Prognostic utility of serum NT-proBNP (fragments 1-76aa and 13-71aa) and galectin-3 in predicting death and re-hospitalisation due to cardiovascular events in patients with heart failure.

Patients with heart failure (HF) are at a higher risk of rehospitalisation. In this study, we investigated the prognostic utility of galectin-3 (Gal-3) and NT-proBNP fragments (1-76aa and 13-71aa) as biomarkers to predict outcomes for patients with HF. We collected blood samples from patients with HF (n = 101). Gal-3 and NT-proBNP fragments (1-76aa and 13-71aa) concentrations were measured by immunoassay. Survival analysis and Cox proportional regression models were used to determine the prognostic utility of Gal-3 and NT-proBNP fragments. In patients with increased baseline levels of NT-proBNP1-76 the time to primary endpoint (cardiovascular death or re-hospitalisation) was significantly shorter (p = 0.0058), but not in patient with increased baseline levels of Gal-3 or NTproBNP13-71. Patients with increased levels of NT-proBNP13-71aa at 1 month showed reduced time to the primary endpoint (p = 0.0123). Our findings demonstrated that Gal-3 and NT-proBNP can be used as prognostic biomarkers to stratify patients with HF.

Proteomic Alterations in Salivary Exosomes Derived from Human Papillomavirus-Driven Oropharyngeal Cancer.

BACKGROUND: Increasing evidence supports the notion that human papillomavirus (HPV) DNA integration onto the human genome can influence and alter the molecular cargo in the exosomes derived from head and neck cancer cells. However, the molecular cargo of salivary exosomes derived from HPV-driven oropharyngeal cancer (HPV-driven OPC) remains unelucidated. METHODS AND MATERIALS: Salivary exosomes morphology and molecular characterizations were examined using the nanoparticle tracking (NTA), western blot analysis, transmission electron microscopy (TEM) and mass spectrometry analysis. RESULTS: We report that HPV16 DNA was detected (80%) in isolated salivary exosomes of HPV-driven OPC patients. Importantly, we demonstrate elevated protein levels of six main glycolytic enzymes [i.e., aldolase (ALDOA), glyceraldehye-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A/B (LDHA and LDHB), phosphoglycerate kinase 1 (PGK1) and pyruvate kinase M1/2 (PKM)] in isolated salivary exosomes of HPV-driven OPC patients, suggesting a novel mechanism underlying the potential role of salivary exosomes in mediating the reciprocal interplay between glucose metabolism and HPV-driven OPC. CONCLUSION: Our data demonstrate the potential diagnostic value of HPV16 DNA and glycolytic enzymes in salivary exosomes in discriminating healthy controls from HPV-driven OPC patients, thereby opening new avenues in the future for clinical translation studies.

Overexpression of miRNA-9 enhances galectin-3 levels in oral cavity cancers.

Oral cavity cancer (OCC) is the predominant subtype of head and neck cancer (HNC) and has up to 50% mortality. Genome-wide microRNA (miR) sequencing data indicates overexpression of miR-9-5p in HNC tumours, however, the biological role of miR-9-5p in OCC is complex; it can either act as a tumour suppressor or an oncomir, regulating many target genes at the post-transcriptional level. We have investigated the overexpression of miR-9-5p in three OCC cell lines. We have evaluated its expression levels and Galectin-3 as potential biomarkers in saliva samples collected from controls and OCC patients. We found that over expression of miR-9-5p in OCC cell lines resulted in a significant reduction in cell proliferation and migration, and an increase in apoptosis, which was paralleled by an increase in Galectin-3 secretion and export of Galectin-3 protein. Our data are consistent with miR-9-5p being a modulator of Galectin-3 via the AKT/γ-catenin pathway. In addition, the positive correlation between the levels of miR-9-5p expression and secreted Galectin-3 in saliva reflects a similar relationship in vivo, and supports the utility of their integrative evaluation in OCC. Our findings indicate that both miR-9-5p and Galectin-3 are critical biomolecules in the progression of OCC.

