Salivary cortisol as a tool for physiological studies and diagnostic strategies

Salivary cortisol is an index of plasma free cortisol and is obtained by a noninvasive procedure. We have been using salivary cortisol as a tool for physiological and diagnostic studies, among them the emergence of circadian rhythm in preterm and term infants. The salivary cortisol circadian rhythm in term and premature infants was established between 8 and 12 postnatal weeks. In the preterm infants the emergence of circadian rhythm was parallel to the onset of sleep rhythm. We also studied the use of salivary cortisol for screening for Cushing's syndrome (CS) in control and obese outpatients based on circadian rhythm and the overnight 1 mg dexamethasone (DEX) suppression test. Salivary cortisol was suppressed to less than 100 ng/dl after 1 mg DEX in control and obese patients. A single salivary cortisol measurement at 23:00 h and again after 1 mg DEX above the 90th percentile of the obese group values had sensitivity and specificity of 93 and 93% (23:00 h), and 91 and 94% (after DEX), respectively. The sensitivity improved to 100% when we combined both parameters. We also studied 11 CS children and 21 age-matched primary obese children for whom salivary cortisol sensitivity and specificity were 100/95% (23:00 h), and 100/95% (1 mg DEX), respectively. Similar to adults, sensitivity and specificity of 100% were obtained by combining 23:00 h and 1 mg DEX. The measurement of salivary cortisol is a useful tool for physiological studies and for the diagnosis of CS in children and adults on an outpatient basis.

The relationship between salivary cortisol concentrations and anxiety in adolescent and non-adolescent pregnant women

The main purpose of the present study was to determine the relationship between salivary cortisol concentrations and self-report anxiety in 50 adolescent and 178 non-adolescent women during the last month of pregnancy. The subjects were randomly selected from a previous study involving women who attended antenatal care from September 1997 to August 2000 at 17 health services in Southeast Brazil. Salivary cortisol was measured with an enzyme immunoassay kit, and anxiety was assessed by the State-Trait Anxiety Inventories (STAI) of Spielberger. After saliva collection, the participants completed the STAI. Mean concentrations of cortisol for both pregnant adolescents (14.17 ± 6.78 nmol/l) and non-adolescents (13.81 ± 8.51 nmol/l) were similar (P = 0.89). Forty-three percent of the pregnant adolescents and 30.5% of the non-adolescents felt anxious at the time of being questioned (State Anxiety Inventory (SAI) scores >40; P = 0.06). Cortisol concentrations in adolescents were negatively related to the SAI scores (r = -0.39; P = 0.01) which assess a temporary condition of anxiety. There was a statistically significant difference in mean cortisol concentrations between adolescents with low (<=40) and high (>40) SAI scores (P = 0.03, t-test), but no differences for non-adolescents. The negative relationship between salivary cortisol concentrations and anxiety scores in adolescents may be due to puberty-related hormone differences during this period of life. Pregnant adolescents may possess unique biological or psychological characteristics compared to adults and non-pregnant adolescents. Thus, we need to know more about the hypothalamic-pituitary-adrenocortical axis of pregnant adolescents.

Blood flow measurements in rats using four color microspheres during blockade of different vasopressor systems

