessary for diabetes for biochemical screening programs.

Second-trimester biochemical screening.

The past years have seen considerable progress in the area of biochemical screening. Increasing data have now clearly shown the advantages of multiple markers, particularly beta-hCG over AFP alone. There continues to be considerable controversy over the best mathematic algorithm and which markers are best (e.g., beta-HCG, uE3, and so forth). There seems to be a plateau of detection frequencies at about 65% to 70% with current methodologies. Further work needs to be done, however, including some new approaches, if there is to be substantial improvement of screening sensitivity. The combination of biochemical with biophysical parameters as discussed elsewhere in this issue represents the next level of sophistication in the attempt to identify the highest proportion of abnormalities with the fewest false-positives.

Biochemical screening.

The goal of screening both low and high risk obstetrics populations for chromosomally abnormal fetuses continues to be developed in many centers worldwide. Advances in the past year have been multifocal in their approach--neural tube detection, the development of new markers for aneuploidy, and attempts to move screening into the first trimester.

Apolipoprotein A-IV. A determinant for binding and uptake of high density lipoproteins by rat hepatocytes.

To identify the role of a specific apoprotein other than apoE which might be responsible for the receptor-mediated uptake of high density lipoprotein (HDL) by rat hepatocytes, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was combined with rat apoE, apoA-I, or apoA-IV to form apoprotein-phospholipid complexes and the complexes were tested for their binding and uptake by primary rat hepatocytes. Apoprotein-POPC complexes were labeled with the specific fluorescent probe, 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine to monitor their uptake by cultured rat hepatocytes at 37 degrees C using digital fluorescence imaging microscopy or were labeled with 125I to study their binding to hepatocytes at 4 degrees C. POPC, either alone or with apoA-I, was not internalized by rat hepatocytes while complexes containing apoE or apoA-IV were taken up by the cells. Specific binding at 4 degrees C was demonstrated for apoE-free HDL, apoA-IV X POPC, and apoE X POPC but not for apoA-I X POPC. The binding of apoE-free HDL was inhibited by apoA-IV X POPC, apoE-free HDL, and apoA-IV + apoA-I X POPC but not by apoA-I X POPC. Binding of apoA-IV X POPC was inhibited by apoE-free HDL, apoA-IV X POPC, and apoA-IV + apoA-I X POPC, but not by apoE X POPC or apoE-enriched HDL. These data indicate that apoA-IV is a ligand responsible for the rat HDL binding to primary rat hepatocytes and that apoA-IV binds to a receptor site distinct from apoE-dependent receptors such as the apoB,E or chylomicron-remnant receptor.

Alpha-fetoprotein.

The past year has seen major challenges to existing maternal serum alpha-fetoprotein testing protocols for both neural tube defects and chromosomal anomalies. These challenges are reviewed along with the physiology of alpha-fetoprotein; the use of amniocentesis, ultrasonography, and additional serum markers in women with elevated alpha-fetoprotein levels; and epidemiologic implications of maternal serum alpha-fetoprotein screening.

Impact of abnormal second-trimester maternal serum single, double, and triple screening on patient choices about prenatal diagnosis.

The development of multiple-marker biochemical screening has increased the percentage of aneuploidies detected for all age groups and has also increased the abnormality/amniocentesis ratio from about 1 in 85 for maternal serum alpha-fetoprotein alone (single screening) to about 1 in 50 for either maternal serum alpha-fetoprotein plus human chorionic gonadotropin (double screening) or maternal serum alpha-fetoprotein combined with human chorionic gonadotropin and unconjugated estriol (triple screening). We evaluated the decisions to have or decline amniocentesis of 985 patients 'at risk' by either single, double, or triple screening, as multiple markers were phased in over a 3-year period. The patient acceptance of the procedure did not change (approximately 80%) either by actual risk or type of biochemical screening. The labeling of 'at risk' status is more important than actual numerical risks, and the patient perception of risk status must be considered in counseling.

Aneuploidy with neural tube defects: another reason for complete evaluation in patients with suspected ultrasound anomalies or elevated maternal serum alpha-fetoprotein.

