Plasma cytokine concentrations of children with autism spectrum disorder and neurotypical siblings.

Several studies of autism spectrum disorder (ASD) have shown cytokine dysregulation in children with ASD, leading to a consideration of the immune theory of the ASD etiopathogenesis and a debate about cytokines as potential biomarkers of ASD. However, the results of these studies are still inconsistent. Overall, studies comparing the cytokine levels of children with ASD and neurotypical siblings achieved relatively different results than studies with control groups of non-siblings. The studies suggest that the immune profile of siblings of individuals with ASD serving as control is more similar to children with ASD than the profile of non-siblings. However, there are still only a few studies with control groups including neurotypical siblings of children with ASD. The aim of our study was to determine whether the concentration of plasma cytokine levels may differentiate children with ASD from their neurotypical siblings. The sample consisted of 40 children with ASD (mean age 7.11 years, SD 2.9) and 21 neurotypical siblings (mean age 7.38, SD 3.3). Levels of 20 cytokines were included into the statistical analysis. A multiple logistic regression model using multiple corrections showed that an increase in log-transformed plasma G-CSF (granulocyte colony stimulating factor) concentration is associated with an increased risk of the child being diagnosed as an ASD case (OR = 4.35, 95% CI 1.77, 10.73). Although the significantly increased concentration of G-CSF suggests a slightly different activity of the immune system of children with ASD, the overall cytokine profile of their siblings appeared to be very similar.

Salivary changes in oxidative stress related to inflammation in oral and gastrointestinal diseases.

OBJECTIVES: The early diagnosis and monitoring of Crohn's disease (CD) and orofacial granulomatosis (OFG) might be facilitated by assaying potential disease biomarkers in saliva. Markers of oxidative stress and inflammation were assayed in salivas from patients with CD, OFG and concurrent OFG and CD (OFG + CD). SUBJECTS: Unstimulated whole mouth saliva was collected from 93 subjects, and immunoglobulin A (IgA), lactoferrin (LF) and myeloperoxidase (MPO) were determined by ELISA. Markers of oxidative stress and antioxidant status were assayed spectrophotometrically. RESULTS: Immunoglobulin A was significantly (p < .03) higher in experimental groups vs the control group. LF was significantly (p < .01) higher in OFG + CD compared to CTRL and CD. Ferric reducing antioxidant power was lower (p ≤ .009) in all experimental groups, and advanced glycation end products were higher (p ≤ .01) in CD and OFG + CD patients. CONCLUSION: Oxidative stress is increased in saliva in CD and OFG. Although MPO, a product of inflammatory cells, was not significantly increased, the other innate immune markers, IgA and LF, which are also secreted by salivary glands, were increased. This study suggests that saliva might be utilized in monitoring CD and OFG but further longitudinal studies focused on analysing a panel of salivary markers are needed.

Autism spectrum disorder and new perspectives on the reliability of second to fourth digit ratio.

The second to fourth digit ratio (2D:4D) is according to previous studies a likely biomarker for prenatal testosterone exposure and its effect on the human brain. It was found to be linked to autism spectrum disorders (ASD). Recently, 2D:4D raised a lot of questions with regard to its stability and autism-related behaviors. Here, we present a cross-sectional study of 2D:4D in boys (N = 91, mean age 7.63) and adults (N = 36 mean age 22.8) with ASD as well as neurotypical students, 506 participants in total. Digit ratio was assessed by taking measurements from digital scans, compared between groups and correlated with the autism quotient. Significant differences were found in the digit ratio of children and adults. Both girls and boys had 2D:4D ratio lower than women and men, both on the right (p = 0.000 in females, p = 0.000 in males) and left hand (p = 0.018 in females, p = 0.011 in males). No significant differences were found in digit ratios between neurotypical subjects and those with ASD nor was there a relationship with the reported autistic traits, which leads us to question the reliability of 2D:4D and its relation to ASD.

Potential of Salivary Biomarkers in Autism Research: A Systematic Review.

The diagnostic process for autism spectrum disorders (ASD) is based on a behavioral analysis of the suspected individual. Despite intensive research, no specific and valid biomarker has been identified for ASD, but saliva, with its advantages such as non-invasive collection, could serve as a suitable alternative to other body fluids. As a source of nucleic acid of both human and microbial origin, protein and non-protein molecules, saliva offers a complex view on the current state of the organism. Additionally, the use of salivary markers seems to be less complicated not only for ASD screening but also for revealing the etiopathogenesis of ASD, since enrolling neurotypical counterparts willing to participate in studies may be more feasible. The aim of the presented review is to provide an overview of the current research performed on saliva in relation to ASD, mutual complementing, and discrepancies that result in difficulties applying the observed markers in clinical practice. We emphasize the methodological limitations of saliva collection and processing as well as the lack of information regarding ASD diagnosis, which is critically discussed.

