Factors Influencing the Measurement of Plasma/Serum Surfactant Protein D Levels by ELISA

Background

Extensive variations in human surfactant protein D (SP-D) levels in circulation as measured by ELISA exist in the published literature. In order to determine the source of these variations, factors influencing the measurement by ELISA were explored.

Materials and Methods

Peripheral blood from healthy individuals was collected into various vacutainers during the same blood draw. Recombinant SP-D was diluted into different matrices and used for a standard curve. Samples were analyzed by capture ELISA using one of two distinct detection antibodies.

Results

The type of matrix had some effects on detection of recombinant SP-D. The type of anticoagulant used and dilution factor had very little effect, except for in plasma collected in EDTA vacutainers. The extent of variation in published values seemed to be due to the ELISA configuration employed, and, in agreement with this, we found that by switching the detection antibody, there was a 50% decrease in the extrapolated SP-D value of serum and plasma samples. Storage of samples resulted in slight changes in measured SP-D levels.

Conclusions

The ELISA configuration employed to measure circulating levels of SP-D has a significant effect on the extrapolated values. In both configurations tested, the use of EDTA as a coagulant resulted in inconsistent values, and we, therefore, suggest the avoidance of this anticoagulant when assaying for SP-D by ELISA. While the demonstrated effects of several factors on measurement of SP-D may not account for all the disparities amongst the previous studies, they stress that variations in methodologies for measuring the same protein can result in very inconsistent results.     

Prediction of Cardiovascular Events in Statin-Treated Stable Coronary Patients of the Treating to New Targets Randomized Controlled Trial by Lipid and Non-Lipid Biomarkers

Several plasma non-lipid biomarkers have been shown to predict major cardiovascular events (MCVEs) in population studies. Our objective was to investigate the relationship between lipid and non-lipid biomarkers levels achieved during statin therapy and the incidence of MCVEs in patients with stable coronary heart disease (CHD). We conducted a substudy of the TNT (Treating to New Targets) study, which was a randomized trial that compared the efficacy of high (80 mg) versus low (10 mg) dose atorvastatin for the secondary prevention of CHD. Fasting plasma levels of standard lipids and of 18 non-lipid biomarkers were obtained after an 8-week run-in period on atorvastatin 10 mg in 157 patients who experienced MCVEs during the 4.9 years of study follow-up and in 1349 controls. MCVE was defined as CHD death, nonfatal, non-procedure-related myocardial infarction, resuscitated cardiac arrest, and fatal or nonfatal stroke. After adjusting for age, sex and treatment arm, plasma levels of high-density lipoprotein (HDL) cholesterol, triglycerides, high-sensitivity C-reactive protein (hsCRP), insulin, neopterin, N-terminal pro-brain natriuretic peptide (BNP), lipoprotein(a) [Lp(a)], and the soluble receptor for advanced glycation end products (sRAGE) were predictive of recurrent MCVEs (P≤0.02 for each doubling of plasma concentration). However, no significant association was observed between the risk of recurrent MCVEs and plasma levels of low-density lipoprotein cholesterol, adiponectin, cystatin C, lipoprotein-associated phospholipase A2, monocyte chemotactic protein-1, matrix metalloproteinase-9, myeloperoxidase, osteopontin, soluble CD40 ligand, soluble intercellular adhesion molecule-1, or soluble vascular cell adhesion molecule-1. After further adjustment for diabetes, hypertension, smoking, and BMI, the relationship between hsCRP, insulin and MCVE were no longer significant, while the relationship between Lp(a), neopterin, NT-proBNP and sRAGE and MCVE remained statistically significant. In conclusion, in patients with CHD treated with atorvastatin, plasma levels of Lp(a), neopterin, NT-proBNP, and sRAGE are associated with the risk of recurrent MCVEs.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00327691","term_id":"NCT00327691"}}NCT00327691.     