The use of colored microspheres to adequately evaluate blood flow changes under different circumstances in the same rat has been validated with a maximum of three different colors due to methodological limitations. The aim of the present study was to validate the use of four different colors measuring four repeated blood flow changes in the same rat to assess the role of vasopressor systems in controlling arterial pressure (AP). Red (150,000), white (200,000), yellow (150,000), and blue (200,000) colored microspheres were infused into the left ventricle of 6 male Wistar rats 1) at rest and 2) after vasopressin (aAVP, 10 µg/kg, iv), 3) renin-angiotensin (losartan, 10 mg/kg, iv), and 4) sympathetic system blockade (hexamethonium, 20 mg/kg, iv) to determine blood flow changes. AP was recorded and processed with a data acquisition system (1-kHz sampling frequency). Blood flow changes were quantified by spectrophotometry absorption peaks for colored microsphere components in the tissues evaluated. Administration of aAVP and losartan slightly reduced the AP (-5.7 ± 0.5 and -7.8 ± 1.2 mmHg, respectively), while hexamethonium induced a 52 ± 3 mmHg fall in AP. The aAVP injection increased blood flow in lungs (78%), liver (117%) and skeletal muscle (>150%), while losartan administration enhanced blood flow in heart (126%), lungs (100%), kidneys (80%), and gastrocnemius (75%) and soleus (94%) muscles. Hexamethonium administration reduced only kidney blood flow (50%). In conclusion, four types of colored microspheres can be used to perform four repeated blood flow measurements in the same rat detecting small alterations such as changes in tissues with low blood flow.

Assessment of tidal volume and thoracoabdominal motion using volume and flow-oriented incentive spirometers in healthy subjects

The objective of the present study was to evaluate incentive spirometers using volume- (Coach and Voldyne) and flow-oriented (Triflo II and Respirex) devices. Sixteen healthy subjects, 24 ± 4 years, 62 ± 12 kg, were studied. Respiratory variables were obtained by respiratory inductive plethysmography, with subjects in a semi-reclined position (45º). Tidal volume, respiratory frequency, minute ventilation, inspiratory duty cycle, mean inspiratory flow, and thoracoabdominal motion were measured. Statistical analysis was performed with Kolmogorov-Smirnov test, t-test and ANOVA. Comparison between the Coach and Voldyne devices showed that larger values of tidal volume (1035 ± 268 vs 947 ± 268 ml, P = 0.02) and minute ventilation (9.07 ± 3.61 vs 7.49 ± 2.58 l/min, P = 0.01) were reached with Voldyne, whereas no significant differences in respiratory frequency were observed (7.85 ± 1.24 vs 8.57 ± 1.89 bpm). Comparison between flow-oriented devices showed larger values of inspiratory duty cycle and lower mean inspiratory flow with Triflo II (0.35 ± 0.05 vs 0.32 ± 0.05 ml/s, P = 0.00, and 531 ± 137 vs 606 ± 167 ml/s, P = 0.00, respectively). Abdominal motion was larger (P < 0.05) during the use of volume-oriented devices compared to flow-oriented devices (52 ± 11% for Coach and 50 ± 9% for Voldyne; 43 ± 13% for Triflo II and 44 ± 14% for Respirex). We observed that significantly higher tidal volume associated with low respiratory frequency was reached with Voldyne, and that there was a larger abdominal displacement with volume-oriented devices.

Correlations between pulse oximetry and peak expiratory flow in acute asthma

Few studies are available concerning correlations between pulse oximetry and peak expiratory flow in children and adolescents with acute asthma. Although the Global Initiative for Asthma states that measurements of lung function and oximetry are critical for the assessment of patients, it is not clear if both methods should necessarily be included in their evaluation. Since there is a significant difference in cost between pulse oximetry equipment and peak expiratory flow devices, we determined whether clinical findings and peak expiratory flow measurements are sufficient to determine the severity of acute asthma. The present prospective observational study was carried out to determine if there is correlation between pulse oximetry and peak expiratory flow determination in 196 patients with acute asthma aged 4 to 15 years diagnosed according to the Global Initiative for Asthma criteria. Patients experiencing their first or second wheezing episode, with fever, related acute or chronic diseases, and unable to perform the peak expiratory flow maneuver were excluded. Measurements of peak expiratory flow and pulse oximetry were performed at admission and after 15 min of each inhaled salbutamol cycle. Correlations obtained by linear regression using the Pearson correlation coefficients (r) were 0.41 (P < 0.0001), 0.53 (P < 0.0001), 0.51 (P < 0.0001), and 0.61 (P < 0.0001) at admission and after the first, second and third cycles of salbutamol, respectively. These correlations showed that one measure cannot substitute the other (Pearson's coefficient <0.7), probably because they evaluate different aspects in the airways, suggesting that peak expiratory flow should not be used alone in the assessment of acute asthma in children and adolescents.