The effects of anthocyanin-rich wheat diet on the oxidative status and behavior of rats.

AIM: To evaluate the effect of food containing anthocyanin-rich wheat on oxidative status and behavior of healthy rats. METHODS: Twenty male rats were divided into the control and anthocyanin group. Oral glucose tolerance test was performed, and proteinuria and creatinine clearance were measured. Behavioral analysis was performed in Phenotyper cages. Serum and tissues were collected to measure the markers of oxidative stress and antioxidant status. RESULTS: Anthocyanins significantly increased total antioxidant capacity in serum (P=0.039), decreased advanced oxidation protein products in the kidney (P=0.002), but increased thiobarbituric acid reactive substances in the kidney compared to the control group. No significant difference between the groups was found in the markers of oxidative stress in the liver and colon, as well as in renal functions and glucose metabolism. The anthocyanin group spent significantly less time in the spotlight zone of the Phenotyper cages (P=0.040), indicating higher anxiety-like behavior. CONCLUSION: Food containing anthocyanin-rich wheat had positive effects on serum antioxidant status and kidney protein oxidation, but increased lipid peroxidation in the kidney and modified animal behavior related to anxiety. The molecular mechanisms leading to observed effects should be further elucidated.

Salivary DNA and markers of oxidative stress in patients with chronic periodontitis.

OBJECTIVES: Previous observational studies have shown that periodontal status is associated with salivary markers of oxidative damage. A direct comparison of periodontitis patients and controls using a wide palette of salivary markers of oxidative stress is lacking. Characteristics of salivary DNA in periodontitis are unknown. The aim of this study was to compare the salivary markers of oxidative stress and characteristics of salivary DNA between patients with chronic periodontitis and periodontitis-free controls. MATERIALS AND METHODS: Saliva was collected from 23 patients with chronic periodontitis and 19 periodontitis-free controls. All participants underwent a clinical periodontal examination. Markers of oxidative and carbonyl stress were measured in saliva. Human and bacterial DNA was quantified, and human DNA integrity was assessed. RESULTS: Salivary thiobarbituric acid-reacting substances were higher in patients than in controls; at least in men, the difference was significant (p < 0.01). In women, patients had significantly lower salivary antioxidant status (p < 0.001). No quantitative differences were found regarding salivary DNA. Tendencies towards reduced DNA integrity were found in periodontitis patients. CONCLUSIONS: The results confirmed the association of salivary thiobarbituric acid-reacting substances with periodontitis. Lipid peroxidation in periodontitis seems to be caused by increased production of reactive oxygen species in men and by decreased antioxidant status in women. Whether lower salivary DNA integrity is involved in the pathogenesis of periodontitis remains to be elucidated. CLINICAL RELEVANCE: Salivary thiobarbituric acid-reacting substances are associated with periodontitis at least on a population level. Sex-specific causes of lipid peroxidation might point towards different pathogenic mechanisms.

Prenatal dietary load of Maillard reaction products combined with postnatal Coca-Cola drinking affects metabolic status of female Wistar rats.

AIM: To assess the impact of prenatal exposure to Maillard reaction products (MRPs) -rich diet and postnatal Coca-Cola consumption on metabolic status of female rats. Diet rich in MRPs and consumption of saccharose/fructose sweetened soft drinks is presumed to impose increased risk of development of cardiometabolic afflictions, such as obesity or insulin resistance. METHODS: At the first day of pregnancy, 9 female Wistar rats were randomized into two groups, pair-fed either with standard rat chow (MRP-) or MRPs-rich diet (MRP+). Offspring from each group of mothers was divided into two groups and given either water (Cola-) or Coca-Cola (Cola+) for drinking ad libitum for 18 days. Oral glucose tolerance test was performed, and circulating markers of inflammation, oxidative stress, glucose and lipid metabolism were assessed. RESULTS: MRP+ groups had higher weight gain, significantly so in the MRP+/Cola- vs MRP-/Cola-. Both prenatal and postnatal intervention increased carboxymethyllysine levels and semicarbazide-sensitive amine oxidase activity, both significantly higher in MRP+/Cola + than in MRP-/Cola-. Total antioxidant capacity was lower in MRP+ groups, with significant decrease in MRP+/Cola + vs MRP-/Cola+. Rats drinking Coca-Cola had higher insulin, homeostatic model assessment of insulin resistance, heart rate, advanced oxidation of protein products, triacylglycerols, and oxidative stress markers measured as thiobarbituric acid reactive substances compared to rats drinking water, with no visible effect of MRPs-rich diet. CONCLUSION: Metabolic status of rats was affected both by prenatal and postnatal dietary intervention. Our results suggest that combined effect of prenatal MRPs load and postnatal Coca-Cola drinking may play a role in development of metabolic disorders in later life.

