Adult Saliva Research Articles

Immunologic characterization of the Bm05br protein using the Rhipicephalus linnaei (Audouin, 1826) species as a tick model

The identification and characterization of tick proteins allow us to discover new physiological targets useful for the development of tick control methods. Bm05br (Brazil Rhipicephalus microplus protein 05) is a protein with unknown function identified in the saliva of R. microplus. Rs05br (Brazil Rhipicephalus sanguineus protein 05), a protein with 99 % similarity to Bm05br, was identified in Rhipicephalus linnaei egg, larval, and nymphal stages, as well as in adult saliva. To improve the knowledge about both proteins, immunological characterization was performed, including antigenicity analysis, vaccination trials, and artificial feeding. The sequence and antigenicity analysis of Bm05br and Rs05br proteins showed that R. linnaei could serve as a tick model for cross-protection studies. The recombinant Bm05br protein was immunogenic. Anti-Bm05br antibodies recognized the homologous protein Rs05br in different stages, organs, and in the saliva of R. linnaei. Although rBm05br did not induce a protective response against infestation in R. linnaei in this study, further experiments could be developed taking into account new formulations and animal models for vaccination. These results also serve as a reference for future research on the function of these proteins in R. microplus and R. linnaei physiology, as well as other species of the genus Rhipicephalus.

Screening for High-Risk Human Papillomavirus Reveals HPV52 and HPV58 among Pediatric and Adult Patient Saliva Samples.

Previous research has demonstrated that the human papillomavirus (HPV) can infect a wide range of human tissues, including those within the oral cavity. High-risk oral HPV strains have been associated with the development and progression of oral cancers, including oral squamous cell carcinomas. Although many studies have examined the prevalence of the high-risk strains HPV16 and HPV18, far fewer have assessed the prevalence of other high-risk HPV strains. An approved study protocol was used to identify HPV52 and HPV58 among clinical samples (n = 87) from a saliva biorepository. Quantitative polymerase chain reaction (qPCR) and validated primers for HPV52 and HPV58 were used to facilitate this screening. This screening demonstrated that a total of n = 4/45 or 8.9% of adult saliva samples harbored high-risk HPV52, and n = 2/45 or 4.4% tested positive for high-risk HPV58. In addition, a total of n = 6/42 or 14.3% of the pediatric saliva samples tested positive for high-risk HPV, including n = 5/42 or 11.9% with HPV52 and n = 3/42 or 7.1% for HPV58. These data demonstrate the presence of the high-risk oncogenic HPV52 and HPV58 strains among both adult and pediatric clinical patient samples. More detailed longitudinal research must be conducted to determine whether this prevalence may be increasing or decreasing over time. In addition, these data strongly support public health prevention efforts, such as knowledge and awareness of the nine-valent HPV vaccine covering additional high-risk strains, including HPV52 and HPV58.

Changes in saliva protein profile throughout Rhipicephalus microplus blood feeding

BackgroundWhen feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding.MethodsTicks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species.ResultsChanges in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as “sialome switching.” This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva.ConclusionsOverall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.Graphical

Performance evaluation of fully automated cobas® 6800 CMV PCR for the detection and quantification of cytomegalovirus DNA in neonatal urine and saliva, and adult urine, saliva, and vaginal secretion.

