High-density lipoprotein and the risk of recurrent venous thromboembolism

BACKGROUND - High-density lipoprotein (HDL) protects against arterial atherothrombosis, but it is unknown whether it protects against recurrent venous thromboembolism. METHODS AND RESULTS - We studied 772 patients after a first spontaneous venous thromboembolism (average follow-up 48 months) and recorded the end point of symptomatic recurrent venous thromboembolism, which developed in 100 of the 772 patients. The relationship between plasma lipoprotein parameters and recurrence was evaluated. Plasma apolipoproteins AI and B were measured by immunoassays for all subjects. Compared with those without recurrence, patients with recurrence had lower mean (±SD) levels of apolipoprotein AI (1.12±0.22 versus 1.23±0.27 mg/mL, P<0.001) but similar apolipoprotein B levels. The relative risk of recurrence was 0.87 (95% CI, 0.80 to 0.94) for each increase of 0.1 mg/mL in plasma apolipoprotein AI. Compared with patients with apolipoprotein AI levels in the lowest tertile (<1.07 mg/mL), the relative risk of recurrence was 0.46 (95% CI, 0.27 to 0.77) for the highest-tertile patients (apolipoprotein AI >1.30 mg/mL) and 0.78 (95% CI, 0.50 to 1.22) for midtertile patients (apolipoprotein AI of 1.07 to 1.30 mg/mL). Using nuclear magnetic resonance, we determined the levels of 10 major lipoprotein subclasses and HDL cholesterol for 71 patients with recurrence and 142 matched patients without recurrence. We found a strong trend for association between recurrence and low levels of HDL particles and HDL cholesterol. CONCLUSIONS - Patients with high levels of apolipoprotein AI and HDL have a decreased risk of recurrent venous thromboembolism. © 2007 American Heart Association, Inc.

The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration

Background The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. Method An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Results Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. Conclusions This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

NT-ProBNP levels in saliva and its clinical relevance to heart failure

Background: Current blood based diagnostic assays to detect heart failure (HF) have large intra-individual and inter-individual variations which have made it difficult to determine whether the changes in the analyte levels reflect an actual change in disease activity. Human saliva mirrors the body's health and well being and similar to 20% of proteins that are present in blood are also found in saliva. Saliva has numerous advantages over blood as a diagnostic fluid which allows for a non-invasive, simple, and safe sample collection. The aim of our study was to develop an immunoassay to detect NT-proBNP in saliva and to determine if there is a correlation with blood levels. Methods: Saliva samples were collected from healthy volunteers (n = 40) who had no underlying heart conditions and HF patients (n = 45) at rest. Samples were stored at -80 degrees C until analysis. A customised homogeneous sandwich AlphaLISA((R)) immunoassay was used to quantify NT-proBNP levels in saliva. Results: Our NT-proBNP immunoassay was validated against a commercial Roche assay on plasma samples collected from HF patients (n = 37) and the correlation was r(2) = 0.78 (p<0.01, y = 1.705 x +1910.8). The median salivary NT-proBNP levels in the healthy and HF participants were <16 pg/mL and 76.8 pg/mL, respectively. The salivary NT-proBNP immunoassay showed a clinical sensitivity of 82.2% and specificity of 100%, positive predictive value of 100% and negative predictive value of 83.3%, with an overall diagnostic accuracy of 90.6%. Conclusion: We have firstly demonstrated that NT-proBNP can be detected in saliva and that the levels were higher in heart failure patients compared with healthy control subjects. Further studies will be needed to demonstrate the clinical relevance of salivary NT-proBNP in unselected, previously undiagnosed populations.

Circulating fragments of N-Terminal Pro-B-Type Natriuretic Peptides in plasma of heart failure patients

BACKGROUND: The use of nonstandardized N-terminal pro-B-type natriuretic peptide (NT-proBNP) assays can contribute to the misdiagnosis of heart failure (HF). Moreover, there is yet to be established a common consensus regarding the circulating forms of NT-proBNP being used in current assays. We aimed to characterize and quantify the various forms of NT-proBNP in the circulation of HF patients. METHODS: Plasma samples were collected from HF patients (n = 20) at rest and stored at -80 degrees C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA (R) immunoassays were developed and validated to quantify 6 fragments of NT-proBNP. RESULTS: Mass spectrometry identified the presence of several N- and C-terminally processed forms of circulating NT-proBNP, with physiological proteolysis between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and Pro75-Arg76. Consistent with this result, AlphaLISA immunoassays demonstrated that antibodies targeting the extreme N or C termini measured a low apparent concentration of circulating NT-proBNP. The apparent circulating NT-proBNP concentration was increased with antibodies targeting nonglycosylated and nonterminal epitopes (P < 0.05). CONCLUSIONS: In plasma collected from HF patients, immunoreactive NT-proBNP was present as multiple N- and C-terminally truncated fragments of the full length NT-proBNP molecule. Immunodetection of NT-proBNP was significantly improved with the use of antibodies that did not target these terminal regions. These findings support the development of a next generation NT-proBNP assay targeting nonterminal epitopes as well as avoiding the central glycosylated region of this molecule. (c) 2013 American Association for Clinical Chemistry

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

The role of endogenous scabies mite complement inhibitors in the development of Streptococcus pyogenes skin infections

This project expands upon the discovery that scabies mites produce protein molecules that interfere with the human complement cascade, disrupting a critical component of the early stages of the host immune response. This is believed to provide an optimal environment for the development of commonly associated secondary bacterial infections. The thesis investigated the effect of two distinct scabies mite proteins, namely SMS B4 and SMIPP-S I1, on the in vitro proliferation of Group A Streptococcus in whole human blood. Additionally, in vitro immunoassays were performed to determine if complement mediated opsonisation and phagocytosis were also disrupted.

