Interactions of Apolipoproteins AI, AII, B and HDL, LDL, VLDL with Polyurethane and Polyurethane-PEO Surfaces.

The lipoproteins (HDL, LDL, VLDL) are important components of blood present in high concentration. Surprisingly, their role in blood-biomaterial interactions has been largely ignored. In previous work apolipoprotein AI (the main protein component of HDL) was identified as a major constituent of protein layers adsorbed from plasma to biomaterials having a wide range of surface properties, and quantitative data on the adsorption of apo AI to a biomedical grade polyurethane were reported. In the present communication quantitative data on the adsorption of apo AI, apo AII and apoB (the latter being a constituent of LDL and VLDL), as well as the lipoprotein particles themselves (HDL, LDL, VLDL), to a biomedical segmented polyurethane (PU) with and without an additive containing poly(ethylene oxide) (material referred to as PEO) are reported. Using radiolabeled apo AI, apo AII, and apoB, adsorption levels on PU from buffer at a protein concentration of 50 µg/mL were found to be 0.34, 0.40, and 0.14 µg/cm(2) (12, 23, and 0.25 nmol/cm(2)) respectively. Adsorption to the PEO surface was <0.02 µg/cm(2) for all three apolipoproteins demonstrating the strong protein resistance of this material. In contrast to the apolipoproteins, significant amounts of the lipoproteins were found to adsorb to the PEO as well as to the PU surface. X-ray photoelectron spectra, following exposure of the surfaces to the lipoproteins, showed a strong phosphorus signal, confirming that adsorption had occurred. It therefore appears that a PEO-containing surface that is resistant to apolipoproteins may be less resistant to the corresponding lipoproteins.

The Canadian healthy infant longitudinal development birth cohort study: biological samples and biobanking.

BACKGROUND: It is hypothesised that complex interactions between genetic and environmental factors give rise to allergy and asthma in childhood. The Canadian Healthy Infant Longitudinal Development (CHILD) study was designed to explore these factors. METHODS: CHILD is a longitudinal, general population birth cohort study following infants from mid-pregnancy to age 5 years. Over this time period, biological samples, questionnaires, clinical measures and environmental data are collected. RESULTS: A total of 3624 families have been recruited, and many thousands of samples and questionnaires have been collected, annotated, and archived. This report outlines the rationale and methodology for collecting and storing diverse biological samples from parents and children in this study, and the mechanisms for their release for analyses. CONCLUSIONS: The CHILD sample and data repository is a tremendous current and future resource and will provide a wealth of information not only informing studies of asthma and allergy, but also potentially in many other aspects of health relevant for Canadian infants and children.

Genetic and treatment-related risk factors associated with external apical root resorption (EARR) concurrent with orthodontia.

OBJECTIVE: As genetic variation accounts for two-thirds of the variation in external apical root resorption (EARR) concurrent with orthodontic treatment, we analyzed the association of selected genetic and treatment-related factors with EARR concurrent with orthodontic treatment. SETTING AND SAMPLE POPULATION: This case-control study of 134 unrelated, orthodontically treated Caucasian individuals was conducted in part at an Indiana Private Practice, Indiana University and the University of Kentucky. METHODS: Utilizing a research data bank containing information from ~1450 orthodontically treated patients, pre- and post-treatment radiographs from 460 individuals were evaluated for EARR of the four permanent maxillary incisors. Sixty-seven unrelated Caucasians with moderate to severe EARR were identified and were age-/sex-matched with orthodontically treated Caucasian controls yielding 38 females and 29 males per group. Factors tested for an association with EARR included the following: 1) treatment duration, 2) extraction of maxillary premolars, 3) numerous cephalometric measurements, and 4) DNA polymorphisms within/near candidate genes in a pathway previously implicated in EARR such as the purinergic-receptor-P2X, ligand-gated ion channel 7 (P2RX7; rs208294, rs1718119, and rs2230912), caspase-1 (CASP1; rs530537, rs580253, and rs554344), interleukin-1 beta (IL1B; rs1143634), interleukin-1 alpha (IL1A; rs1800587), and interleukin-1 receptor antagonist (IL1RA; rs419598) genes. Stepwise logistic regression was utilized to identify the factors significantly associated (significance taken at or less than the layered Bonferroni correction alpha) with the occurrence of EARR. RESULTS: A long length of treatment and the presence of specific genotypes for P2RX7 SNP rs208294 were significantly associated with EARR. CONCLUSION: EARR occurrence was associated with both genetic and treatment-related variables, which together explained 25% of the total variation associated with EARR in the sample tested.