Alterations in Antioxidant Status and Erythrocyte Properties in Children with Autism Spectrum Disorder.

Erythrocytes are responsible for the transport of oxygen within the organism, which is particularly important for nerve tissues. Erythrocyte quality has been shown to be deteriorated in oxidative stress conditions. In this study, we measured the same series of oxidative stress markers in plasma and erythrocytes to compare the differences between neurotypical children (controls) and children with autism spectrum disorder (ASD). We also focused on erythrocyte properties including their deformability, osmotic resistance, Na,K-ATPase activity, nitric oxide levels and free radical levels in children with ASD and controls. Greater oxidative damage to proteins and lipids was observed in the erythrocytes than in the plasma of ASD subjects. Additionally, antioxidant enzymes were more active in plasma samples from ASD children than in their erythrocytes. Significantly higher nitric oxide level and Na,K-ATPase enzyme activity were detected in erythrocytes of ASD individuals in comparison with the controls. Changes in oxidative status could at least partially contribute to the deterioration of erythrocyte morphology, as more frequent echinocyte formation was detected in ASD individuals. These alterations are most probably responsible for worsening the erythrocyte deformability observed in children with ASD. We can conclude that abnormalities in antioxidant status and erythrocyte properties could be involved in the pathomechanisms of ASD and eventually contribute to its clinical manifestations.

The Relationship of Steroid Hormones, Genes Related to Testosterone Metabolism and Behavior in Boys With Autism in Slovakia.

OBJECTIVE: Purpose of the study was to identify the relationship among actual plasmatic levels of steroid hormones and behavioral manifestations in boys with autism and to assess the genetic contribution to these manifestations. METHODS: 172 boys with autism under 10 years of age and 135 neurotypical boys attended the study. ADI-R and ADOS-2 were used to evaluate the core symptom severities. Problem behavior was assessed using BPI-01 questionnaire. Levels of testosterone, estradiol, dehydroepiandrosterone, dehydroepiandrosterone-sulfate and sex hormone binding globulin (SHBG) were measured in plasma of autistic boys. Three SNPs (in ESR1, SHBG, SRD5A2 genes) and one STR in AR gene (number of CAG repeats in first exon) were assessed. Hormonal levels and number of CAG repeats in AR gene were used for correlation analysis with behavioral measures. Genotype and allelic frequencies were compared among autistic and neurotypical boys. RESULTS: We found negative relationship among SHBG levels and restricted, repetitive behaviors (measured by ADOS-2) and positive relationship among actual testosterone levels and frequency of stereotyped behavior (measured by BPI-01). CONCLUSION: Actual levels of SHBG and testosterone are related to severities of restricted and repetitive behaviors in boys with autism. Mechanisms of action of these hormones in brain require further investigation.

Comparing the impact of the first and second wave of COVID-19 lockdown on Slovak families with typically developing children and children with autism spectrum disorder.

LAY ABSTRACT: A global pandemic caused by a new coronavirus (severe acute respiratory syndrome coronavirus 2) affected everyday lives of all people, including individuals with special needs, such as autism spectrum disorder. The aim of this research was to compare the mental health of families with children with autism spectrum disorder to families with typically developing children, and between the first and the second wave of COVID-19 outbreak in Slovakia. This mainly included symptoms of depression, anxiety, and stress of parents and problem behavior or sleeping difficulties of their children. The research sample consisted of 332 parents (155 of which have children with autism spectrum disorder), 179 surveyed during the first wave and 153 during the second wave. Online parent questionnaire was created, including demographic and specific topic questions, Depression Anxiety and Stress Scale-42 questionnaire, and internalizing and externalizing maladaptive behavior subscales from Vineland Adaptive Behavior Scales. Our results show that during the first wave, parents of autism spectrum disorder children suffered high levels of anxiety. During the second wave, both groups of parents suffered increased anxiety, stress, and depression, but especially severe for parents of children with autism spectrum disorder. Internalizing maladaptive behavior of autistic children grew significantly between the waves. Parental depression, anxiety, and stress were interconnected with maladaptive behavior of both autism spectrum disorder and typically developing children, suggesting the importance of the therapy options for whole families.