Relationship between p16 expression and prognosis in different anatomic subsites of OSCC.

BACKGROUND: p16 has often been found to be overexpressed in patients oral squamous cell carcinoma (OSCC), but its prognostic value between anatomic subsites is still unclear. OBJECTIVE: The aim of this study was to investigate the diagnostic and prognostic values of p16 in OSCC originating from tongue, gingiva or buccal mucosa. METHODS: A total of 147 OSCC patients with tumors arising from the tongue, gingiva or buccal mucosa were enrolled in this study. p16 expression was detected using immunohistochemistry (IHC), and the presence of HPV16 was determined by real-time PCR in p16 positive patients. The correlation of p16 expression with the clinical parameters was evaluated. RESULTS: Only one p16 positive patient with a cut off value of 25% and 75% was HPV16 positive. Although overall survival (OS), recurrence free survival (RFS) and metastasis free survival (MFS) had no significant differences between the p16 positive and negative patients, p16 negative patients (cut off value 25%) had more RFS in the buccal mucosa cancer (p= 0.03) than the p16-positive patients. CONCLUSIONS: The prevalence of HPV16 in Chinese OSCC patients was low. p16 overexpression decoupled from HPV infection was not a prognostic marker for OSCC patients except for patients with the buccal mucosa cancer.

Development of Paper-Based Analytical Devices for Minimizing the Viscosity Effect in Human Saliva.

Rationale: Saliva as a sample matrix is rapidly gaining interest for disease diagnosis and point-of-care assays because it is easy to collect (non-invasive) and contains many health-related biomarkers. However, saliva poses particular problems relative to more common urine and blood matrices, which includes low analyte concentrations, lack of understanding of biomolecule transportation and inherent viscosity variability in human samples. While several studies have sought to improve assay sensitivity, few have addressed sample viscosity specifically. The goal of this study is to minimize the effect of sample viscosity on paper-based analytical devices (PADs) for the measurement of pH and nitrite in human saliva. Methods: PADs were used to measure salivary pH from 5.0 to 10.0 with a universal indicator consisting of chlorophenol red, phenol red and phenolphthalein. Nitrite determination was performed using the Griess reaction. Artificial saliva with viscosity values between 1.54 and 5.10 mPa∙s was tested on the proposed PAD. To ensure the proposed PADs can be tailored for use in-field analysis, the devices were shipped to Australia and tested with human specimens. Results: Initial experiments showed that viscosity had a significant impact on the calibration curve for nitrite; however, a more consistent curve could be generated when buffer was added after the sample, irrespective of sample viscosity. The linear range for nitrite detection was 0.1 to 2.4 mg/dL using the improved method. The nitrite measurement in artificial saliva also showed a good correlation with the standard spectrophotometry method (p=0.8484, paired sample t-test, n=20). Measured pH values from samples with varying viscosities correlated well with the results from our pH meter. Conclusions: The inherent variation of salivary viscosity that impacts nitrite and pH results can be addressed using a simple washing step on the PAD without the need for complex procedures.

Salivary miRNA panel to detect HPV-positive and HPV-negative head and neck cancer patients.

Head and neck squamous cell carcinomas (HNSCC) are a heterogeneous group of tumours that originate predominantly from the oral cavity, pharynx and larynx. Our aim was to determine whether salivary miRNA expression levels can diagnose these cancer subtypes. Saliva samples were collected from healthy controls (n=113, smoker and non-smokers), HPV-positive (n=54) and HPV-negative (n=47) HNSCC patients. The miRNA expression levels in saliva was quantified using qPCR. The potential of salivary miRNAs to discriminate these groups of patients was evaluated using multiple logistic regression with ROC analysis and a 10-fold cross-validation analysis. Salivary miRNA-9, -127, -134, -191, -222 and -455 were shown to discriminate a control group from a HPV-negative HNSCC patient group with a sensitivity of 60% and a specificity of 94%; whilst salivary miRNA-9,-134, -196b, -210, and -455 were the most parsimonious subset discriminating a control group from a HPV-positive HNSCC group, with a sensitivity of 65% and a specificity of 95%. Furthermore, miRNA-9, -134, -196b, -210 and -455 as a panel, was the most parsimonious subset to discriminate HPV-positive HNSCC patients from HPV-negative HNSCC patients. In addition, the expression levels of miRNA-9, -127, -196a, -196b, -210, -222 and -455 were significantly increased in the saliva collected from early stage HNSCC patients compared to controls. A future multi-centre confirmatory study is warranted to test the diagnostic performance of these salivary miRNA prior to clinical implementation.