Some recent reports have suggested that invasive testing is unnecessary when ultrasound either confirms or refutes a neural tube defect (NTD). However, counseling for recurrence risks and the possibilities for in utero therapy would be significantly altered by an aneuploid karyotype. We report our experience with 53 pregnancies affected by NTD in which we found 13.2% of these fetuses with abnormal chromosomes. In view of the higher than previously published incidence of aneuploidy, we believe that fetal karyotypes are essential in the evaluation of all fetuses with NTDs.

2nd-trimester maternal serum marker results are not altered by delayed analysis.

OBJECTIVE: The goal of the study was to evaluate the significance of delayed laboratory analysis of maternal serum alpha-fetoprotein, beta-subunit of human chorionic gonadotropin, and unconjugated estriol for prenatal screening. METHODS: Biochemical analysis of 30 consecutive biochemical screening specimens of maternal serum alpha-fetoprotein, beta-subunit of human chorionic gonadotropin, and unconjugated estriol was performed immediately upon arrival to the laboratory, 7 days later, and again 14 days after maternal blood was drawn. Differences among the results of the three sets of biochemical studies were evaluated by one-way analysis of variance for repeated measures. RESULTS: No significant differences were found among the results of immediate assays as compared with those at a 7- or a 14-day delay for all three biochemical markers. CONCLUSIONS: Our data suggest that up to a 14-day delay in the performance of the 2nd-trimester maternal serum biochemical screening assays will not alter the results significantly. The results of maternal serum screening are, thus, clinically valid even if the laboratory assays were performed several days after maternal blood was drawn.

Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.

Cardiac valves arise from endocardial cushions, specialized regions of the developing heart that are formed by an endothelial-to-mesenchymal cell transdifferentiation. Whether and to what extent this transdifferentiation is retained in mature heart valves is unknown. Herein we show that endothelial cells from mature valves can transdifferentiate to a mesenchymal phenotype. Using induction of alpha-smooth muscle actin (alpha-SMA), an established marker for this process, two distinct pathways of transdifferentiation were identified in clonally derived endothelial cell populations isolated from ovine aortic valve leaflets. alpha-SMA expression was induced by culturing clonal endothelial cells in medium containing either transforming growth factor-beta or low levels of serum and no basic fibroblast growth factor. Cells induced to express alpha-SMA exhibited markedly increased migration in response to platelet-derived growth factor-BB, consistent with a mesenchymal phenotype. A population of the differentiated cells co-expressed CD31, an endothelial marker, along with alpha-SMA, as seen by double-label immunofluorescence. Similarly, this co-expression of endothelial markers and alpha-SMA was detected in a subpopulation of cells in frozen sections of aortic valves, suggesting the transdifferentiation may occur in vivo. Hence, the clonal populations of valvular endothelial cells described here provide a powerful in vitro model for dissecting molecular events that regulate valvular endothelium.

Conformational properties of human and rat apolipoprotein A-IV.

Apolipoprotein A-IV has been isolated from four sources: human and rat lymph and plasma. Conformational properties of the rat and human apoA-IV in solution and denaturation changes induced by guanidine hydrochloride (Gnd X HCl) were studied using circular dichroic and fluorescence spectroscopy, and analytical sedimentation equilibrium ultracentrifugation. We have shown that both rat and human apoA-IV have similar secondary structure with negative maxima in the circular dichroic spectra at 222 nm and 207 nm. Furthermore, we have found no significant difference in the alpha-helical content of the apoA-IV from rat plasma (33%), rat lymph (37%), human plasma (35%), or human lymph (35%). Our denaturation studies with Gnd X HCl demonstrated reversibility and the fact that each apoA-IV had a tendency to self-associate in solution and the self-association could be disrupted by low concentrations of Gnd X HCl (less than or equal to 0.4 M). Unfolding of the secondary structure of each apoA-IV occurred at higher concentrations of Gnd X HCl (midpoint less than or equal to 1.0 M). The apparent free energy of denaturation of the four apoA-IV proteins calculated from changes in the circular dichroic spectra upon addition of increasing concentrations of Gnd X HCl varied in a range from 3.0 to 4.2 kcal/mol. The fluorescence experiments revealed that apoA-IV from all sources had a maximum fluorescence emission at 342.5 nm, which shifted to the red region upon addition of increasing concentrations of Gnd X HCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Logistic regression generated probability estimates for trisomy 21 outcomes from serum alpha fetoprotein and beta human chorionic gonadotrophin: simplification with increased specificity.