Association between Environmental Tobacco Smoke Exposure and Adaptive Behavior in Individuals with Autism Spectrum Disorder.

The study focuses on current issues of adaptive behavior in individuals with autism spectrum disorder (ASD) and on the possible risk factor of environmental tobacco smoke (ETS). Children examined at the Academic Research Center for Autism (ARCA) in Bratislava were involved in the study. The study sample included 84 children (71 boys) with ASD (average age 5.35 years) and a non-ASD group of 24 children (20 boys; average age 8.10 years). The "ETS Questionnaire" focused on the detection of parental smoking habits and other ETS exposures. The concentrations of cotinine in urine were measured by ELISA kit. A significant delay in adaptive behavior of children with ASD in comparison with the non-ASD group was identified. The significant differences were in adaptive behavior, communication, and everyday skills. Children with ASD were more likely to be exposed to ETS, especially in the household. Good agreement was found between objective and subjective ETS exposure indicators (kappa = 0.613). Self-reported exposure to ETS corresponded significantly with the median levels of urinary cotinine. In addition to evaluation and assessment of the quality of adaptive behavior, an important goal of further research should be to identify, investigate, and eliminate environmental factors that interfere with adaptive behavior.

The acute effect of psychosocial stress on the level of oxidative stress in children.

The effect of chronic stress on oxidative stress (OS) is commonly discussed while the effect of acute stress situation is not fully examined yet. The present study was aimed to analyse whether acute psychosocial strain causes changes in OS and antioxidant status. Unstimulated saliva was collected from 46 healthy prepubertal children during the control and stress day. On the stress day, collection was performed before and after a stress situation induced by the Trier social stress test. Saliva collection during the control day imitated the stress day without the stress strain. Samples were used for analysis of lipid peroxidation, thiobarbituric acid reactive substances (TBARS), advanced glycation end products (AGEs) and markers of antioxidant status, total antioxidant capacity (TAC) and ferric reducing antioxidant power (FRAP). On the stress day, increased level of FRAP was observed in the second saliva collection in comparison with the first collection. Within the same day, no significant changes in the levels of TBARS, AGEs and TAC were observed in samples taken before and after stress strain. Significantly higher levels of TBARS were observed on stress day in comparison to control day. In summary, acute psychosocial stress caused increase of FRAP during the stress day. TBARS did not increase during the stress day in the second sample but it was higher compared to the control day. None of the interactions with gender were statistically significant. It appears the short-term exposure to stress could potentially stimulate antioxidant activity.

Alteration of the steroidogenesis in boys with autism spectrum disorders.

The etiology of autism spectrum disorders (ASD) remains unknown, but associations between prenatal hormonal changes and ASD risk were found. The consequences of these changes on the steroidogenesis during a postnatal development are not yet well known. The aim of this study was to analyze the steroid metabolic pathway in prepubertal ASD and neurotypical boys. Plasma samples were collected from 62 prepubertal ASD boys and 24 age and sex-matched controls (CTRL). Eighty-two biomarkers of steroidogenesis were detected using gas-chromatography tandem-mass spectrometry. We observed changes across the whole alternative backdoor pathway of androgens synthesis toward lower level in ASD group. Our data indicate suppressed production of pregnenolone sulfate at augmented activities of CYP17A1 and SULT2A1 and reduced HSD3B2 activity in ASD group which is partly consistent with the results reported in older children, in whom the adrenal zona reticularis significantly influences the steroid levels. Furthermore, we detected the suppressed activity of CYP7B1 enzyme readily metabolizing the precursors of sex hormones on one hand but increased anti-glucocorticoid effect of 7α-hydroxy-DHEA via competition with cortisone for HSD11B1 on the other. The multivariate model found significant correlations between behavioral indices and circulating steroids. From dependent variables, the best correlation was found for the social interaction (28.5%). Observed changes give a space for their utilization as biomarkers while reveal the etiopathogenesis of ASD. The aforementioned data indicate a direction of the future research with a focus on the expression and functioning of genes associated with important steroidogenic enzymes in ASD patients from early childhood to adrenarche.

Wood and Its Impact on Humans and Environment Quality in Health Care Facilities.

