Estrogen metabolizing enzymes in endometrium and endometriosis

BACKGROUND Estradiol (E-2) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)l in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS Aromatase and 17 beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17 beta-HSD, EST and STS were readily detectable. Only 17 beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17 beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. CONCLUSIONS In endometriosis lesions, the balance is tilted in favor of enzymes producing E2. This is due to a suppression of types 2 and 4 17 beta-HSD, and an increased expression of aromatase and type 1 17 beta-HSD in ectopic endometrium.

Effects of selective oestrogen receptor modulators on proliferation in tissue cultures of pre- and postmenopausal human endometrium

We characterised the effects of selective oestrogen receptor modulators (SERM) in explant cultures of human endometrium tissue. Endometrium tissues were cultured for 24 h in Millicell-CM culture inserts in serum-free medium in the presence of vehicle,17 beta-estradiol (17 beta-E2,1 nM), oestrogen receptor (ER) antagonist ICI 164.384 (40 nM), and 4-OH-tamoxifen (40 nM), raloxifene (4 nM), lasofoxifene (4 nM)and acolbifene (4 nM). Protein expression of ER alpha, ER beta 1 and Ki-67 were evaluated by immunohistochemistry (IHC). The proliferative fraction was assessed by counting the number of Ki-67 positive cells. Nuclear staining of ER( and ER(1 was observed in the glandular epithelium and stroma of pre- and postmenopausal endometrium. ER(1 protein was also localized in the endothelial cells of blood vessels. Treating premenopausal endometrium tissue with 17 beta-E2 increased the fraction of Ki-67 positive cells (p < 0.001) by 55% in glands compared to the control. Raloxifene (4 nM) increased (p < 0.05) the Ki-67 positive fraction. All other SERMS did not affect proliferation in this model. Treating postmenopausal endometrium with 17(-E2 increased (p < 0.001) the fraction of Ki-67 positive cells by 250% in glands compared to the control. A similar effect was also seen for 4-OH-tamoxifen, whereas the rest of SERMs did not stimulate proliferation. We demonstrated that oestradiol increases the fraction of proliferating cells in short term explant cultures of postmenopausal endometrium. In addition, we were able to reveal the agonistic properties of 4-OH-tamoxifen and confirm that raloxifene and next-generation SERMs acolbifene and lasofoxifene were neutral on the human postmenopausal endometrium. (C) 2008 Elsevier Ltd. All rights reserved.

One-step homogeneous C-reactive protein assay for saliva

Background: Cardiovascular disease is the leading cause of death in the world. Human C-reactive protein (CRP) has been used in the risk assessment of coronary events. Human saliva mirrors the body's health and well-being and is non-invasive, easy to collect and ideal for third world countries as well as for large patient screening. The aim was to establish a saliva CRP reference range and to demonstrate the clinical utility of salivary CRP levels in assessing the coronary events in a primary health care setting. Methods: We have used a homogeneous bead based assay to detect CRP levels in human saliva. We have developed a rapid 15 min (vs 90 min), sequential, one-step assay to detect CRP in saliva. Saliva was collected from healthy volunteers (n = 55, ages 20-70 years) as well as from cardiac patients (n = 28, ages 43-86 years). Results: The assay incubation time was optimised from 90 min to 15 mm and generated a positive correlation (n = 29, range 10-2189 pg/mL, r2 = 0.94; Passing Bablok slope 0.885. Intercept 0, p>0.10), meaning we could decrease the incubation time and produce equivalent results with confidence. The mean CRP level in the saliva of healthy human volunteers was 285 pg/mL and in cardiac patients was 1680 pg/mL (p<0.01). Analysis of CRP concentrations in paired serum and saliva samples from cardiac patients gave a positive correlation (r2 = 0.84, p<0.001) and the salivary CRP concentration capable of distinguishing healthy from diseased patients. Conclusions: The results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events. (C) 2011 Elsevier B.V. All rights reserved.

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.