Retrograde amnesia gradients: effects of direct cortical stimulation.

Electrical stimulation was delivered bilaterally to either the anterior or posterior cortex in rats from 0.1 second to 4 hours after a single training trial on an inhibitory avoidance task. As indicated by a retention test given 24 hours later, the length of the retrograde amnesia gradients ranged from 5 seconds to 240 minutes, depending on the brain region stimulated and the intensity of the stimulating current. The stimulation intensity that was threshold for amnesia varied directly with the length of the interval between training and treatment.

Application of proteomics to the study of cardiovascular biology.

Proteomics involves the integration of a number of technologies with the aim of analyzing the complete complement of proteins expressed by a biological system in response to various stimuli and/or under different physiological or pathophysiological conditions. Recent technical improvements to the methods employed for protein separation and protein identification have resulted in a dramatic increase in the number of proteomics-based research projects. More importantly, it has become readily apparent that examining changes in the proteome offers insight into understanding cellular and molecular mechanisms that cannot be obtained through genomic analysis. There are numerous examples of cardiovascular functions whose molecular pathways are mediated through post-translational processes such as phosphorylation. The use of proteomics offers the ability to simultaneously monitor the changes in protein expression and/or cell signaling pathways in response to such conditions as cardiac hypertrophy and heart failure. Together with complementary genomic data, proteomics-based research can greatly increase our understanding of cardiovascular biology.

Development of behavioral tolerance: a search for subcellular mechanisms.

Development of behavioral tolerance is one of the processes by which living organisms adjust to changes in their internal and external environments. The search for neurochemical mechanisms underlying such processes requires the testing of many hypotheses. The present study was designed to examine the possible involvement of certain subcellular events. The concentrations of acetylcholine (ACh) and choline (Ch), the high-affinity transport of Ch, and the rate of synthesis of ACh were measured in synaptosomes prepared from the brains of rats. The assays were made at critical times during the acute changes in behavior induced by administration of the anticholinesterase, di-isopropylfluorophosphate, and during the development of behavioral tolerance to this compound as chronicity of administration continued. No statistically significant differences were found among treatment groups in the total concentration of ACh or Ch, the synthesis of ACh, or the high-affinity transport of Ch. These results, plus evidence from previous experiments, indicate that the development of behavioral tolerance does not relate to the factors studied. Consequently, alternative mechanisms should be considered. In addition to changes in cholinergic (muscarinic) receptors already shown to occur concomitantly with the development of behavioral tolerance, it is suggested that the possible involvement of mechanisms controlling release of ACh should be studied.

Prenatal serum alpha-fetoprotein screening for neural tube defects.

Twenty-two cases of open neural tube defect were found in a population of 17,703 unselected pregnancies (1.2 per 1000) within the Long Island, New York, region. Voluntary screening of maternal serum alpha-fetoprotein levels identified 20 of the 22 cases (91%). Six hundred ninety-two participants demonstrated serial elevations in maternal serum alpha-fetoprotein. Of this group, which was designated at increased risk for open neural tube defect, 24% had underestimated gestational age, 13% had multiple gestations, and 53% were candidates for amniocentesis. In the amniocentesis group, the detection yield of neural tube defect was 20 per 365 (5.5%). Neither false-negative amniotic fluid evaluations nor termination of normal pregnancy due to false-positive amniotic fluid levels occurred. Perinatal outcome data, including pregnancy complications, date, mode of delivery, sex, birth weight, Apgar score, and congenital malformations of the neonate other than neural tube defect, are reported for the first 9300 consecutive participants of the 17,703 population study. These data identify a correlation between rate of perinatal loss and maternal serum alpha-fetoprotein levels.

Amniography in the prenatal diagnosis of neural tube defects.

Amniography for the visualization and confirmation of suspected neural tube defect was performed in 9 midtrimester gravidas. In all cases, amniotic fluid alpha-feto protein (AFP) was abnormally elevated. Four cases of anencephaly and one of spina bifida were demonstrated by amniography. These pregnancies were terminated and the defects were confirmed by gross pathologic examination. In 4 remaining cases, amniography was normal. Three of these pregnancies proceeded to term, culminating in the birth of a normal child. The fourth patient had spontaneous abortion of a normal fetus at 23 weeks of gestation. The experience reported here suggests that amniography is an important adjunctive diagnostic technique in the prenatal diagnosis of neural tube defect, and if used correctly, may significantly reduce the chance of false-positive diagnosis.