Role of nitric oxide and prostaglandin in the maintenance of cortical and renal medullary blood flow

This study was undertaken in anesthetized dogs to evaluate the relative participation of prostaglandins (PGs) and nitric oxide (NO) in the maintenance of total renal blood flow (TRBF), and renal medullary blood flow (RMBF). It was hypothesized that the inhibition of NO should impair cortical and medullary circulation because of the synthesis of this compound in the endothelial cells of these two territories. In contrast, under normal conditions of perfusion pressure PG synthesis is confined to the renal medulla. Hence PG inhibition should predominantly impair the medullary circulation. The initial administration of 25 µM kg-1 min-1 NG-nitro-L-arginine methyl ester produced a significant 26% decrease in TRBF and a concomitant 34% fall in RMBF, while the subsequent inhibition of PGs with 5 mg/kg meclofenamate further reduced TRBF by 33% and RMBF by 89%. In contrast, the initial administration of meclofenamate failed to change TRBF, while decreasing RMBF by 49%. The subsequent blockade of NO decreased TRBF by 35% without further altering RMBF. These results indicate that initial PG synthesis inhibition predominantly alters the medullary circulation, whereas NO inhibition decreases both cortical and medullary flow. This latter change induced by NO renders cortical and RMBF susceptible to a further decrease by PG inhibition. However, the decrease in medullary circulation produced by NO inhibition is not further enhanced by subsequent PG inhibition.

Normal flow-mediated vasodilatation of the brachial artery and carotid artery intima-media thickness in subclinical hypothyroidism

Subclinical hypothyroidism (SHT) is a disease for which exact therapeutic approaches have not yet been established. Previous studies have suggested an association between SHT and coronary heart disease. Whether this association is related to SHT-induced changes in serum lipid levels or to endothelial dysfunction is unclear. The aim of this study was to determine endothelial function measured by the flow-mediated vasodilatation of the brachial artery and the carotid artery intima-media thickness (IMT) in a group of women with SHT compared with euthyroid subjects. Triglycerides, total cholesterol, HDL-C, LDL-C, apoprotein A (apo A), apo B, and lipoprotein(a) were also determined. Twenty-one patients with SHT (mean age: 42.4 ± 10.8 years and mean thyroid-stimulating hormone (TSH) levels: 8.2 ± 2.7 µIU/mL) and 21 euthyroid controls matched for body mass index, age and atherosclerotic risk factors (mean age: 44.2 ± 8.5 years and mean TSH levels: 1.4 ± 0.6 µIU/mL) participated in the study. Lipid parameters (except HDL-C and apo A, which were lower) and IMT values were higher in the common carotid and carotid bifurcation of SHT patients with positive serum thyroid peroxidase antibodies (TPO-Ab) (0.62 ± 0.2 and 0.62 ± 0.16 mm for the common carotid and carotid bifurcation, respectively) when compared with the negative TPO-Ab group (0.55 ± 0.24 and 0.58 ± 0.13 mm, for common carotid and carotid bifurcation, respectively). The difference was not statistically significant. We conclude that minimal thyroid dysfunction had no adverse effects on endothelial function in the population studied. Further investigation is warranted to assess whether subclinical hypothyroidism, with and without TPO-Ab-positive serology, has any effect on endothelial function.