Traditional Gaussian approaches to biochemical screening make calculations very cumbersome. We have sought to develop a logistic regression mathematical model for the calculation of risk for trisomy 21 in multiple marker screening that would be more "user friendly." Biochemical screening data from 15,444 patients with confirmed normal outcomes and 28 with trisomy 21 were analyzed by logistic regression analysis (LRA) to produce a direct mathematical approach to risk calculation that combines the improvements in sensitivity and specificity already demonstrated by us with extreme value cumulative function surface fittings, but which can be extended for other exploratory variables. LRA yields a 71.4% sensitivity with a 95% specificity which is comparable to our previously published discriminant aneuploidy detection (DADs) approach; both are an improvement over the commonly used Gaussian approach of risk assessment. In our study population, LRA performed as well as DADs; both performed better than the Gaussian method, and LRA is a simpler approach.

Multiple marker screening in multifetal gestations: failure to predict adverse pregnancy outcomes.

The objective of the study was to assess whether the association known in singleton pregnancies of high maternal serum alpha-fetoprotein (AFP) or human chorionic gonadotropin (HCG) with adverse outcomes applies also to multifetal gestations. Maternal serum AFP and HCG were evaluated in 207 multifetal pregnancies. High values were defined as a maternal serum AFP of > 4.5 and a HCG of > 4.0 multiples of the median (MoM), with appropriate adjustments. Results were correlated with premature delivery, stillbirths, or pregnancy termination for fetal anomalies. There were 10 stillbirths, 7 terminations of pregnancy for fetal anomalies, and 66 premature deliveries in the study group. Maternal serum AFP was somewhat higher in abnormal pregnancies than in those with normal outcome (3.4 vs. 2.5 MoM, respectively, NS). A high AFP level was found in 6% of pregnancies with adverse outcomes and in 4% of uncomplicated gestations (NS). High HCG values, also observed in 5% of cases, were all associated with normal outcome. Multiple marker screening suggested an increased risk for aneuploidy in 9% of patients, all of whom were euploid on amniocentesis karyotypes. Maternal serum screening in multiple gestations is confounded by the differing contributions of fetuses, and abnormal results cannot reliably predict adverse pregnancy outcomes.

MOMs (multiples of the median) and DADs (discriminant aneuploidy detection): improved specificity and cost-effectiveness of biochemical screening for aneuploidy with DADs.

OBJECTIVE: Our purpose was to assess the efficacy of double- and triple-screening paradigms for Down syndrome and to develop a more logical, statistical approach to risk prediction that will decrease the cost of screening and allow the incorporation of new parameters appropriately weighted for their contribution. STUDY DESIGN: Data from 24,504 patients who had biochemical screening for Down syndrome by single (alpha-fetoprotein), double (alpha-fetoprotein, beta-human chorionic gonadotropin), or triple screening (alpha-fetoprotein, beta-human chorionic gonadotropin, unconjugated estriol) who had complete outcome information were analyzed by (1) existing gaussian-based methods, (2) the Glasgow ratio method, and (3) a new statistical approach (i.e., directly adjusted data sets for discriminant aneuploidy detection [DADs]) RESULTS: By use of individual risk-based thresholds for "at risk" status, both double and triple screening performed far better than single screening, but the percentages of patients at risk varied widely. When the percentages at risk were held constant, the sensitivity of double and triple screenings was similar, suggesting that there are no benefits of using estriol as a third marker. For 25,000 patients the use of only alpha-fetoprotein and beta-human chorionic gonadotropin would save about $500,000, with no decrease in sensitivity. With the DADs approach a statistically sound model giving more stable estimates was developed that permits each factor to be analyzed for its own explained proportion of variance and allows each parameter to have different weighting. For this data set the same sensitivity was seen with, conservatively, a 1% reduction in the percentage of patients at risk, which would reduce by 250 the number of amniocenteses, at a further savings of about $400,000. CONCLUSIONS: By use of existing methods, double screening is equally as effective as triple screening, so that the expense of estriol is unnecessary. The DADs approach, by allowing for variable weighting of parameters, lowers the at risk percentage and will permit a much more flexible approach as new parameters become available. Changing to DADs and eliminating estriol should achieve higher specificity for the same sensitivity and save, conservatively, about $900,000 in this series. Extrapolated nationally, if confirmed, the annual savings could approach $72,000,000.