Laboratory testing for cytomegalovirus (CMV) in bodily fluids is essential to manage congenital and prenatal CMV infection. The rapid and fully automated cobas® CMV PCR is approved only for the testing of plasma in transplant patients. To evaluate the performance of the cobas® CMV to detect and quantify CMV DNA in neonatal and adult female urine, saliva, and vaginal secretion, the limit of detection (LoD), limit of quantification (LoQ), imprecision, linearity, PCR efficiency, bias, analytical specificity, cross-reactivity, and cross-contamination of the cobas® CMV for urine, saliva, and vaginal secretion was determined. The performance of the assay was evaluated prospectively with two laboratory-developed PCR assays using neonatal and adult urine, saliva swabs, and vaginal swabs. The LoD and LoQ were 31 and 100 IU/mL, respectively, for urine, and 81 and 100 IU/mL, respectively, for vaginal secretion. The LoD and LoQ for saliva were the same (200 IU/mL). The cobas® CMV was precise (coefficient of variation ≤10%), linear (R2 ≥ 0.995), and efficient (1.07 and 1.09) between 100 and 250,000 IU/mL for the sample types. The bias and analytical specificity was <±0.30 log10 IU/mL and 100%, respectively. Cross-reactivity with non-CMV pathogens was not detected. Cross-contamination rate was 0.28%. The diagnostic accuracy, sensitivity, and specificity of the cobas® CMV for neonatal urine and saliva were ≥95.0%, ≥93.3%, and ≥90.4%, respectively. The overall percent agreement for adult urine, saliva, and vaginal secretion was 86.6%, 94.5%, and 89.4%, respectively. Taken together, the cobas® CMV demonstrated acceptable analytical and diagnostic performance, and is suitable for routine diagnostic laboratory investigation of CMV infection in neonates and adults.

Tooth Formation as Experimental Model to Study Chemotherapy on Tissue Development: Effect of a Specific Dose of Temozolomide/Veliparib.

Background: Chemotherapy treatment of cancer in children can influence formation of normal tissues, leading to irreversible changes in their structure and function. Tooth formation is susceptible to several types of chemotherapy that induce irreversible changes in the structure of enamel, dentin and dental root morphology. These changes can make the teeth more prone to fracture or to caries when they have erupted. Recent studies report successful treatment of brain tumors with the alkylating drug temozolomide (TMZ) in combination with veliparib (VLP) in a glioblastoma in vivo mouse model. Whether these drugs also affect tooth formation is unknown. Aim: In this study the effect of TMZ/VLP on incisor formation was investigated in tissue sections of jaws from mice and compared with mice not treated with these drugs. Materials and method: The following aspects were studied using immunohistochemistry of specific protein markers including: (1) proliferation (by protein expression of proliferation marker Ki67) (2) a protein involved in paracellular ion transport (expression of tight junction (TJ) protein claudin-1) and (3) in transcellular passage of ions across the dental epithelium (expression of Na+, K+ 2Cl- cotransporter/NKCC1). Results: Chemotherapy with TMZ/VLP strongly reduced immunostaining for claudin-1 in distal parts of maturation ameloblasts. No gross changes were found in the treated mice, either in cell proliferation in the dental epithelium at the cervical loop or in the immunostaining pattern for NKCC1 in (non-ameloblastic) dental epithelium. The salivary glands in the treated mice contained strongly reduced immunostaining for NKCC1 in the basolateral membranes of acinar cells. Discussion/Conclusions: Based on the reduction of claudin-1 immunostaining in ameloblasts, TMZ/VLP may potentially influence forming enamel by changes in the structure of TJs structures in maturation ameloblasts, structures that are crucial for the selective passage of ions through the intercellular space between neighboring ameloblasts. The strongly reduced basolateral NKCC1 staining seen in fully-grown salivary glands of TMZ/VLP-treated mice suggests that TMZ/VLF could also influence ion transport in adult saliva by the salivary gland epithelium. This may cause treated children to be more susceptible to caries.

LL-37 transports immunoreactive cGAMP to activate STING signaling and enhance interferon-mediated host antiviral immunity.