Anandamide injected into the lateral ventricle of the brain inhibits submandibular salivary secretion by attenuating parasympathetic neurotransmission

Our objective was to determine the effect of arachidonylethanolamide (anandamide, AEA) injected intracerebroventricularly (icv) into the lateral ventricle of the rat brain on submandibular gland (SMG) salivary secretion. Parasympathetic decentralization (PSD) produced by cutting the chorda tympani nerve strongly inhibited methacholine (MC)-induced salivary secretion while sympathetic denervation (SD) produced by removing the superior cervical ganglia reduced it slightly. Also, AEA (50 ng/5 µL, icv) significantly decreased MC-induced salivary secretion in intact rats (MC 1 µg/kg: control (C), 5.3 ± 0.6 vs AEA, 2.7 ± 0.6 mg; MC 3 µg/kg: C, 17.6 ± 1.0 vs AEA, 8.7 ± 0.9 mg; MC 10 µg/kg: C, 37.4 ± 1.2 vs AEA, 22.9 ± 2.6 mg). However, AEA did not alter the significantly reduced salivary secretion in rats with PSD, but decreased the slightly reduced salivary secretion in rats with SD (MC 1 µg/kg: C, 3.8 ± 0.8 vs AEA, 1.4 ± 0.6 mg; MC 3 µg/kg: C, 14.7 ± 2.4 vs AEA, 6.9 ± 1.2 mg; P < 0.05; MC 10 µg/kg: C, 39.5 ± 1.0 vs AEA, 22.3 ± 0.5 mg; P < 0.001). We showed that the inhibitory effect of AEA is mediated by cannabinoid type 1 CB1 receptors and involves GABAergic neurotransmission, since it was blocked by previous injection of the CB1 receptor antagonist AM251 (500 ng/5 µL, icv) or of the GABA A receptor antagonist, bicuculline (25 ng/5 µL, icv). Our results suggest that parasympathetic neurotransmission from the central nervous system to the SMG can be inhibited by endocannabinoid and GABAergic systems.

Reversible flow of cholesteryl ester between high-density lipoproteins and triacylglycerol-rich particles is modulated by the fatty acid composition and concentration of triacylglycerols

We determined the influence of fasting (FAST) and feeding (FED) on cholesteryl ester (CE) flow between high-density lipoproteins (HDL) and plasma apoB-lipoprotein and triacylglycerol (TG)-rich emulsions (EM) prepared with TG-fatty acids (FAs). TG-FAs of varying chain lengths and degrees of unsaturation were tested in the presence of a plasma fraction at d > 1.21 g/mL as the source of CE transfer protein. The transfer of CE from HDL to FED was greater than to FAST TG-rich acceptor lipoproteins, 18% and 14%, respectively. However, percent CE transfer from HDL to apoB-containing lipoproteins was similar for FED and FAST HDL. The CE transfer from HDL to EM depended on the EM TG-FA chain length. Furthermore, the chain length of the monounsaturated TG-containing EM showed a significant positive correlation of the CE transfer from HDL to EM (r = 0.81, P < 0.0001) and a negative correlation from EM to HDL (r = -041, P = 0.0088). Regarding the degree of EM TG-FAs unsaturation, among EMs containing C18, the CE transfer was lower from HDL to C18:2 compared to C18:1 and C18:3, 17.7%, 20.7%, and 20%, respectively. However, the CE transfer from EMs to HDL was higher to C18:2 than to C18:1 and C18:3, 83.7%, 51.2%, and 46.3%, respectively. Thus, the EM FA composition was found to be the rate-limiting factor regulating the transfer of CE from HDL. Consequently, the net transfer of CE between HDL and TG-rich particles depends on the specific arrangement of the TG acyl chains in the lipoprotein particle core.

5-Bromo-2’-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae)

5-Bromo-2’-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.

Do whole-body vibration exercise and resistance exercise modify concentrations of salivary cortisol and immunoglobulin A?