Within-Day Baseline Variation in Salivary Biomarkers in Healthy Men.

Saliva is an easily accessible sample and offers practical and noninvasive biomarker solutions as an alternative to blood and urine-based diagnostics. Saliva contains a plethora of biomolecules such as nucleic acids, hormones, proteins, and electrolytes. On the other hand, little is known on the extent to which the biomolecules in saliva vary over time within a given person. This baseline information is crucial for future development of robust saliva-based diagnostics. We have collected unstimulated whole mouth saliva from 20 healthy young men at four times during the day, including before and after a meal. We measured the salivary cortisol, testosterone, C-reactive protein (CRP), stability of genomic DNA (gDNA) and DNA methylation levels of APC, P16INK4a, and PCQAP in these samples. We found that the salivary CRP, DNA methylation, and CD44 gDNA levels did not vary significantly across four time points (p > 0.05) while the salivary cortisol and testosterone levels significantly varied from the morning collection to the afternoon collection (p < 0.05). Furthermore, salivary cortisol levels were significantly affected by eating (p < 0.05). Our study offers a within-person baseline temporal assessment of several clinically relevant biomolecules and diagnostics, and suggests that salivary cortisol and testosterone levels vary over time in a given day whereas CRP and DNA methylation of tumor suppressor genes and CD44 amplification are stable throughout the day. Future research and clinical applications of salivary biomarkers and diagnostics should take into consideration their temporal variations.

Salivary DNA methylation panel to diagnose HPV-positive and HPV-negative head and neck cancers.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with a typical 5 year survival rate of <40 %. DNA methylation in tumour-suppressor genes often occurs at an early stage of tumorigenesis, hence DNA methylation can be used as an early tumour biomarker. Saliva is an ideal diagnostic medium to detect early HNSCC tumour activities due to its proximity to tumour site, non-invasiveness and ease of sampling. We test the hypothesis that the surveillance of DNA methylation in five tumour-suppressor genes (RASSF1α, p16 INK4a , TIMP3, PCQAP/MED15) will allow us to diagnose HNSCC patients from a normal healthy control group as well as to discriminate between Human Papillomavirus (HPV)-positive and HPV-negative patients. METHODS: Methylation-specific PCR (MSP) was used to determine the methylation levels of RASSF1α, p16 INK4a , TIMP3 and PCQAP/MED15 in DNA isolated from saliva. Statistical analysis was carried out using non-parametric Mann-Whitney's U-test for individually methylated genes. A logistic regression analysis was carried out to determine the assay sensitivity when combing the five genes. Further, a five-fold cross-validation with a bootstrap procedure was carried out to determine how well the panel will perform in a real clinical scenario. RESULTS: Salivary DNA methylation levels were not affected by age. Salivary DNA methylation levels for RASSF1α, p16 INK4a , TIMP3 and PCQAP/MED15 were higher in HPV-negative HNSCC patients (n = 88) compared with a normal healthy control group (n = 122) (sensitivity of 71 % and specificity of 80 %). Conversely, DNA methylation levels for these genes were lower in HPV-positive HNSCC patients (n = 45) compared with a normal healthy control group (sensitivity of 80 % and specificity of 74 %), consistent with the proposed aetiology of HPV-positive HNSCCs. CONCLUSIONS: Salivary DNA tumour-suppressor methylation gene panel has the potential to detect early-stage tumours in HPV-negative HNSCC patients. HPV infection was found to deregulate the methylation levels in HPV-positive HNSCC patients. Large-scale double-blinded clinical trials are crucial before this panel can potentially be integrated into a clinical setting.