Human saliva as a tool to investigate intimate partner violence

Human saliva mirrors body’s health and well-being and many of the biomolecules present in blood or urine can also be found in salivary secretions. However, biomolecular concentrations in saliva are usually one tenth to one thousandth of the levels in blood (Pfaffe et al., 2011). Sensitive detection technology platforms are therefore required to detect biomolecules in saliva. Another road block to the advancement of salivary diagnostics is the lack of information related to healthy state saliva vs. a diseased saliva, baseline levels and reference ranges and diurnal variations. Saliva has numerous advantages over blood or urine as a diagnostic fluid: (a) the non-invasive nature of sample collection and the simple, safe, painless and cost-effective methods to collect it; (b) unskilled personnel can collect saliva samples at multiple time points; and (c) the total protein concentration is approximately a quarter of that is present in plasma, which makes it easier to investigate low abundance proteins (Pfaffe et al., 2011). Currently, saliva assays are routinely used to determine, diseases such as HIV, drugs and substances of abuse to provide information on exposure and give qualitative information on the type of illicit drug used (Kintz et al., 2009), cortisol levels for diagnosing Cushing’s syndrome (Doi et al., 2008), and use for biomonitoring of exposure to chemicals (Caporossi et al., 2010) to measure hormones (Gröschl, 2009)....

Evaluation of saliva collection devices for the analysis of proteins

Background: Human saliva mirrors the body's health and can be collected non-invasively, does not require specialized skills and is suitable for large population based screening programs. The aims were twofold: to evaluate the suitability of commercially available saliva collection devices for quantifying proteins present in saliva and to provide levels for C-reactive protein (CRP), myoglobin, and immunoglobin E (IgE) in saliva of healthy individuals as a baseline for future studies. Methods: Saliva was collected from healthy volunteers (n = 17, ages 18-33 years). The following collection methods were evaluated: drool; Salimetrics (R) Oral Swab (SOS); Salivette (R) Cotton and Synthetic (Sarstedt) and Greiner Bio-One Saliva Collection System (GBO SCS (R)). We used AlphaLISA (R) assays to measure CRP, IgE and myoglobin levels in human saliva. Results: Significant (p<0.05) differences in the salivary flow rates were observed based on the method of collection, Le. salivary flow rates were significantly lower (p<0.05) in unstimulated saliva (Le. drool and SOS), when compared with mechanically stimulated methods (p<0.05) (Salivette (R) Cotton and Synthetic) and acid stimulated method (p<0.05) (SCS (R)). Saliva collected using SOS yielded significantly (p<0.05) lower concentrations of myoglobin and CRP, whilst, saliva collected using the Salivette (R) Cotton and Synthetic swab yielded significantly (p<0.05) lower myoglobin and IgE concentrations respectively. Conclusions: The results demonstrated significantly relevant differences in analyte levels based on the collection method. Significant differences in the salivary flow rates were also observed depending on the saliva collection method. The data provide preliminary baseline values for salivary CRP, myoglobin, and IgE levels in healthy participants and based on the collection method. (C) 2012 Elsevier B.V. All rights reserved.

Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry

Saliva contains a number of biochemical components which may be useful for diagnosis/monitoring of metabolic disorders, and as markers of cancer or heart disease. Saliva collection is attractive as a non-invasive sampling method for infants and elderly patients. We present a method suitable for saliva collection from neonates. We have applied this technique for the determination of salivary nucleotide metabolites. Saliva was collected from 10 healthy neonates using washed cotton swabs, and directly from 10 adults. Two methods for saliva extraction from oral swabs were evaluated. The analytes were then separated using high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The limits of detection for 14 purine/pyrimidine metabolites were variable, ranging from 0.01 to 1.0 mu M. Recovery of hydrophobic purine/pyrimidine metabolites from cotton tips was consistently high using water/acetonitrile extraction (92.7-111%) compared with water extraction alone. The concentrations of these metabolites were significantly higher in neonatal saliva than in adults. Preliminary ranges for nucleotide metabolites in neonatal and adult saliva are reported. Hypoxanthine and xanthine were grossly raised in neonates (49.3 +/- 25.4; 30.9 +/- 19.5 mu M respectively) compared to adults (4.3 +/- 3.3; 4.6 +/- 4.5 mu M); nucleosides were also markedly raised in neonates. This study focuses on three essential details: contamination of oral swabs during manufacturing and how to overcome this; weighing swabs to accurately measure small saliva volumes; and methods for extracting saliva metabolites of interest from cotton swabs. A method is described for determining nucleotide metabolites using HPLC with photo-diode array or MS/MS. The advantages of utilising saliva are highlighted. Nucleotide metabolites were not simply in equilibrium with plasma, but may be actively secreted into saliva, and this process is more active in neonates than adults. (C) 2013 Elsevier B.V. All rights reserved.