Cyclic 2',3'-GMP-AMP (cGAMP) binds to and activates stimulator of interferon genes (STING), which then induces interferons to drive immune responses against tumors and pathogens. Exogenous cGAMP produced by infected and malignant cells and synthetic cGAMP used in immunotherapy must traverse the cell membrane to activate STING in target cells. However, as an anionic hydrophilic molecule, cGAMP is not inherently membrane permeable. Here, we show that LL-37, a human host defense peptide, can function as a transporter of cGAMP. LL-37 specifically binds cGAMP and efficiently delivers cGAMP into target cells. cGAMP transferred by LL-37 activates robust interferon responses and host antiviral immunity in a STING-dependent manner. Furthermore, we report that LL-37 inducers vitamin D3 and sodium butyrate promote host immunity by enhancing endogenous LL-37 expression and its mediated cGAMP immune response. Collectively, our data uncover an essential role of LL-37 in innate immune activation and suggest new strategies for immunotherapy.

Comparability of microbiota of swabbed and spit saliva.

In general, saliva is used for microbiota analysis in longitudinal studies, and several collection methods are being used. Using a robust sample collection procedure is important, as it may influence salivary composition. This study explored the comparability of the microbiota of swabbed and spit saliva. Twenty‐two females participated in this cross‐sectional study. The bacterial composition of the three saliva samples (swab collected by the participant (SW‐P), swab collected by the researcher (SW‐R), and spit (SP) was assessed by 16S rRNA gene amplicon sequencing. The bacterial composition of the swabbed and the spit saliva was significantly different irrespective of the operator, and Shannon diversity was significantly higher in spit saliva than in SW‐P and SW‐R. The salivary microbiota of spit and swabbed adult saliva differs significantly. Research on microbial composition therefore requires collection of similar saliva sample types in all study participants.

A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva

BackgroundThe argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential functions in tick feeding and host defence modulation, which may contribute to host infection by tick-borne pathogens. Hence, these proteins could be candidate antigen targets for the development of vaccines aimed at the control and prevention of O. erraticus infestations and the diseases this tick transmits. The objective of the present work was to obtain and characterise the proteome of the saliva of O. erraticus adult ticks as a means to identify and select novel salivary antigen targets.MethodsA proteomics informed by transcriptomics (PIT) approach was applied to analyse samples of female and male saliva separately using the previously obtained O. erraticus sialotranscriptome as a reference database and two different mass spectrometry techniques, namely liquid chromatography–tandem mass spectrometry (LC–MS/MS) in data-dependent acquisition mode and sequential window acquisition of all theoretical fragment ion spectra MS (SWATH-MS).ResultsUp to 264 and 263 proteins were identified by LC–MS/MS in the saliva of O. erraticus female and male ticks, respectively, totalling 387 non-redundant proteins. Of these, 224 were further quantified by SWATH-MS in the saliva of both male and female ticks. Quantified proteins were classified into 23 functional categories and their abundance compared between sexes. Heme/iron-binding proteins, protease inhibitors, proteases, lipocalins and immune-related proteins were the categories most abundantly expressed in females, while glycolytic enzymes, protease inhibitors and lipocalins were the most abundantly expressed in males. Ninety-seven proteins were differentially expressed between the sexes, of which 37 and 60 were overexpressed in females and males, respectively.ConclusionsThe PIT approach demonstrated its usefulness for proteomics studies of O. erraticus, a non-model organism without genomic sequences available, allowing the publication of the first comprehensive proteome of the saliva of O. erraticus reported to date. These findings confirm important quantitative differences between sexes in the O. erraticus saliva proteome, unveil novel salivary proteins and functions at the tick–host feeding interface and improve our understanding of the physiology of feeding in O. erraticus ticks. The integration of O. erraticus sialoproteomic and sialotranscriptomic data will drive a more rational selection of salivary candidates as antigen targets for the development of vaccines aimed at the control of O. erraticus infestations and the diseases it transmits.Graphical

Saliva is inferior to nose and throat swabs for SARS-CoV-2 detection in children.

