Saliva, a bodily fluid with recognized and potential diagnostic applications

Human whole saliva is a bodily fluid that can be obtained easily by noninvasive techniques. Specimens can be collected by the patient also at home in order to monitor health status and variations of several analytes of clinical interest. The contributions to whole saliva include secretions from salivary glands and, among others, from the gingival crevicular fluid that derives from the epithelial mucosa. Therefore, saliva is currently a relevant diagnostic fluid for many substances, including steroids, nonpeptide hormones, therapeutic drugs, and drugs of abuse. This review at first briefly describes the different contributions to whole saliva. A section illustrates the procedures for the collection, handling, and storage of salivary specimens. Another section describes the present use of whole saliva for diagnostic purposes and its specific utilization for the diagnosis of several local and systemic diseases. The final sections illustrate the future opportunities offered by various not conventional techniques with a focus on the most recent –omic investigations. It describes the various issues that have to be taken into account to avoid false positives and negatives, such as the strength of the experimental plan, the adequacy of the number of samples under study, and the proper choice of controls

Top-Down Proteomics of Human Saliva Highlights Anti-inflammatory, Antioxidant, and Antimicrobial Defense Responses in Alzheimer Disease

Alzheimer disease (AD) is the most prevalent neurodegenerative disease in the elderly, characterized by accumulation in the brain of misfolded proteins, inflammation, and oxidative damage leading to neuronal cell death. By considering the viewpoint that AD onset and worsening may be influenced by environmental factors causing infection, oxidative stress, and inflammatory reaction, we investigated the changes of the salivary proteome in a population of patients with respect to that in healthy controls (HCs). Indeed, the possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. Moreover, the oral cavity continuously established adaptative and protective processes toward exogenous stimuli. In the present study, qualitative/quantitative variations of 56 salivary proteoforms, including post-translationally modified derivatives, have been analyzed by RP-HPLC-ESI-IT-MS and MS/MS analyses, and immunological methods were applied to validate MS results. The salivary protein profile of AD patients was characterized by significantly higher levels of some multifaceted proteins and peptides that were either specific to the oral cavity or also expressed in other body districts: (i) peptides involved in the homeostasis of the oral cavity; (ii) proteins acting as ROS/RNS scavengers and with a neuroprotective role, such as S100A8, S100A9, and their glutathionylated and nitrosylated proteoforms; cystatin B and glutathionylated and dimeric derivatives; (iii) proteins with antimicrobial activity, such as α-defensins, cystatins A and B, histatin 1, statherin, and thymosin β4, this last with a neuroprotective role at the level of microglia. These results suggested that, in response to injured conditions, Alzheimer patients established defensive mechanisms detectable at the oral level. Data are available via ProteomeXchange with identifier PXD021538

Salivary Cystatin D Interactome in Patients with Systemic Mastocytosis: An Exploratory Study

Mastocytosis, a rare blood disorder characterized by the proliferation of clonal abnormal mast cells, has a variegated clinical spectrum and diagnosis is often difficult and delayed. Recently we proposed the cathepsin inhibitor cystatin D-R26 as a salivary candidate biomarker of systemic mastocytosis (SM). Its C26 variant is able to form multiprotein complexes (mPCs) and since protein–protein interactions (PPIs) are crucial for studying disease pathogenesis, potential markers, and therapeutic targets, we aimed to define the protein composition of the salivary cystatin D-C26 interactome associated with SM. An exploratory affinity purification-mass spectrometry method was applied on pooled salivary samples from SM patients, SM patient subgroups with and without cutaneous symptoms (SM+C and SM−C), and healthy controls (Ctrls). Interactors specifically detected in Ctrls were found to be implicated in networks associated with cell and tissue homeostasis, innate system, endopeptidase regulation, and antimicrobial protection. Interactors distinctive of SM−C patients participate to PPI networks related to glucose metabolism, protein S-nitrosylation, antibacterial humoral response, and neutrophil degranulation, while interactors specific to SM+C were mainly associated with epithelial and keratinocyte differentiation, cytoskeleton rearrangement, and immune response pathways. Proteins sensitive to redox changes, as well as proteins with immunomodulatory properties and activating mast cells, were identified in patients; many of them were involved directly in cytoskeleton rearrangement, a process crucial for mast cell activation. Although preliminary, these results demonstrate that PPI alterations of the cystatin D-C26 interactome are associated with SM and provide a basis for future investigations based on quantitative proteomic analysis and immune validation