High-yeld RNA-extraction method for saliva

BACKGROUND: The use of salivary diagnostics is increasing because of its noninvasiveness, ease of sampling, and the relatively low risk of contracting infectious organisms. Saliva has been used as a biological fluid to identify and validate RNA targets in head and neck cancer patients. The goal of this study was to develop a robust, easy, and cost-effective method for isolating high yields of total RNA from saliva for downstream expression studies. METHODS: Oral whole saliva (200 mu L) was collected from healthy controls (n = 6) and from patients with head and neck cancer (n = 8). The method developed in-house used QIAzol lysis reagent (Qiagen) to extract RNA from saliva (both cell-free supernatants and cell pellets), followed by isopropyl alcohol precipitation, cDNA synthesis, and real-time PCR analyses for the genes encoding beta-actin ("housekeeping" gene) and histatin (a salivary gland-specific gene). RESULTS: The in-house QIAzol lysis reagent produced a high yield of total RNA (0.89 -7.1 mu g) from saliva (cell-free saliva and cell pellet) after DNase treatment. The ratio of the absorbance measured at 260 nm to that at 280 nm ranged from 1.6 to 1.9. The commercial kit produced a 10-fold lower RNA yield. Using our method with the QIAzol lysis reagent, we were also able to isolate RNA from archived saliva samples that had been stored without RNase inhibitors at -80 degrees C for >2 years. CONCLUSIONS: Our in-house QIAzol method is robust, is simple, provides RNA at high yields, and can be implemented to allow saliva transcriptomic studies to be translated into a clinical setting.

Saliva proteome research: current status and future outlook

Human saliva harbours proteins of clinical relevance and about 30% of blood proteins are also present in saliva. This highlights that saliva can be used for clinical applications just as urine or blood. However, the translation of salivary biomarker discoveries into clinical settings is hampered by the dynamics and complexity of the salivary proteome. This review focuses on the current status of technological developments and achievements relating to approaches for unravelling the human salivary proteome. We discuss the dynamics of the salivary proteome, as well as the importance of sample preparation and processing techniques and their influence on downstream protein applications; post-translational modifications of salivary proteome and protein: protein interactions. In addition, we describe possible enrichment strategies for discerning post-translational modifications of salivary proteins, the potential utility of selected-reaction-monitoring techniques for biomarker discovery and validation, limitations to proteomics and the biomarker challenge and future perspectives. In summary, we provide recommendations for practical saliva sampling, processing and storage conditions to increase the quality of future studies in an emerging field of saliva clinical proteomics. We propose that the advent of technologies allowing sensitive and high throughput proteome-wide analyses, coupled to well-controlled study design, will allow saliva to enter clinical practice as an alternative to blood-based methods due to its simplistic nature of sampling, non-invasiveness, easy of collection and multiple collections by untrained professionals and cost-effective advantages.

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((R)) 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.

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((R)) 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 novel saliva-based microRNA biomarker panel to detect head and neck cancers

Background MicroRNAs (miRNAs) are known to play an important role in cancer development by post-transcriptionally affecting the expression of critical genes. The aims of this study were two-fold: (i) to develop a robust method to isolate miRNAs from small volumes of saliva and (ii) to develop a panel of saliva-based diagnostic biomarkers for the detection of head and neck squamous cell carcinoma (HNSCC). Methods Five differentially expressed miRNAs were selected from miScript™ miRNA microarray data generated using saliva from five HNSCC patients and five healthy controls. Their differential expression was subsequently confirmed by RT-qPCR using saliva samples from healthy controls (n = 56) and HNSCC patients (n = 56). These samples were divided into two different cohorts, i.e., a first confirmatory cohort (n = 21) and a second independent validation cohort (n = 35), to narrow down the miRNA diagnostic panel to three miRNAs: miR-9, miR-134 and miR-191. This diagnostic panel was independently validated using HNSCC miRNA expression data from The Cancer Genome Atlas (TCGA), encompassing 334 tumours and 39 adjacent normal tissues. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic capacity of the panel. Results On average 60 ng/μL miRNA was isolated from 200 μL of saliva. Overall a good correlation was observed between the microarray data and the RT-qPCR data. We found that miR-9 (P <0.0001), miR-134 (P <0.0001) and miR-191 (P <0.001) were differentially expressed between saliva from HNSCC patients and healthy controls, and that these miRNAs provided a good discriminative capacity with area under the curve (AUC) values of 0.85 (P <0.0001), 0.74 (P < 0.001) and 0.98 (P < 0.0001), respectively. In addition, we found that the salivary miRNA data showed a good correlation with the TCGA miRNA data, thereby providing an independent validation. Conclusions We show that we have developed a reliable method to isolate miRNAs from small volumes of saliva, and that the saliva-derived miRNAs miR-9, miR-134 and miR-191 may serve as novel biomarkers to reliably detect HNSCC. © 2014 International Society for Cellular Oncology.