It is important to identify children and adolescents infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as they are often asymptomatic and may unintentionally spread the virus. However, information on the best sampling methods are limited. Most analyse nose and throat swabs with real-time polymerase chain reaction (RT-PCR), but these tests are uncomfortable and young children may not co-operate, increasing sub-optimal sample collection and false-negative results. Detecting SARS-CoV-2 in adult saliva, using RT-PCR1,2 has shown promise. However, young children struggle to produce saliva spontaneously and there have been conflicting results about using this method for children.3,4 In contrast, oral swabs cause minimal discomfort, do not generate aerosols, collect adequate viral material5 and can be used by parents or day care staff without personal protective equipment.

Levels of Salivary Enzymes of Apolygus Lucorum (Hemiptera: Miridae), From 1st Instar Nymph to Adult, and Their Potential Relation to Bug Feeding.

In recent years, Apolygus lucorum has caused increasing damage to cotton and fruit trees in China. The salivary enzymes secreted by A. lucorum when sucking on host plants induce a series of biochemical reactions in plants, and the pre-oral digestion benefits the bug feeding. In this study, the food intake of A. lucorum from 1st instar nymphs to adults was measured, and the corresponding salivary activity of pectinase, amylase, cellulase, protease, polyphenol oxidase and peroxidase was determined. Daily food intake varied with developmental stage, peaking in 3rd and 4th instar nymphs. Pectinase, amylase, cellulase and protease were detected in both nymphal and adult saliva of A. lucorum, while neither polyphenol oxidase nor peroxidase was detected. Protease activity varied with food intake peaking at the 3rd-4th instar, and then slightly decreasing at the 5th instar. Levels of pectinase, amylase and cellulase increased significantly with the daily feeding level until the 3rd instar, corresponding with increasing damage to host plants. The activity of both cellulase and protease had a significant linear relationship with the average daily food intake. The increasing activity of enzymes in saliva explain stage-specific impacts of A. lucorum on the host plants, and suggest that optimal management of A. lucorum would be confined to its control threshold prior to the peak of daily feeding in the 3rd instar.

Ectodysplasin A in Biological Fluids and Diagnosis of Ectodermal Dysplasia

The tumor necrosis factor (TNF) family ligand ectodysplasin A (EDA) is produced as 2 full-length splice variants, EDA1 and EDA2, that bind to EDA receptor (EDAR) and X-linked EDA receptor (XEDAR/EDA2R), respectively. Inactivating mutations in Eda or Edar cause hypohidrotic ectodermal dysplasia (HED), a condition characterized by malformations of the teeth, hair and glands, with milder deficiencies affecting only the teeth. EDA acts early during the development of ectodermal appendages—as early as the embryonic placode stage—and plays a role in adult appendage function. In this study, the authors measured EDA in serum, saliva and dried blood spots. The authors detected 3- to 4-fold higher levels of circulating EDA in cord blood than in adult sera. A receptor binding-competent form of EDA1 was the main form of EDA but a minor fraction of EDA2 was also found in fetal bovine serum. Sera of EDA-deficient patients contained either background EDA levels or low levels of EDA that could not bind to recombinant EDAR. The serum of a patient with a V262F missense mutation in Eda, which caused a milder form of X-linked HED (XLHED), contained low levels of EDA capable of binding to EDAR. In 2 mildly affected carriers, intermediate levels of EDA were detected, whereas a severely affected carrier had no active EDA in the serum. Small amounts of EDA were also detectable in normal adult saliva. Finally, EDA could be measured in spots of wild-type adult or cord blood dried onto filter paper at levels significantly higher than that measured in EDA-deficient blood. Measurement of EDA levels combined with receptor-binding assays might be of relevance to aid in the diagnosis of total or partial EDA deficiencies.

Monitoring of human herpesviruses-6 and -7 DNA in saliva samples during the acute and convalescent phases of exanthem subitum.