A Catalog of Coding Sequence Variations in Salivary Proteins’ Genes Occurring during Recent Human Evolution

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins’ gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5′-3′ untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes

Investigation by top-down high-performance liquid chromatography-mass spectrometry of glutathionylation and cysteinylation of salivary S100A9 and cystatin B in preterm newborns

Glutathionylation and cysteinylation can be involved in the protection of critical cysteines from irreversible oxidative damages. S100A9 long and cystatin B, proteins highly represented in the saliva of preterm and at-term newborns, can undergo these modifications. Levels of S100A9 long and cystatin B and their glutathionylated and cysteinylated derivatives have been determined by a top-down platform based on high-performance liquid chromatography-electrospray ionization-mass spectrometry in 100 salivary samples serially collected from 17 preterm newborns with different postconceptional age at birth (178-226 days) and in 90 salivary samples collected from at-term newborns and babies. Results showed that: (1) S100A9 long and cystatin B were mainly present as unmodified forms in extremely preterm newborn; (2) the percentage of the S-thiolated derivatives of both proteins increased with increasing the postconceptional age; (3) the greatest variation occurred up to about 280 days of postconceptional age. Interestingly, differences in the levels of the S-thiolated derivatives only depended on the postconceptional age and not on whether the infant was born preterm or at-term. Inadequate levels of cysteine and glutathione might be responsible for the low level of S-thiolated derivatives measured in preterm newborns. Data are available via ProteomeXchange with identifier PXD025517

Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles

The Melanin-Concentrating Hormone (MCH) System Modulates Behaviors Associated with Psychiatric Disorders

Deficits in sensorimotor gating measured by prepulse inhibition (PPI) of the startle have been known as characteristics of patients with schizophrenia and related neuropsychiatric disorders. PPI disruption is thought to rely on the activity of the mesocorticolimbic dopaminergic system and is inhibited by most antipsychotic drugs. These drugs however act also at the nigrostriatal dopaminergic pathway and exert adverse locomotor responses. Finding a way to inhibit the mesocorticolimbic- without affecting the nigrostriatal-dopaminergic pathway may thus be beneficial to antipsychotic therapies. The melanin-concentrating hormone (MCH) system has been shown to modulate dopamine-related responses. Its receptor (MCH1R) is expressed at high levels in the mesocorticolimbic and not in the nigrostriatal dopaminergic pathways. Interestingly a genomic linkage study revealed significant associations between schizophrenia and markers located in the MCH1R gene locus. We hypothesize that the MCH system can selectively modulate the behavior associated with the mesocorticolimbic dopamine pathway. Using mice, we found that central administration of MCH potentiates apomorphine-induced PPI deficits. Using congenic rat lines that differ in their responses to PPI, we found that the rats that are susceptible to apomorphine (APO-SUS rats) and exhibit PPI deficits display higher MCH mRNA expression in the lateral hypothalamic region and that blocking the MCH system reverses their PPI deficits. On the other hand, in mice and rats, activation or inactivation of the MCH system does not affect stereotyped behaviors, dopamine-related responses that depend on the activity of the nigrostriatal pathway. Furthermore MCH does not affect dizocilpine-induced PPI deficit, a glutamate related response. Thus, our data present the MCH system as a regulator of sensorimotor gating, and provide a new rationale to understand the etiologies of schizophrenia and related psychiatric disorders

Receptor Activation and Inositol Lipid Hydrolysis in Neural Tissues

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66228/1/j.1471-4159.1987.tb05618.x.pd