Transmitted/Founder Simian Immunodeficiency Virus Envelope Sequences in Vesicular Stomatitis and Semliki Forest Virus Vector Immunized Rhesus Macaques

Identification of transmitted/founder simian immunodeficiency virus (SIV) envelope sequences responsible for infection may prove critical for understanding HIV/SIV mucosal transmission. We used single genome amplification and phylogenetic analyses to characterize transmitted/founder SIVs both in the inoculum and in immunized-infected rhesus monkeys. Single genome amplification of the SIVsmE660 inoculum revealed a maximum diversity of 1.4%. We also noted that the consensus sequence of the challenge stock differed from the vaccine construct in 10 amino acids including 3 changes in the V4 loop. Viral env was prepared from rhesus plasma in 3 groups of 6 immunized with vesicular stomatitis virus (VSV) vectors and boosted with Semliki forest virus (SFV) replicons expressing (a) SIVsmE660 gag-env (b) SIVsmE660 gag-env plus rhesus GM-CSF and (c) control influenza hemagglutinin protein. Macaques were immunized twice with VSV-vectors and once with SFV vector and challenged intrarectally with 4000 TCID₅₀. Single genome amplification characterized the infections of 2 unprotected animals in the gag-env immunized group, both of which had reduced acute plasma viral loads that ended as transient infections indicating partial immune control. Four of 6 rhesus were infected in the gag-env + GM-CSF group which demonstrated that GM-CSF abrogated protection. All 6 animals from the control group were infected having high plasma viral loads. We obtained 246 full-length envelope sequences from SIVsmE660 infected macaques at the peak of infection and determined the number of transmitted/founder variants per animal. Our analysis found that 2 of 2 gag-env vaccinated but infected macaques exhibited single but distinct virus envelope lineages whereas rhesus vaccinated with gag-env-GM-CSF or HA control exhibited both single and multiple env lineages. Because there were only 2 infected animals in the gag-env vaccinated rhesus compared to 10 infected rhesus in the other 2 groups, the significance of finding single env variants in the gag-env vaccinated group could not be established.     

Increased β-Cyanoalanine Nitrilase Activity Improves Cyanide Tolerance and Assimilation in Arabidopsis

Plants naturally produce cyanide （CN） which is maintained at low levels in their cells by a process of rapid assimilation. However, high concentrations of environmental CN associated with activities such as industrial pollution are toxic to plants. There is thus an interest in increasing the CN detoxification capacity of plants as a potential route to phytoremediation. Here, Arabidopsis seedlings overexpressing the Pseudomonas fluorescens β-cyanoalanine nitrilase pinA were compared with wild-type and a β-cyanoalanine nitrilase knockout line （△Atnit4） for growth in the presence of exogenous CN. After incubation with CN, ＋PfpinA seedlings had increased root length, increased fresh weight, and decreased leaf bleaching compared with wild-type, indicating increased CN tolerance. The increased tolerance was achieved without an increase in β-cyanoalanine synthase activity, the other enzyme in the cyanide assimilation pathway, suggesting that nitrilase activity is the limiting factor for cyanide detoxification. Labeling experiments with [^13C] KCN demonstrated that the altered CN tolerance could be explained by differences in flux from CN to Asn caused by altered β-cyanoalanine nitrilase activity. Metabolite profiling after CN treatment provided new insight into downstream metabolism, revealing onward metabolism of Asn by the photorespiratory nitrogen cycle and accumulation of aromatic amino acids.     

Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation

Oxidative damage to vascular cell membrane phospholipids causes physicochemical changes in membrane structure and lipid organization, contributing to atherogenesis. Oxidative stress combined with hyperglycemia has been shown to further increase the risk of vascular and metabolic diseases. In this study, the effects of glucose on oxidative stress-induced cholesterol domain formation were tested in model membranes containing polyunsaturated fatty acids and physiologic levels of cholesterol. Membrane structural changes, including cholesterol domain formation, were characterized by small angle X-ray scattering (SAXS) analysis and correlated with spectrophotometrically-determined lipid hydroperoxide levels. Glucose treatment resulted in a concentration-dependent increase in lipid hydroperoxide formation, which correlated with the formation of highly-ordered cholesterol crystalline domains (unit cell periodicity of 34 Å) as well as a decrease in overall membrane bilayer width. The effect of glucose on lipid peroxidation was further enhanced by increased levels of cholesterol. Treatment with free radical-scavenging agents inhibited the biochemical and structural effects of glucose, even at elevated cholesterol levels. These data demonstrate that glucose promotes changes in membrane organization, including cholesterol crystal formation, through lipid peroxidation.     