A pilot study to demonstrate diagnostic potential of galectin-3 levels in saliva.

AIM: Heart failure (HF) affects millions of older individuals in both developed and low/middle-income countries. Serum galectin-3 levels have been shown to have prognostic value. However, its use as a diagnostic biomarker has not been explored. The aim was to establish a saliva galectin-3 reference range and to demonstrate the potential diagnostic utility of salivary and serum galectin-3 levels in assessing HF. METHODS: Blood and saliva samples were collected from age-matched healthy controls (n=51) and patients with HF (n=63). Customised immunoassays were developed to quantify salivary galectin-3 levels. The diagnostic performances of these assays were evaluated by receiver operator characteristic (ROC) curves analysis. RESULTS: The galectin-3 concentrations were significantly elevated in saliva and serum samples of patients with HF compared with controls (p<0.001 and p<0.0001, respectively). Using ROC curve analysis, both serum and salivary galectin-3 gave area under the curve (AUC)=0.86 and AUC=0.73, respectively. There was also a significant correlation (r=0.4, p<0.01) between serum and salivary galectin-3 levels. CONCLUSIONS: For the first time, we have quantified galectin-3 levels in human saliva and have demonstrated potential clinical utility in diagnosing HF. Further, larger multicentre clinical trials are needed before salivary galectin-3 levels can be implemented in a clinical setting.

A pilot study to compare the detection of HPV-16 biomarkers in salivary oral rinses with tumour p16(INK4a) expression in head and neck squamous cell carcinoma patients.

BACKGROUND: Human papilloma virus-16 (HPV-16) infection is a major risk factor for a subset of head and neck squamous cell carcinoma (HNSCC), in particular oropharyngeal squamous cell carcinoma (OPSCC). Current techniques for assessing the HPV-16 status in HNSCC include the detection of HPV-16 DNA and p16(INK4a) expression in tumor tissues. When tumors originate from hidden anatomical sites, this method can be challenging. A non-invasive and cost-effective alternative to biopsy is therefore desirable for HPV-16 detection especially within a community setting to screen at-risk individuals. METHODS: The present study compared detection of HPV-16 DNA and RNA in salivary oral rinses with tumor p16(INK4a) status, in 82 HNSCC patients using end-point and quantitative polymerase chain reaction (PCR). RESULTS: Of 42 patients with p16(INK4a)-positive tumours, 39 (sensitivity = 92.9 %, PPV = 100 % and NPV = 93 %) had oral rinse samples with detectable HPV-16 DNA, using end-point and quantitative PCR. No HPV-16 DNA was detected in oral rinse samples from 40 patients with p16(INK4a) negative tumours, yielding a test specificity of 100 %. For patients with p16(INK4a) positive tumours, HPV-16 mRNA was detected using end-point reverse transcription PCR (RT-PCR) in 24/40 (sensitivity = 60 %, PPV = 100 % and NPV = 71 %), and using quantitative RT-PCR in 22/40 (sensitivity = 55 %, PPV = 100 % and NPV = 69 %). No HPV-16 mRNA was detected in oral rinse samples from the p16(INK4a)-negative patients, yielding a specificity of 100 %. CONCLUSIONS: We demonstrate that the detection of HPV-16 DNA in salivary oral rinse is indicative of HPV status in HNSCC patients and can potentially be used as a diagnostic tool in addition to the current methods.

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.

DNA Methylation at the Novel CpG Sites in the Promoter of MED15/PCQAP Gene as a Biomarker for Head and Neck Cancers.

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P < 0.05 and Mann-Whitney test P < 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P < 0.01) and 0.63 (P < 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC.

Profiling of immunoglobulins in resting and mechanically stimulated saliva.