The amounts of the DNAs of human herpesviruses-6 (HHV-6) and -7 (HHV-7) in saliva samples were monitored during the acute and convalescent phases of exanthem subitum (ES) to elucidate the kinetics of virus shedding after ES. A total of 247 saliva samples were collected from 17 children (5 males and 12 females: 8-31 months old at onset). The monitoring period ranged from 152 to 721 days after onset, and in 15 children it was longer than 1 year. Among the 17 cases, 16 were attributed to HHV-6B, while a single case was attributed to HHV-7. Detection rates and average amounts of HHV-6 DNA in saliva samples after ES attributed to HHV-6B were low in the acute phase, increased to the maximum in the convalescent phase at 3-7 months, and then decreased. In addition, to investigate the source of infection, saliva samples from the older siblings (age 3-9 years) and parents of ES patients and children with a history of ES were also examined. The detection rate of HHV-6 DNA in saliva samples from 3- to 9-year-old children was significantly higher than the rate in adult saliva samples. Taken together, these findings suggest that the saliva of children in the convalescent phase of ES might be a more likely source of HHV-6 infection than that of adults. J. Med. Virol. 89:696-702, 2017. © 2016 Wiley Periodicals, Inc.

Proteomic characterization of the acid-insoluble fraction of whole saliva from preterm human newborns

The acid-insoluble salivary proteome obtained by addition of TFA to whole human saliva from adults, preterm and at-term newborns has been analysed by 2-DE in order to evidence differences among the three groups, and integrate data previously obtained on the acid-soluble fraction. 2-DE spots differentially expressed among the three groups were submitted to in-gel tryptic digestion and the peptide mixtures analysed by high resolution HPLC–ESI–MS/MS. By this strategy, we identified 3 over-expressed proteins in at-term newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100A6, S100A7, S100A9, prolactin-inducible protein, Ig kappa chain, cystatin SN, cystatin S/SA and α-amylase 1). Four spots, already detected but not characterized by other authors in human saliva 2-DE, were attributed to different protein species of S100A9 (long-type and long-type monophosphorylated, short-type and short-type monophosphorylated) by MS/MS analysis of tryptic peptides and sequential staining of 2-DE gels with Pro-Q Diamond, for specific detection of phosphoproteins, and total protein SYPRO Ruby stain. SignificanceDifferential protein expression analysis of the acid insoluble fraction of saliva from preterm, at-term newborns and adults has been performed in this study by coupling 2-DE analysis and high-resolution tandem mass spectrometry in order to complete the information previously obtained by top-down LC–MS only on the acid-soluble proteome. Several proteins identified in the acid insoluble fraction of both preterm newborn and adult saliva are not of glandular origin, being only prolactin-inducible protein, salivary cystatins, α-amylase and polymeric immunoglobulin receptor exclusive of salivary glands.Three proteins resulted increased in at-term newborns with respect to preterm newborns and adults: BPI fold-containing family A member 1, two proteoforms of annexin A1 and keratin type 1 cytoskeletal 13, while several proteins were significantly increased in adults.

Prevalence of blood contamination in adult saliva and quantitative measurement of salivary hemoglobin level by using an anti-human hemoglobin monoclonal antibody

Prevalence of blood contamination in adult saliva and quantitative measurement of salivary hemoglobin level by using an anti-human hemoglobin monoclonal antibody

Breastmilk-Saliva Interactions Boost Innate Immunity by Regulating the Oral Microbiome in Early Infancy.

IntroductionXanthine oxidase (XO) is distributed in mammals largely in the liver and small intestine, but also is highly active in milk where it generates hydrogen peroxide (H2O2). Adult human saliva is low in hypoxanthine and xanthine, the substrates of XO, and high in the lactoperoxidase substrate thiocyanate, but saliva of neonates has not been examined.ResultsMedian concentrations of hypoxanthine and xanthine in neonatal saliva (27 and 19 μM respectively) were ten-fold higher than in adult saliva (2.1 and 1.7 μM). Fresh breastmilk contained 27.3±12.2 μM H2O2 but mixing baby saliva with breastmilk additionally generated >40 μM H2O2, sufficient to inhibit growth of the opportunistic pathogens Staphylococcus aureus and Salmonella spp. Oral peroxidase activity in neonatal saliva was variable but low (median 7 U/L, range 2–449) compared to adults (620 U/L, 48–1348), while peroxidase substrate thiocyanate in neonatal saliva was surprisingly high. Baby but not adult saliva also contained nucleosides and nucleobases that encouraged growth of the commensal bacteria Lactobacillus, but inhibited opportunistic pathogens; these nucleosides/bases may also promote growth of immature gut cells. Transition from neonatal to adult saliva pattern occurred during the weaning period. A survey of saliva from domesticated mammals revealed wide variation in nucleoside/base patterns.Discussion and ConclusionDuring breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors. Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral–and hence gut–microbiota. Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