A single bout of resistance exercise (RE) induces hormonal and immune responses, playing an important role in a long-term adaptive process. Whole-body vibration (WBV) has also been shown to affect hormonal responses. Evidence suggests that combining WBV with RE may amplify hormonal and immune responses due to the increased neuromuscular load. Therefore, the aim of this study was to evaluate salivary cortisol (Scortisol) and salivary IgA (SIgA) concentrations following a RE session combined or not with WBV. Nine university students (22.9 ± 5.1 years, 175.8 ± 5.2 cm, and 69.2 ± 7.3 kg) performed five sets of squat exercise (70% one-repetition-maximum) combined (R+V30) or not (R) with WBV at 30 Hz. Saliva samples were obtained before and after exercise. Subjects also rated their effort according to the Borg CR-10 scale (RPE). Data were analyzed by a mixed model. RPE was higher after R+V30 (8.3 ± 0.7) compared to R (6.2 ± 0.7). However, Scortisol (pre: 10.6 ± 7.6 and 11.7 ± 7.6, post: 8.3 ± 6.3 and 10.2 ± 7.2 ng/mL for R and R+V30, respectively) and SIgA concentrations (pre: 98.3 ± 22.6 and 116.1 ± 51.2, post: 116.6 ± 64.7 and 143.6 ± 80.5 µg/mL for R and R+V30, respectively) were unaffected. No significant correlations were observed between Scortisol and RPE (r = 0.45, P = 0.22; r = 0.30, P = 0.42, for R and R+V30, respectively). On the basis of these data, neither protocol modified salivary cortisol or IgA, although RPE was higher after R+V30 than R.

Expression of beclin 1 in primary salivary adenoid cystic carcinoma and its relation to Bcl-2 and p53 and prognosis

Beclin 1 plays a critical role in autophagy and functions as a haploinsufficient tumor suppressor. The expression and prognostic significance of beclin 1 in head and neck adenoid cystic carcinoma (ACC) are largely unexplored. Therefore, we investigated the expression of beclin 1, Bcl-2, and p53 in head and neck ACC tissue. Tissue samples from 35 cases (15 females, 20 males) of head and neck ACC were utilized for immunohistochemistry. Beclin 1 expression was observed in 32 cases (91.4%) and considered to be high in 15 cases (42.9%) and low in 20 cases (57.1%). Beclin 1 expression was significantly correlated with a histological growth pattern (P=0.046) and histological grade (P=0.037). Beclin 1 expression was inversely correlated with Bcl-2 expression (P=0.013) and significantly associated with overall survival (P=0.006). Bcl-2 and p53 expression were observed in 21 cases (60.0%) and 16 cases (45.7%). Bcl-2 expression was significantly correlated with perineural invasion (P=0.041) and not associated with overall survival (P=0.053). p53 expression was directly correlated with beclin 1 expression (P=0.044). Our results indicated that beclin 1 may be a novel, promising prognostic factor for clinical outcome in head and neck ACC patients and may play a part in the development of head and neck ACC by interacting with Bcl-2 and p53.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Salivary testosterone and immunoglobulin A were increased by resistance training in adults with Down syndrome

This study was designed to assess the influence of resistance training on salivary immunoglobulin A (IgA) levels and hormone profile in sedentary adults with Down syndrome (DS). A total of 40 male adults with DS were recruited for the trial through different community support groups for people with intellectual disabilities. All participants had medical approval for participation in physical activity. Twenty-four adults were randomly assigned to perform resistance training in a circuit with six stations, 3 days per week for 12 weeks. Training intensity was based on functioning in the eight-repetition maximum (8RM) test for each exercise. The control group included 16 age-, gender-, and BMI-matched adults with DS. Salivary IgA, testosterone, and cortisol levels were measured by ELISA. Work task performance was assessed using the repetitive weighted-box-stacking test. Resistance training significantly increased salivary IgA concentration (P=0.0120; d=0.94) and testosterone levels (P=0.0088; d=1.57) in the exercising group. Furthermore, it also improved work task performance. No changes were seen in the controls who had not exercised. In conclusion, a short-term resistance training protocol improved mucosal immunity response as well as salivary testosterone levels in sedentary adults with DS.

Effects of 5-aza-2′deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECKgene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.