A multimarker approach to diagnose and stratify heart failure

Background We have previously demonstrated that circulating NT-proBNP is truncated at the N and C termini. Aims of this study are three-fold: firstly to determine whether the NT-proBNP levels correlate with NYHA functional classes when measuring with different antibody pairs; secondly to evaluate the diagnostic potential of ProBNP and; thirdly to investigate whether combining NT-proBNP assays with or without ProBNP would lead to better diagnostic accuracies. Methods Plasma samples were collected from healthy controls (n = 52) and HF patients (n = 46). Customized AlphaLISA® immunoassays were developed and validated to measure the concentrations of proBNP and NT-proBNP (with antibodies targeting 13–45, 13–76, 28–76). The diagnostic performance and predictive value of proBNP and NT-proBNP assays and their combinations were evaluated. Results Plasma proBNP assay showed acceptable diagnostic performance. NT-proBNP13–76 assay is useful in diagnosing and stratifying HF patients. The diagnostic performance of NT-proBNP13–76 demonstrated improvement over commercial NT-proBNP tests. The combination of NT-proBNP13–76 with NT-proBNP28–76 assays gave the best diagnostic assay performance. Conclusion Our results demonstrate that while there is major heterogeneity in circulating NT-proBNP, specific epitopes of the peptides are extraordinarily stable, providing ideal targets for clinically useful diagnostic assays. Future new clinical diagnostic clinical trials should include a multimarker approach rather than using a single marker to diagnose HF.

A potential epigenetic marker mediating serum folate and vitamin B12 levels contributes to ischemic stroke risk

Background and Purpose Stroke is a multifactorial disease that may be associated with aberrant DNA methylation profiles.We investigated epigenetic dysregulation for the MTHFR gene among ischaemic stroke patients. Methods Cases (n=297) and controls (n=110) were recruited after obtaining signed written informed consent, following a screening process against the inclusion/exclusion criteria. Serum vitamin metabolites (folate, vitamin B12 and homocysteine) were determined using immunoassays and methylation profiles for CpGs A and B in the MTHFR gene were determined using bisulfitepyrosequencing method. Results Methylation of MTHFR significantly increased the susceptibility risk for ischemic stroke. In particular, CpG A outperformed CpG B in mediating folate and vitamin B12 levels to increase ischemic stroke susceptibility risks by 4.73 fold. CpGs A and B were not associated with either serum homocysteine levels or ischemic stroke severity. Conclusion CpG A is a potential epigenetic marker in mediating serum folate and vitamin B12 to contribute to ischemic stroke.

Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth

The early and accurate assessment of burns is essential to inform patient treatment regimens; however, this first critical step in clinical practice remains a challenge for specialist burns clinicians worldwide. In this regard, protein biomarkers are a potential adjunct diagnostic tool to assist experienced clinical judgement. Free circulating haemoglobin has previously shown some promise as an indicator of burn depth in a murine animal model. Using blister fluid collected from paediatric burn patients, haemoglobin abundance was measured using semi-quantitative Western blot and immunoassays. Although a trend was observed in which haemoglobin abundance increased with burn wound severity, several patient samples deviated significantly from this trend. Further, it was found that haemoglobin concentration decreased significantly when whole cells, cell debris and fibrinous matrix was removed from the blister fluid by centrifugation; although the relationship to depth was still present. Statistical analyses showed that haemoglobin abundance in the fluid was more strongly related to the time between injury and sample collection and the time taken for spontaneous re-epithelialisation. We hypothesise that prolonged exposure to the blister fluid microenvironment may result in an increased haemoglobin abundance due to erythrocyte lysis, and delayed wound healing

Clinical relevance of MTHFR, eNOS, ACE, and ApoE gene polymorphisms and serum vitamin profile among Malay patients with ischemic stroke

Background The purpose of this study was threefold. First, it was to determine the relationship between serum vitamin profiles and ischemic stroke. The second purpose was to investigate the association of methylenetetrahydrofolate reductase (MTHFR), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE), and apolipoprotein-E (ApoE) gene polymorphisms with ischemic stroke and further correlate with serum vitamin profiles among ischemic stroke patients. The third purpose of the study was to highlight the interaction of MTHFR and eNOS haplotypes with serum vitamin profiles and ischemic stroke risks. Methods Polymorphisms of these genes were analyzed in age-, sex-, and ethnicity-matched case–controls (n = 594); serum vitamin profiles were determined using immunoassays. Results The MTHFR 677C>T, 1298A>C, eNOS intron 4a/b, and ApoE polymorphisms were significantly associated with the increased risk of ischemic stroke. Elevated serum homocysteine and vitamin B12 levels were associated with MTHFR 677C>T and eNOS intron 4a/b polymorphisms. The ApoE and eNOS −786T>C polymorphisms were associated with increased serum vitamin B12 levels. However, none of the polymorphisms influenced serum folate levels except for the MTHFR 1298A>C. Different patterns of MTHFR and eNOS haplotypes tend to affect serum vitamin profiles to different degrees, which contribute to either different susceptibility risk or protective effect on ischemic stroke. Overall, increased levels of serum homocysteine and vitamin B12 levels were associated with higher risk of ischemic stroke in the investigated population. Conclusions The present study suggests that the genotypes and haplotypes of MTHFR 677C>T and eNOS intron 4a/b polymorphisms are potential serum biomarkers in the pathophysiological processes of ischemic stroke, by modulating homocysteine and vitamin B12 levels.