Saliva from nymph and adult females of Haemaphysalis longicornis: a proteomic study.

BackgroundHaemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods.MethodsH. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences.ResultsA total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products.ConclusionsThis study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0918-y) contains supplementary material, which is available to authorized users.

Salivary testosterone measurement by liquid chromatography tandem mass spectrometry in adult males and females

Salivary testosterone (Sal-T) may be a useful surrogate of serum free testosterone. The study aims were to use a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to determine whether Sal-T concentrations accurately reflect Sal-T concentrations in both sexes and to investigate practical aspects of sample collection. Saliva and serum samples were collected in 104 male and 91 female subjects. A more sensitive LC-MS/MS assay was developed to enable Sal-T quantitation in the low concentrations found in females. Saliva (200 µL) was extracted with 1 mL of methyl-tert-butyl ether following the addition of D5-testosterone. Quantitation was performed using a Waters TQ-S mass spectrometer. The assay achieved a lower limit of quantification of 5 pmol/L, sufficiently sensitive to measure testosterone in female saliva. Sal-T showed a diurnal variation but samples taken at weekly and monthly intervals showed no significant differences. Sal-T was stable at ambient temperature for up to 5 days, after freeze-thawing and 3 years frozen storage. Reference intervals for Sal-T were 93-378 pmol/L in males and 5-46 pmol/L in females. Sal-T correlated significantly with serum calculated free-T in males (r = 0.71, P < 0.001) and in females (r = 0.39, P < 0.001). These results confirm that testosterone can be reliably and accurately measured by LC-MS/MS in both adult male and female saliva samples. These results lay the foundation for further exploration of the clinical application of Sal- T as a reliable alternative to serum testosterone in the diagnosis and management of androgen disorders and assessment of androgen status in clinical research.

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 1.0μM. 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±19.5μM respectively) compared to adults (4.3±3.3; 4.6±4.5μM); 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 photodiode 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.

HPLC‐ESI‐MS and MS/MS structural characterization of multifucosylated N‐glycoforms of the basic proline‐rich protein IB‐8a CON1+ in human saliva

This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1(+), a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1(+) and the variant IB-8a CON1(-), lacking of the glycosylation site, have been also detected in human saliva.

Human infant saliva peptidome is modified with age and diet transition

In order to describe developmental changes in human salivary peptidome, whole saliva was obtained from 98 infants followed longitudinally at 3 and 6months of age. Data on teeth eruption and diet at the age of 6months were also recorded. Salivary peptide extracts were characterised by label-free MALDI-MS. Peptides differentially expressed between the two ages, and those significantly affected by teeth eruption or introduction of solid foods were identified by MALDI TOF-TOF and LC ESI MS-MS. Out of 81 peaks retained for statistical analysis, 26 were overexpressed at the age of 6months. Exposure to solid foods had a more pronounced effect on profiles (overexpression of nine peaks) than teeth eruption (overexpression of one peak). Differential peaks corresponded to fragments of acidic and basic PRPs, statherin and histatin. Comparison with existing knowledge on adult saliva peptidome revealed that proteolytic processing of salivary proteins is qualitatively quite comparable in infants and in adults. However, age and diet are modulators of salivary peptidome in human infants.