Comparing periodontitis biomarkers in saliva, oral rinse and gingival crevicular fluid: A pilot study.

AIM: To explore the feasibility of screening for periodontitis by measuring biomarkers, namely total proteolytic activity (TPA), matrix metalloproteinase (MMP)-8, chitinase, lysozyme or their combination, in saliva, oral rinse and gingival crevicular fluid (GCF). MATERIAL AND METHODS: Subjects were recruited among healthy/gingivitis individuals and untreated periodontitis patients in Academic Centre for Dentistry Amsterdam (ACTA). All participants donated samples of unstimulated whole saliva, oral rinse and GCF. The protein concentrations and MMP-8 levels were determined by ELISA. Enzymatic activities were measured using appropriate fluorogenic substrates. RESULTS: In oral rinse samples, periodontitis patients (n = 19) exhibited significantly higher concentrations of MMP-8 and TPA than controls (n = 20). MMP-8 in combination with chitinase explained 88% of the variance and assigned a subject to control or periodontitis group, with best accuracy (87.2%) in oral rinse. CONCLUSIONS: The combination of MMP-8 and chitinase in the current oral rinse procedure has the potential to discriminate periodontitis from periodontal health/gingivitis.

Histatin-1, a histidine-rich peptide in human saliva, promotes cell-substrate and cell-cell adhesion.

Histatins (Hsts) are histidine-rich peptides exclusively present in the saliva of higher primates. In this study, we explored the effects of Hsts on cell-substrate and cell-cell adhesion. Histatin (Hst)-1 caused a significant (>2-fold) increase (EC50 = 1 µM) in the ability of human adherent cells to attach and spread, even in conditions that impaired cell spreading. Other tested Hsts did not stimulate cell spreading, indicating a specific effect of Hst1. The effect of Hst1 on cell-cell adhesion was investigated by using transepithelial resistance (TER) measurements in the human cell line Caco-2, a widely used model for the epithelial layer. We found that 10 µM Hst1 caused a 20% increase in TER compared to the negative control, indicating a function for Hst1 in intercellular cell adhesion and epithelial integrity. A role for Hst1 in both cell-substrate and cell-cell adhesion is highly conceivable, because these 2 modes of adhesion are closely related via shared components and connected signaling pathways.

Different wound healing properties of dermis, adipose, and gingiva mesenchymal stromal cells.

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis-, adipose-, and gingiva-derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin-derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation.

Complement activation by salivary agglutinin is secretor status dependent.

After mucosal damage or gingival inflammation, complement proteins leak into the oral cavity and mix with salivary proteins such as salivary agglutinin (SAG/gp-340/DMBT1). This protein is encoded by the gene Deleted in Malignant Brain Tumors 1 (DMBT1), and it aggregates bacteria, viruses and fungi, and activates the lectin pathway of the complement system. In the lectin pathway, carbohydrate structures on pathogens or altered self cells are recognized. SAG is highly glycosylated, partly on the basis of the donor's blood group status. Whereas secretors express Lewis b, Lewis y, and antigens from the ABO-blood group system on SAG, non-secretors do not. Through mannose-binding lectin (MBL) binding and C4 deposition assays, we aimed to identify the chemical structures on SAG that are responsible for complement activation. The complement-activating properties of SAG were completely abolished by oxidation of its carbohydrate moiety. SAG-mediated activation of complement was also inhibited in the presence of saccharides such as fucose and Lewis b carbohydrates, and also after pretreatment with the fucose-binding lectin, Anguilla anguilla agglutinin. Complement activation was significantly (p<0.01) higher in secretors than in non-secretors. Our results suggest that fucose-rich oligosaccharide sidechains, such as Lewis b antigens, are involved in the activation of complement by SAG.

Growth of Candida albicans in human saliva is supported by low-molecular-mass compounds.