The paper presents the application of natural materials, especially wood, which are relevant for human well-being in built environments of health, social, and day care facilities. These properties were tested by a complex methodology in a case study in the wooden waiting room at National Oncology Institute in Bratislava. In this space, experimental tests of physiological responses were further executed on 50 volunteers moving in the waiting room for 20 min. In this article, the EEG (electroencephalograph) (four persons) and emotions from the faces of all our volunteers before entering and after a stay in a wooden waiting room were recorded. Specifically, the ECG (electrocardiograph), heart rate (HR), and respiration activity were measured by using our own designed ECG holter (40 persons), and also blood pressure and cortisol levels were observed. The usage of wooden materials verifies their regenerative and positive impact on the human nervous system, through the appealing aesthetics (color, texture, and structures), high contact comfort, pleasant smell, possibility to regulate air humidity, volatile organic compound emissions (VOC-emissions), and acoustic well-being in the space.

Salivary creatinine and urea are higher in an experimental model of acute but not chronic renal disease.

Plasma creatinine and urea are commonly used markers of kidney function in both acute and chronic renal failure. The needed repeated blood collection is associated with pain, stress and might lead to infections. Saliva has the potential to be a non-invasive alternative diagnostic fluid. The use of saliva in clinical practice is limited, since many factors affect the concentration of salivary biomarkers. The aim of our study was to analyze salivary creatinine and urea in the animal models of acute and chronic renal disease. Bilateral nephrectomy and adenine nephropathy were induced in adult male mice. Both, plasma creatinine and urea were higher in animals with renal failure compared to controls. Salivary creatinine was higher by 81% and salivary urea by 43% in comparison to the control group, but only in animals with bilateral nephrectomy and not in adenine nephropathy. Our results indicate that the increase of salivary creatinine and urea depends on the experimental model of renal failure and its severity. Further studies are needed to monitor the dynamics of salivary markers of renal function and to reveal determinants of their variability.

Oxidative stress in the oral cavity is driven by individual-specific bacterial communities.

The term "bacterial dysbiosis" is being used quite extensively in metagenomic studies, however, the identification of harmful bacteria often fails due to large overlap between the bacterial species found in healthy volunteers and patients. We hypothesized that the pathogenic oral bacteria are individual-specific and they correlate with oxidative stress markers in saliva which reflect the inflammatory processes in the oral cavity. Temporally direct and lagged correlations between the markers and bacterial taxa were computed individually for 26 volunteers who provided saliva samples during one month (21.2 ± 2.7 samples/volunteer, 551 samples in total). The volunteers' microbiomes differed significantly by their composition and also by their degree of microbiome temporal variability and oxidative stress markers fluctuation. The results showed that each of the marker-taxa pairs can have negative correlations in some volunteers while positive in others. Streptococcus mutans, which used to be associated with caries before the metagenomics era, had the most prominent correlations with the oxidative stress markers, however, these correlations were not confirmed in all volunteers. The importance of longitudinal samples collections in correlation studies was underlined by simulation of single sample collections in 1000 different combinations which produced contradictory results. In conclusion, the distinct intra-individual correlation patterns suggest that different bacterial consortia might be involved in the oxidative stress induction in each human subject. In the future, decreasing cost of DNA sequencing will allow to analyze multiple samples from each patient, which might help to explore potential diagnostic applications and understand pathogenesis of microbiome-associated oral diseases.

Blood Contamination in Saliva: Impact on the Measurement of Salivary Oxidative Stress Markers.

Salivary oxidative stress markers represent a promising tool for monitoring of oral diseases. Saliva can often be contaminated by blood, especially in patients with periodontitis. The aim of our study was to examine the impact of blood contamination on the measurement of salivary oxidative stress markers. Saliva samples were collected from 10 healthy volunteers and were artificially contaminated with blood (final concentration 0.001-10%). Next, saliva was collected from 12 gingivitis and 10 control patients before and after dental hygiene treatment. Markers of oxidative stress were measured in all collected saliva samples. Advanced oxidation protein products (AOPP), advanced glycation end products (AGEs), and antioxidant status were changed in 1% blood-contaminated saliva. Salivary AOPP were increased in control and patients after dental treatment (by 45.7% and 34.1%, p < 0.01). Salivary AGEs were decreased in patients after microinjury (by 69.3%, p < 0.001). Salivary antioxidant status markers were decreased in both control and patients after dental treatment (p < 0.05 and p < 0.01). One % blood contamination biased concentrations of salivary oxidative stress markers. Saliva samples with 1% blood contamination are visibly discolored and can be excluded from analyses without any specific biochemic detection of blood constituents. Salivary markers of oxidative stress were significantly altered in blood-contaminated saliva in control and patients with gingivitis after dental hygiene treatment.