Maternal serum and amniotic fluid alpha-fetoprotein as a marker of acute fetal distress in a midtrimester abortion model.

Maternal serum (MS) and amniotic fluid (AF) alpha-fetoprotein (alpha-FP) levels were serially determined in 18 cases of elective midtrimester abortion. Prostaglandin F2alpha (PGF2alpha) and 20% NaCl were used as the abortifacients in 2 groups of 9 patients, respectively. The time from instillation to abortion (IAT) was accurately recorded in all cases. A marked 260-600% increase in MS alpha-FP occurred prior to fetal demise in both groups. Amniotic fluid alpha-FP content remained largely unchanged for the first 6 hours following intraamniotic prostaglandin injection. A 50% INCREASE WAS OBSERVED IN THE AF alpha-FP content in the group with 20% NaCl-induced abortion (after an initial dilutionary drop). The results of this investigation confirm the value of alpha-FP in MS as a marker of impending fetal demise. This rise is not caused by a prior alpha-FP change in the AF. The data suggest a major fetomaternal transplacental route for alpha-FP.

Origin of amniotic fluid alpha-fetoprotein in normal and defective pregnancies.

Alpha-fetoprotein was determined by electroimmunodiffusion and radioimmunoassay in 109 neonatal urine samples and 94 amniotic fluid samples. The samples were obtained from newborns and from pregnancies ranging in gestational age from 20 to 40 weeks. When alpha-fetoprotein values of neonatal urine and amniotic fluid were correspondingly correlated with gestational age, almost identical declining curves could be drawn. Twenty-one cerebrospinal fluid samples from newborns ranging from 25 to 40 weeks of gestation were similarly determined. No correlation between cerebrospinal fluid alpha-fetoprotein and gestational age could be demonstrated. It is concluded that fetal urine is the major source of alpha-fetoprotein in the amniotic fluid of normal pregnancy. In pregnancies associated with neural tube defects, alpha-fetoprotein elevation is probably not due to the leakage of cerebrospinal fluid into the amniotic cavity.

Rapid assay--possible application in the diagnosis of premature rupture of the membranes.

Premature rupture of the membranes frequently presents a diagnostic dilemma to the clinician. The presence of alpha-fetoprotein (AFP) in amniotic fluid suggests a possible solution. A rapid, easy-to-use latex agglutination test for AFP was evaluated on 99 amniotic fluid samples of known AFP concentration. Body fluids (maternal serum, urine, vaginal secretions, and seminal fluid) that commonly interfere with other tests were also studied. The sensitivity for amniotic fluids from gestations less than 39 weeks was 93%, and specificity was 94%. The test is most accurate under 35 weeks, when diagnosis is critical. Equivocal results may be resolved by using radioimmunoassay. The results suggest that a rapid assay for AFP may be useful in the diagnosis of ruptured membranes. It has few interfering factors, is safe, and requires no elaborate equipment or training.

Amniotic fluid alpha-fetoprotein as a marker in prenatal diagnosis of neural tube defects.

The prenatal diagnosis of anencephaly and spina bifida (neural tube defect, NTD) through amniotic fluid analysis for alpha-fetoprotein (AFP) is gradually gaining clinical recognition. AFP concentrations were determined in 237 amniotic fluids from normal pregnancies ranging between 7 and 42 weeks of gestation. A steady decline in AFP from 26 mug/ml at 7-9 weeks to 155 ng/ml at term is observed. AFP concentration was determined in 35 amniotic fluids from 33 confirmed neural tube defective pregnancies. In 14 cases where amniotic fluid was examined prior to the 26th week of gestation. AFP was markedly elevated when compared with the normal range of the same gestational period. In 21 amniotic fluids past the 26th week, 17 cases (85-) had markedly elevated AFP levels; however, 2 cases of anencephaly, 1 of spina bifida, and 1 of hydrocephaly gave levels within the normal range. It is concluded that elevated AFP in the amniotic fluid is a reliable but nonspecific marker for open neural tube defects prior to the 26th week of pregnancy, but may become normal after the 26th week in a small percentage of patients.