Validation of an immunoassay to measure plasminogen-activator inhibitor-1 concentrations in human saliva

Introduction We have previously shown that the concentrations of D-dimer are significantly elevated in saliva compared with plasma. Saliva offers several advantages compared with blood analysis. We hypothesised that human saliva contains plasminogen activator inhibitor-1 (PAI-1) and that the concentrations are not affected by the time of saliva collection. The aim was to adopt and validate an immunoassay to quantify PAI-1 concentrations in saliva and to determine whether saliva collection time has an influence in the measurement. Materials and methods Two saliva samples (morning and afternoon) from the same day were collected from healthy subjects (N = 40) who have had no underlying heart conditions. A customized AlphaLISA® immunoassay (PerkinElmer®, MA, USA) was adopted and used to quantify PAI-1 concentrations. We validated the analytical performance of the customized immunoassay by calculating recovery of known amount of analyte spiked in saliva. Results: The recovery (95.03%), intra- (8.59%) and inter-assay (7.52%) variations were within the acceptable ranges. The median salivary PAI-1 concentrations were 394 pg/mL (interquartile ranges (IQR) 243.4-833.1 pg/mL) in the morning and 376 (129.1-615.4) pg/mL in the afternoon and the plasma concentration was 59,000 (24,000-110,000) pg/mL. Salivary PAI-1 did not correlate with plasma (P = 0.812). Conclusions The adopted immunoassay produced acceptable assay sensitivity and specificity. The data demonstrated that saliva contains PAI-1 and that its concentration is not affected by the time of saliva collection. There is no correlation between salivary and plasma PAI-1 concentrations. Further studies are required to demonstrate the utility of salivary PAI-1 in CVD risk factor studies.

Cuelure but not zingerone make the sex pheromone of male Bactrocera tryoni (Tephritidae: Diptera) more attractive to females

In tephritid fruit flies of the genus Bactrocera Macquart, a group of plant derived compounds (sensu amplo ‘male lures') enhance the mating success of males that have consumed them. For flies responding to the male lure methyl eugenol, this is due to the accumulation of chemicals derived from the male lure in the male rectal gland (site of pheromone synthesis) and the subsequent release of an attractive pheromone. Cuelure, raspberry ketone and zingerone are a second, related group of male lures to which many Bactrocera species respond. Raspberry ketone and cuelure are both known to accumulate in the rectal gland of males as raspberry ketone, but it is not known if the emitted male pheromone is subsequently altered in complexity or is more attractive to females. Using Bactrocera tryoni as our test insect, and cuelure and zingerone as our test chemicals, we assess: (i) lure accumulation in the rectal gland; (ii) if the lures are released exclusively in association with the male pheromone; and (iii) if the pheromone of lure-fed males is more attractive to females than the pheromone of lure-unfed males. As previously documented, we found cuelure was stored in its hydroxyl form of raspberry ketone, while zingerone was stored largely in an unaltered state. Small but consistent amounts of raspberry ketone and β-(4-hydroxy-3-methoxyphenyl)-propionic acid were also detected in zingerone-fed flies. Males released the ingested lures or their analogues, along with endogenous pheromone chemicals, only during the dusk courtship period. More females responded to squashed rectal glands extracted from flies fed on cuelure than to glands from control flies, while more females responded to the pheromone of calling cuelure-fed males than to control males. The response to zingerone treatments in both cases was not different from the control. The results show that male B. tryoni release ingested lures as part of their pheromone blend and, at least for cuelure, this attracts more females.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.