Saliva plays a key role in the maintenance of a stable oral microflora. It contains antimicrobial compounds but also functions as a substrate for growth of bacteria under conditions of low external nutrient supply. Besides bacteria, yeasts, in particular Candida albicans, commonly inhabit the oral cavity. Under immunocompromised conditions, instantaneous outgrowth of this yeast occurs in oral carriers of C. albicans, suggesting that this yeast is able to survive in the oral cavity with saliva as sole source of growth substrate. The aim of the present study was to identify the salivary constituents that are used by C. albicans for growth and survival in saliva. In addition, we have explored the effect of growth in saliva on the susceptibility of C. albicans to histatin 5, a salivary antifungal peptide. It was found that C. albicans was able to grow in human saliva without addition of glucose, and in the stationary phase could survive for more than 400 h. Candida albicans grown in saliva was more than 10 times less susceptible for salivary histatin 5 than C. albicans cultured in Sabouraud medium.

Interindividual variation, correlations, and sex-related differences in the salivary biochemistry of young healthy adults.

A cross-sectional observational study was conducted to evaluate interindividual biochemical variation in unstimulated whole saliva in a population of 268 systemically healthy young students, 18-30 yr of age, with no apparent caries lesions or periodontal disease. Salivary flow rate, protein content, pH, buffering capacity, mucins MUC5B and MUC7, albumin, secretory IgA, cystatin S, lactoferrin, chitinase, amylase, lysozyme, and proteases were measured using ELISAs and enzymatic activity assays. Significant differences were found between male and female subjects. Salivary pH, buffering capacity, protein content, MUC5B, secretory IgA, and chitinase activity were all lower in female subjects compared with male subjects, whereas MUC7 and lysozyme activity were higher in female subjects. There was no significant difference between sexes in salivary flow rate, albumin, cystatin S, amylase, and protease activity. Principal component analysis (PCA) and spectral clustering (SC) were used to assess intervariable relationships within the data set and to identify subgroups. Spectral clustering identified two clusters of participants, which were subsequently described. This study provides a comprehensive overview of the distribution and inter-relations of a set of important salivary biochemical variables in a systemically healthy young adult population, free of apparent caries lesions and periodontal disease. It highlights significant gender differences in salivary biochemistry.

Sphingoid bases inhibit acid-induced demineralization of hydroxyapatite.

Calcium hydroxyapatite (HAp), the main constituent of dental enamel, is inherently susceptible to the etching and dissolving action of acids, resulting in tooth decay such as dental caries and dental erosion. Since the prevalence of erosive wear is gradually increasing, there is urgent need for agents that protect the enamel against erosive attacks. In the present study we studied in vitro the anti-erosive effects of a number of sphingolipids and sphingoid bases, which form the backbone of sphingolipids. Pretreatment of HAp discs with sphingosine, phytosphingosine (PHS), PHS phosphate and sphinganine significantly protected these against acid-induced demineralization by 80 ± 17%, 78 ± 17%, 78 ± 7% and 81 ± 8%, respectively (p < 0.001). On the other hand, sphingomyelin, acetyl PHS, octanoyl PHS and stearoyl PHS had no anti-erosive effects. Atomic force measurement revealed that HAp discs treated with PHS were almost completely and homogeneously covered by patches of PHS. This suggests that PHS and other sphingoid bases form layers on the surface of HAp, which act as diffusion barriers against H(+) ions. In principle, these anti-erosive properties make PHS and related sphingosines promising and attractive candidates as ingredients in oral care products.

Sortase A as a tool to functionalize surfaces.

A widely accepted approach to combat surface fouling is based on the prevention of biofoulants to attach to a surface by the functionalization with poly(ethylene glycol) (PEG). The goal of this study was to generate a proof of concept for the enzymatic coupling of PEG to a peptide precoated surface by using the enzyme Sortase A (SrtA). A hydrophobic polystyrene surface was primed with anchoring peptide P3 equipped with a pentaglycine acceptor motif for SrtA, to enable subsequent transpeptidation with either biotin or a PEG-tail containing the sortase recognition motif LPETG. High levels of surface-bound biotin were detected only in cases with biotin-LPETG and SrtA. Little if any reactivity was detected in wells treated with the SrtA scrambled motif EGLTP, or in the absence of SrtA. Conjugation of PEG resulted in a significant decrease of bacterial adherence to the surface.

Identification of the hydroxyapatite-binding domain of salivary agglutinin.