PIP: A steady decline in alpha fetoprotein (AFP) levels was observed in single specimens of amniotic fluid (AF) from 237 patients, ranging from 26 mcg/ml at 7-9 weeks to 155 ng/ml at term. All pregnancies tested were normal. 35 AF specimens from 33 confirmed neural tube defective pregnancies were assayed for AFP. Very high levels of AFP were found in 13/14 fluids examined before Week 26 of gestation. A value of 23 mcg/ml was determined in 1 sample where the infant had skin-covered encephalocele. A fluid taken from the same patient at 34 weeks fell to 6.4 mcg of AFP. 21 AF samples from patients past the 26th week of pregnancy were analyzed. Of these, 1 case of spina bifida and 2 of anecephaly gave no detectable levels of AFP by electroimmunodiffusion. By radioimmunoassay, however, these samples measured 3700, 256, and 700 ng/ml. 1 case of hydroencephaly, examined at 33 weeks, had an AFP level of 1.5 mcg/ml. A sharp drop in AFP from 353.6 at 15 weeks to 10.4 mcg/ml at 29 weeks was noted in the only serially examined open neural tube defective pregnancy.

Amniotic fluid adenosine 3',5' - monophosphate in prostaglandin-induced midtrimester abortions.

Cyclic adenosine 3'5-monophosphate (cAMP) was serially measured in the amniotic fluid (AF) of 9 patients undergoing midtrimester abortions induced by intraamniotic prostaglandins. The results demonstrate an increase in AF cAMP ranging from 2- to 7-fold within the 6 hours of observation. The fetal heart tones were closely monitored by a Doppler instrument and the time from injection of abortifacient to fetal demise (IDT) and to fetal expulsion (IAT) was accurately recorded. No correlation between the rate of AF cAMP increase and IDT or IAT could be demonstrated. The concentration of cAMP in amniotic fluid obtained from patients with fetal demise in utero was lower than that found in AF of fetuses of corresponding gestational age where the fetus was alive. The significance of AF cAMP as a potential indicator of fetal distress is discussed.

First-trimester Down syndrome screening using dried blood biochemistry and nuchal translucency.

OBJECTIVE: To assess the effectiveness of free beta-hCG, pregnancy-associated plasma protein A, and nuchal translucency in a prospective first-trimester prenatal screening study for Down syndrome and trisomy 18. METHODS: Risks were calculated for Down syndrome and trisomy 18 based on maternal age and biochemistry only (n = 10,251), nuchal translucency only (n = 5809), and the combination of nuchal translucency and biochemistry (n = 5809). RESULTS: The study population included 50 Down syndrome and 20 trisomy 18 cases. Nuchal translucency measurement was done on 33 Down syndrome and 13 trisomy 18 cases. Down syndrome screening using combined biochemistry and ultrasound resulted in a false-positive rate of 4.5% (95% confidence interval [CI] 3.9%, 5.2%) and detection rate of 87.5% (95% CI 47%, 100%) in patients under age 35 years. In older patients, the false-positive rate was 14.3% (95% CI 12.7%, 15. 8%) and detection rate was 92% (95% CI 74%, 99%). For trisomy 18 screening, the false-positive rate was 0.4% (95% CI 0.24%, 0.69%) and detection rate was 100% (95% CI 40%, 100%) in younger patients, whereas in older patients the false-positive rate was 1.4% (95% CI 0. 9%, 2.0%) and detection rate was 100% (95% CI 66%, 100%). Using modeling, at a fixed 5% false-positive rate, the Down syndrome detection rate was 91%. Conversely, at a fixed 70% Down syndrome detection rate, the false-positive rate was 1.4%. CONCLUSION: First-trimester screening for Down syndrome and trisomy 18 is effective and offers substantial benefits to clinicians and patients.

Early prenatal diagnosis of soft-tissue malformations.

This study presents 3 cases of severe soft-tissue malformations of the fetus. Prenatal diagnosis was established early in the second trimester, and the accuracy of the diagnosis was confirmed by pathological examination in each instance. A clinically oriented work-up plan for early prenatal diagnosis is proposed.

Regulation of apolipoprotein B biogenesis in human hepatocytes: posttranscriptional control mechanisms that determine the hepatic production of apolipoprotein B-containing lipoproteins.