Transduction of Cellular Sequence by a Human Immunodeficiency Virus Type 1-Derived Vector

During studies examining the rate of human immunodeficiency virus type 1 (HIV-1) mutation in a single cycle of replication, the 5' long terminal repeat of one progeny provirus was found to contain an insertion of 147 bp including an entire tRNA sequence as well as an additional 66 bp insertion of nonviral origin. Database searches revealed that 65 of 66 bp aligned with the human CpG island sequence found on chromosomes 6, 14, and 17. Therefore it seems probable that it is of human cellular sequence origin and was transduced by HIV-1. This is the first demonstration that HIV-1 can capture a cellular sequence. The site of integration of the parental provirus was mapped to chromosome 1p32.1. Sequence with homology to the transduced CpG island was not found on chromosome 1, suggesting that the transduced cellular sequence was not linked to the site of viral integration.     

Hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic field conditions

The frequency of hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic conditions was assessed in field experiments, where R. raphanistrum were randomly planted at two different densities into large plots of B. napus. An acetolacate synthase (ALS)-inhibiting herbicide-resistant trait was used to detect potential hybrid individuals. No hybrids were detected amongst 25,000 seedlings grown from seed collected from R. raphanistrum plants. Two hybrids were obtained from more than 52-million B. napus seedlings. Both hybrids were characterised as amphidiploids (AACCRrRr, 2n = 56) and were fertile. The frequency of hybridisation into B. napus in this experiment using male-fertile B. napus was 4 × 10^–8. Received: 31 August 2000 / Accepted: 23 January 2001     

Surface feeding and aggressive behaviour of diploid and triploid brown trout Salmo trutta during allopatric pair‐wise matchings

Diploid and triploid brown trout Salmo trutta were acclimated for 6 weeks on two feeding regimes (floating and sinking). Thereafter, aggression and surface feeding response were compared between pairs of all diploid, all triploid and diploid and triploid S. trutta in an experimental stream. In each pair‐wise matching, fish of similar size were placed in allopatry and rank was determined by the total number of aggressive interactions recorded. Dominant individuals initiated more aggression than subordinates, spent more time defending a territory and positioned themselves closer to the surface food source (Gammarus pulex), whereas subordinates occupied the peripheries. In cross ploidy trials, diploid S. trutta were more aggressive than triploid, and dominated their sibling when placed in pair‐wise matchings. Surface feeding, however, did not differ statistically between ploidy irrespective of feeding regime. Triploids adopted a sneak feeding strategy while diploids expended more time defending a territory. In addition, we also tested whether triploids exhibit a similar social dominance to diploids when placed in allopatry. Although aggression was lower in triploid pairs than in the diploid and triploid pairs, a dominance hierarchy was also observed between individuals of the same ploidy. Dominant triploid fish were more aggressive and consumed more feed items than subordinate individuals. Subordinate fish displayed a darker colour index than dominant fish suggesting increased stress levels. Dominant triploid fish, however, appeared to be more tolerant of subordinate individuals and did not display the same degree of invasive aggression as seen in the diploid and diploid or diploid and triploid matchings. These novel findings suggest that sterile triploid S. trutta feed similarly but are less aggressive than diploid trout. Future studies should determine the habitat choice of triploid S. trutta after release and the interaction between wild fish and triploids during the breeding season prior to utilization of triploids as an alternative management strategy within freshwater fisheries.     

SIVcpz cross-species transmission and viral evolution toward HIV-1 in a humanized mouse model

HIV-1 evolved from its progenitor SIV strains, but details are lacking on its adaptation to the human host. We followed the evolution of SIVcpz in humanized mice to mimic cross-species transmission. Increasing viral loads, CD4⁺ T-cell decline, and non-synonymous mutations were seen in the entire genome reflecting viral adaptation.