The salivary agglutinin glycoprotein (SAG) is present in saliva but is also part of the salivary pellicle, playing a seemingly paradoxical role with regard to bacterial homeostasis. On the one hand, SAG aggregates bacteria in solution, thereby preventing bacterial colonization. On the other hand, when bound to the tooth surface, SAG facilitates bacterial colonization and microbial growth. The protein part of SAG is predominantly composed of conserved scavenger receptor cysteine-rich (SRCR) domains. Previously it was found that bacterial binding and aggregation is mediated via a single peptide loop, designated SRCRP2 (P2), within the SRCR domains of SAG. The current data suggest that the SRCR domains also harbour a hydroxyapatite (HA)-binding moiety, SRCRP3 (P3). The observation that P2 and P3 individually play unique roles in the function of SAGs contributes to our understanding of the dual role of SAGs in bacterial binding. Inspired by the bacterial-modulating capacity of SAGs, we created a P3-polyethylene glycol (PEG) conjugate. It was found that a P3 coating resulted in an increased antifouling activity of 20% compared with the uncoated surface in vitro. An additional PEG moiety resulted in an antifouling activity of up to 40% and 30% for Streptococcus mutans and Staphylococcus epidermidis, respectively.

The influence of oral bacteria on epithelial cell migration in vitro.

Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study, Porphyromonas gingivalis was identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminating P. gingivalis in delayed healing of the ulcerations. Therefore, it was tested whether P. gingivalis and its secreted products could inhibit the migration of oral epithelial cells in an in vitro scratch assay. To compare, the oral bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia, and Streptococcus mitis were included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope. P. gingivalis, P. nigrescens, and secretions of P. gingivalis strongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% for P. gingivalis and 20% for P. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing.

Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis.

Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10(5) CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO(2) showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

Membrane interaction and antibacterial properties of two mildly cationic peptide diastereomers, bombinins H2 and H4, isolated from Bombina skin.

Bombinins H are mildly cationic antimicrobial peptides isolated from the skin of the anuran genus Bombina, the fire-bellied toad. Some members of this peptide family coexist in skin secretions as diastereomers in which a single D: -amino acid (alloisoleucine or leucine) is incorporated as a result of the post-translational modification of the respective gene-encoded L-amino acid. Here we report on the antimicrobial properties and membrane interactions of bombinins H2 and H4. The latter differs from H2 by the presence of a D-alloisoleucine at the second N-terminal position. Specifically, we have evaluated the antimicrobial activity of H2 and H4 against a large panel of reference and clinical isolates of Gram-negative and Gram-positive bacteria; performed membrane permeation assays on both intact cells and model membranes (lipid monolayers and liposomes) mimicking the composition of the plasma membrane of Gram-negative/positive bacteria; used biochemical tools, such as trypsin-encapsulated liposomes and capillary electrophoresis, to monitor the peptides' ability to translocate through the membrane of liposomes mimicking Escherichia coli inner membrane. The results revealed interesting relationships between the presence of a single D: -amino acid in the sequence of an antimicrobial peptide and its target microbial cell selectivity/membrane-perturbing activity.

Patterns in consumption of potentially erosive beverages among adolescent school children in the Netherlands.

AIM: To determine the frequency of intake and patterns in consumption of potentially erosive beverages in school children in the Netherlands. METHODS: A cross-sectional, single centre study was performed among 502 school children in Rotterdam, in age varying between 12 and 19 years. Data on consumption of soft drinks, energy drinks, sports drinks and alcopops were obtained through a self-reported questionnaire. Gender- and age-related differences in consumption were analysed with Chi-square, Kruskal-Wallis and Mann-Whitney tests. Associations between variables were investigated with Chi-square tests and Spearman's rank order correlation analysis. RESULTS: Boys consumed soft drinks, energy drinks and sports drinks more frequently than girls, and on average also consumed higher amounts of these drinks. No gender-related differences were observed in alcopop consumption. Consumption of all drinks was most frequent at 14- or 15-year of age, with the exception of alcopops which was most frequent by 16-year-old school children. Significant positive associations were observed between the consumption of soft drinks, energy drinks and/or sports drinks. Alcopop consumption was only associated with consumption of energy drinks. CONCLUSION: Consumption of soft drinks, energy drinks, sports drinks and alcopops by school children is related to age and gender. The significant positive associations between the consumption of these drinks suggest that a subgroup of school children exists with a high cumulative intake of these potentially erosive drinks.