BACKGROUND: Dysregulation of salivary immunoglobulins has been implicated in illnesses ranging from periodontal disease to HIV aids and malignant cancers. Despite these advances there is a lack of agreement among studies with regard to the salivary immunoglobulin levels in healthy controls. METHODOLOGY: Resting and mechanically stimulated saliva samples and matching serum samples were collected from healthy individuals (n = 33; 40-55 years of age; gender: 23 female, 10 male). A matrix-matched AlphaLISA(®) assay was developed to determine the concentrations of IgG1 and IgG4 in serum and saliva samples. CONCLUSION: Clear relationships were observed in the flow rate and concentration of each immunoglobulin in the two types of saliva. This study affirms the need to establish and standardize collection methods before salivary IgGs are used for diagnostic purposes.

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. PURPOSE OF WORK: 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.

Quantification of D-dimer levels in human saliva.

BACKGROUND: Plasma D-dimer tests are currently used to exclude deep vein thrombosis and pulmonary embolism. Human saliva has numerous advantages over blood as a diagnostic sample. The aims of our study were to develop a reliable immunoassay to detect D-dimer levels in saliva, and to determine the correlation between salivary and blood D-dimer levels. RESULTS/METHODOLOGY: Saliva and blood samples were collected from 40 healthy volunteers. We developed a AlphaLISA® immunoassay with acceptable analytical performances to quantify D-dimer levels in the samples. The median salivary D-dimer levels were 138.1 ng/ml (morning) and 140.7 ng/ml (afternoon), and the plasma levels were 75.0 ng/ml. Salivary D-dimer levels did not correlate with plasma levels (p = 0.61). CONCLUSION: For the first time, we have quantified D-dimer levels and found twofold increase in saliva (p < 0.05) than in plasma. Further studies are required to demonstrate the clinical relevance/utility of salivary D-dimer in patients with confirmed deep vein thrombosis and/or pulmonary embolism.

Research on the dynamic hysteresis loop model of the residence times difference (RTD)-fluxgate.

Based on the core hysteresis features, the RTD-fluxgate core, while working, is repeatedly saturated with excitation field. When the fluxgate simulates, the accurate characteristic model of the core may provide a precise simulation result. As the shape of the ideal hysteresis loop model is fixed, it cannot accurately reflect the actual dynamic changing rules of the hysteresis loop. In order to improve the fluxgate simulation accuracy, a dynamic hysteresis loop model containing the parameters which have actual physical meanings is proposed based on the changing rule of the permeability parameter when the fluxgate is working. Compared with the ideal hysteresis loop model, this model has considered the dynamic features of the hysteresis loop, which makes the simulation results closer to the actual output. In addition, other hysteresis loops of different magnetic materials can be explained utilizing the described model for an example of amorphous magnetic material in this manuscript. The model has been validated by the output response comparison between experiment results and fitting results using the model.

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 °C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA® 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.

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 ∼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°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× +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.

Antitumor effect of an antibody against gastrointestinal cancer-associated antigen CA19.9.

Despite tremendous efforts in developing cancer vaccines and monoclonal antibodies (mAbs), only a few passive or active immunotherapeutic modalities have succeeded. The failure may be due to the existence of immunosuppressive factors, which caused anergy of patients' immune system. In this study, we discovered that CA19.9 is one such immunosuppressive factor, which could inhibit the proliferation of activated human lymphocytes. However, this inhibition could be reversed by a mAb, LC44, which was specifically generated against CA19.9. An in vitro cytotoxic experiment showed that mAb LC44 could form an immunocomplex with CA19.9, which subsequently induced the production of cytotoxic T-cells reacting to CA19.9-positive cancer cells. In addition, mAb LC44 could mediate the complement-dependent cytotoxicity to CA19.9-positive cancer cells. mAb LC44 showed an antitumor effect in immunodeficient mice with colon cancer burden in the presence or absence of CA19.9. Based on the observations from this study, we postulated that the mAb, LC44, could be a promising antitumor agent for gastrointestinal cancer and worthy of further investigation.