OBJECTIVES: Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins appears to be a common cause of hyperlipoproteinemia in humans. Patients with overproduction states secrete denser cholesterol ester-rich lipoprotein particles which are highly atherogenic. The formation of apoB particles involves a very complex process that requires the coordinated synthesis and assembly of apoB, triglycerides, cholesterol esters, phospholipids, and other components. ApoB expression is an important prerequisite for the assembly and secretion of apoB particles. Evidence to date appears to suggest that apoB expression is regulated posttranscriptionally. ApoB secretion rate is determined at the levels of apoB translocation into the endoplasmic reticulum (ER) as well as degradation within the ER. RESULTS AND HYPOTHESIS: Based on available data, we postulate that the rate of apoB particle secretion is determined at the critical point where newly-synthesized apoB interacts with core lipids, particularly triglycerides. The supply of these lipids determines the rate of translocation of the apoB molecule across the ER membrane and into the ER lumen. Lipidation of apoB facilitates its proper folding, its assembly into a lipoprotein particle, and its extracellular secretion. In the absence of lipids, apoB is misfolded resulting in the abortion of ER translocation and subsequent degradation by an apoB specific protease. CONCLUSIONS: The balance between intracellular degradation and extracellular secretion determines the rate at which the human liver secretes apoB particles.

Genetic predisposition to external apical root resorption in orthodontic patients: linkage of chromosome-18 marker.

External apical root resorption (EARR) is a common orthodontic treatment sequela. Previous studies implicate a substantial genetic component for EARR. Using a candidate gene approach, we investigated possible linkage of EARR associated with orthodontic treatment with the TNSALP, TNFalpha, and TNFRSF11A gene loci. The sample was comprised of 38 American Caucasian families with a total of 79 siblings who completed comprehensive orthodontic treatment. EARR was assessed by means of pre- and post-treatment radiographs. Buccal swab cells were collected for extraction and analysis of DNA. No evidence of linkage was found with EARR and the TNFalpha and TNSALP genes. Non-parametric sibling pair linkage analysis identified evidence of linkage (LOD = 2.5; p = 0.02) of EARR affecting the maxillary central incisor with the microsatellite marker D18S64 (tightly linked to TNFRSF11A). This indicates that the TNFRSF11A locus, or another tightly linked gene, is associated with EARR.

Dual Analyte immunoassay--a new approach to neural tube defect and Down's syndrome screening.

A microtiter plate based Dual Analyte enzyme-immunoassay method for the simultaneous measurement of alpha-fetoprotein (AFP) and Free-beta human chorionic gonadotrophin (hCG) was evaluated. This rapid assay, which has application in both Neural Tube Defect screening and Down's screening, shows good precision with between assay coefficients of variation between 5 and 7.5% for AFP and 3.7 to 5.8% for Free-beta(hCG). Correlation with single analyte procedures is good, with correlation coefficients being greater than 0.91 in both cases. Clinical discrimination in detecting both types of abnormalities is not compromised by this new simultaneous Dual Analyte assay. We conclude that the Dual Analyte approach, which combines analytes achieving the highest known detection efficiency, will bring about improvements in the efficiency of screening, reduce costs and improve report turnaround, all leading to better quality of patient care.

Free beta-chorionic gonadotropin: a cross-reactivity study of two immunometric assays used in prenatal maternal serum screening for Down's syndrome.

We have evaluated the cross-reactivity characteristics of two distinct immunometric assays for the measurement of free beta-human chorionic gonadotropin (free beta). These maternal serum assays have been used in the initial studies which evaluated free beta as a marker in the prenatal detection of Down's syndrome. It has been suggested that free beta assays are subject to substantial potential for cross-reactivity. To confirm that free beta was the analyte responsible for enhanced detection efficiency both non-competitive and competitive cross-reactivity evaluations were undertaken. These studies demonstrated acceptably small cross-reactivity to other glycoprotein hormones or their beta components. We conclude that properly designed free beta assays will provide specific analyte measurement and improved detection efficiency in Down's syndrome screening.

First trimester biochemical screening for trisomy 21: the role of free beta hCG, alpha fetoprotein and pregnancy associated plasma protein A.

The potential efficacy of screening for trisomy 21 in the first trimester, using maternal serum markers alpha fetoprotein, free beta human chorionic gonadotropin, unconjugated oestriol and pregnancy associated plasma protein A, was studied in an unselected population of women between the seventh and fourteenth week of gestation. Using a combination of alpha fetoprotein and free beta human chorionic gonadotropin, 53% of affected pregnancies could be identified at a false positive rate of 5%. Unconjugated oestriol and pregnancy associated plasma protein A levels were lower in cases of trisomy 21, but their inclusion with other markers did not significantly improve detection rate. Monitoring the same pregnancies also in the second trimester showed that screening in the first trimester identified the same cases as in the second. We conclude that first trimester screening using free beta human chorionic gonadotropin and alpha fetoprotein, is a viable possibility and will lead to detection rates in excess of 50%. Prospective studies are needed to confirm these observations.