The role of salivary histatin and the human cathelicidin LL-37 in wound healing and innate immunity.

Antimicrobial peptides are multifunctional in innate immunity and wound repair of multicellular organisms. We were the first to discover that histatins, a family of salivary antimicrobial peptides, enhance epithelial cell migration, suggesting a role in oral wound healing. It is unknown whether histatins display innate-immunity activities, similar to other antimicrobial peptides such as LL-37. Therefore, we compared the effect of Histatin-2 and LL-37 on several activities within the context of wound healing and innate immunity. We found that Histatin-2 enhances fibroblast migration, but only weakly induces proliferation. LL-37 enhances both fibroblast migration and proliferation, but only at a narrow concentration optimum (approximately 1 microm). At higher concentrations LL-37 causes cell death, whereas Histatin-2 is not cytotoxic. Both peptides do not alter fibroblast-to-myofibroblast differentiation. Histatin-2 does not alter interleukin-8 (IL-8) expression and lipopolysaccharide (LPS)-elevated cytokine and chemokine expression. In contrast, LL-37 induces IL-8 expression, but dampens the LPS-induced immune response. Neither Histatin-2 nor LL-37 affects human-neutrophil migration. Histatins are, unlike other antimicrobial peptides, not cytotoxic or proinflammatory. It seems that they are important for the initial stage of wound healing in which fast wound coverage is important for healing without infection, inflammation, or fibrosis development. Interestingly, these characteristics are more typical for the mouth than for skin.

Structure-activity analysis of histatin, a potent wound healing peptide from human saliva: cyclization of histatin potentiates molar activity 1,000-fold.

Wounds in the mouth heal faster and with less scarification and inflammation than those in the skin. Saliva is thought to be essential for the superior oral wound healing, but the involved mechanism is still unclear. We have previously discovered that a human-specific peptide, histatin, might be implicated in the wound-healing properties of saliva. Here we report that histatin enhances reepithelialization in a human full-skin wound model closely resembling normal skin. The peptide does not stimulate proliferation but induces cell spreading and migration, two key initiating steps in reepithelialization. Activation of cells by histatin requires a G-protein-coupled receptor that activates the ERK1/2 pathway. Using a stepwise-truncation method, we determined the minimal domain (SHREFPFYGDYGS) of the 38-mer-parent peptide that is required for activity. Strikingly, N- to C-terminal cyclization of histatin-1 potentiates the molar activity approximately 1000-fold, indicating that the recognition of histatin by its cognate receptor requires a specific spatial conformation of the peptide. Our results emphasize the importance of histatin in human saliva for tissue protection and recovery and establish the experimental basis for the development of synthetic histatins as novel skin wound-healing agents.

Salivary Histatin 1 and 2 Are Targeted to Mitochondria and Endoplasmic Reticulum in Human Cells.

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control-scrambled F-Hst1 (F-Hstscr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotrackerTM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-trackerTM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

All-trans retinoic acid and human salivary histatin-1 promote the spreading and osteogenic activities of pre-osteoblasts in vitro.

Cell-based bone tissue engineering techniques utilize both osteogenic cells and biomedical materials, and have emerged as a promising approach for large-volume bone repair. The success of such techniques is highly dependent on cell adhesion, spreading, and osteogenic activities. In this study, we investigated the effect of co-administration of all-trans retinoic acid (ATRA) and human salivary peptide histatin-1 (Hst1) on the spreading and osteogenic activities of pre-osteoblasts on bio-inert glass surfaces. Pre-osteoblasts (MC3T3-E1 cell line) were seeded onto bio-inert glass slides in the presence and absence of ATRA and Hst1. Cell spreading was scored by measuring surface areas of cellular filopodia and lamellipodia using a point-counting method. The distribution of fluorogenic Hst1 within osteogenic cells was also analyzed. Furthermore, specific inhibitors of retinoic acid receptors α, ß, and γ, such as ER-50891, LE-135, and MM-11253, were added to identify the involvement of these receptors. Cell metabolic activity, DNA content, and alkaline phosphatase (ALP) activity were assessed to monitor their effects on osteogenic activities. Short-term (2 h) co-administration of 10 µm ATRA and Hst1 to pre-osteoblasts resulted in significantly higher spreading of pre-osteoblasts compared to ATRA or Hst1 alone. ER-50891 and LE-135 both nullified these effects of ATRA. Co-administration of ATRA and Hst1 was associated with significantly higher metabolic activity, DNA content, and ALP activity than either ATRA or Hst1 alone. In conclusion, co-administration of Hst1 with ATRA additively stimulated the spreading and osteogenicity of pre-osteoblasts on bio-inert glass surfaces in vitro.

Histatins are the major wound-closure stimulating factors in human saliva as identified in a cell culture assay.

Wounds in the oral cavity heal much faster than skin lesions. Among other factors, saliva is generally assumed to be of relevance to this feature. Rodent saliva contains large amounts of growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF). In humans, however, the identity of the involved compounds has remained elusive, especially since EGF and NGF concentrations are approximately 100,000 times lower than those in rodent saliva. Using an in vitro model for wound closure, we examined the properties of human saliva and the fractions that were obtained from saliva by high-performance liquid chromotography (HPLC) separation. We identified histatin 1 (Hst1) and histatin 2 (Hst2) as major wound-closing factors in human saliva. In contrast, the d-enantiomer of Hst2 did not induce wound closure, indicating stereospecific activation. Furthermore, histatins were actively internalized by epithelial cells and specifically used the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, thereby enhancing epithelial migration. This study demonstrates that members of the histatin family, which up to now were implicated in the antifungal weaponry of saliva, exert a novel function that likely is relevant for oral wound healing.

Identification of salivary components that induce transition of hyphae to yeast in Candida albicans.

Candida albicans, the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphae and hyphae filaments. The hyphae form is considered the most invasive form of the fungus. The purpose of this study is to investigate the effect of saliva on hyphae growth of C. albicans. Candida albicans hyphae were inoculated in Roswell Park Memorial Institute medium with whole saliva, parotid saliva or buffer mimicking the saliva ion composition, and cultured for 18 h at 37 degrees C under aerobic conditions with 5% CO(2). Whole saliva and parotid saliva induced transition to yeast growth, whereas the culture with buffer remained in the hyphae form. Parotid saliva was fractionated on a reverse-phase C8 column and each fraction was tested for inducing transition to yeast growth. By immunoblotting, the salivary component in the active fraction was identified as statherin, a phosphoprotein of 43 amino acids that has been implicated in remineralization of the teeth. Synthetically made statherin induced transition of hyphae to yeast. By deletion of five amino acids at the negatively charged N-terminal site (DpSpSEE), yeast-inducing activity and binding to C. albicans were increased. In conclusion, statherin induces transition to yeast of C. albicans hyphae and may thus contribute to the oral defense against candidiasis.

Please try harder! The influence of hearing status and evaluative feedback during listening on the pupil dilation response, saliva-cortisol and saliva alpha-amylase levels.

The pupil dilation response is sensitive both to listening effort and the emotional significance of a task. We aimed to assess the influence of evaluative feedback on the pupil dilation response using a speech reception threshold (SRT) task. Besides the pupil dilation response, we acquired subjective ratings and two physiological biomarkers sensitive to stress: cortisol and alpha-amylase levels as determined in saliva samples. We included 34 participants with normal hearing (mean age = 52 years, age range 25-67 years) and 29 age-matched participants with mild-to-moderate hearing loss (mean age = 52 years, age range 23-64 years). Half of the participants performed a standard SRT test without feedback, and the other half performed an SRT test in which they did receive feedback and were urged to perform better. The SRT conditions targeted 50% and 71% correct reception of the sentences. Pupil size was recorded and saliva samples were obtained and participants rated their experience of the task. Participants with hearing loss performed more poorly on the SRT test than participants with normal hearing participants receiving feedback had better SRTs in the 71% intelligibility condition and higher peak pupil dilation in both intelligibility conditions than participants who did not receive feedback, irrespective of hearing status. Saliva cortisol level and alpha-amylase activity reflected the usual diurnal patterns but showed no effects of hearing status or feedback. Finally, participants who received feedback experienced more difficulties than those who did not receive feedback, irrespective of hearing status. This study underlines the importance of taking into account the influence of task instructions and feedback in a speech perception task as these factors may influence the experienced difficulties, listening effort, and task performance.