Evaluation of 16S rRNA primer sets for characterisation of microbiota in paediatric patients with autism spectrum disorder.

intestinal microbiota is becoming a significant marker that reflects differences between health and disease status also in terms of gut-brain axis communication. Studies show that children with autism spectrum disorder (ASD) often have a mix of gut microbes that is distinct from the neurotypical children. Various assays are being used for microbiota investigation and were considered to be universal. However, newer studies showed that protocol for preparing DNA sequencing libraries is a key factor influencing results of microbiota investigation. The choice of DNA amplification primers seems to be the crucial for the outcome of analysis. In our study, we have tested 3 primer sets to investigate differences in outcome of sequencing analysis of microbiota in children with ASD. We found out that primers detected different portion of bacteria in samples especially at phylum level; significantly higher abundance of Bacteroides and lower Firmicutes were detected using 515f/806r compared to 27f/1492r and 27f*/1495f primers. So, the question is whether a gold standard of Firmicutes/Bacteroidetes ratio is a valuable and reliable universal marker, since two primer sets towards 16S rRNA can provide opposite information. Moreover, significantly higher relative abundance of Proteobacteria was detected using 27f/1492r. The beta diversity of sample groups differed remarkably and so the number of observed bacterial genera.

The effect of long-term hypogonadism on body composition and morphometry of aged male Wistar rats.

Clinical studies show that hypogonadism in the aging male is associated with obesity and osteoporosis. Experimental studies are mostly conducted on relatively young adult animals and the induced hypogonadism lasts for a relatively short time. The present study aimed to describe the effect of long-term hypogonadism beginning in puberty on body composition, morphometry, and bone mineral density in aged male rats. Morphometric measurements and dual-energy X-ray absorptiometry were conducted at the age of 30 months on control and gonadectomized males. Long-term hypogonadism did not affect body weight, but led to a higher fat mass (by 26 %), lower lean mass (by 44 %), shorter body length (by 9 %), and anogenital distance (by 26 %), as well as to lower tail circumference (by 15 %) in comparison to control males. Lower bone mineral density (by 13 %) and bone mineral content (by 15 %) were observed in gonadectomized males. Results showing sarcopenic obesity and osteoporosis in this model of long-term hypogonadism might mimic the situation in aging males better than the widely used short-term hypogonadism induced in young animals. The morphometric analysis could potentially be a useful tool to study normal weight obesity without the need for specific equipment.

The effect of saliva stimulation on the secretion of cortisol during stress and physiological conditions.

OBJECTIVES: Many studies use stimulated saliva for the assessment of cortisol. However, it is not yet clear how stimulation affects the flow of specific markers. The aim was to assess whether stimulation of salivation affects the physiological flow of cortisol during a stressing day as compared to an ordinary day. The second aim was to show how the normalising factor affects the outcome of the study. METHODS: Stimulated saliva was taken from 42 children at 8:00 a.m. and 12:00 a.m. on two separate days one month apart. During the first day, the children were exposed to stress situation, while the second day was considered a control day. The concentration of cortisol was analysed using ELISA. RESULTS: The highest level of cortisol was observed in the morning of the stress day (p 0.99). CONCLUSION: Based on our results, the examination of the cortisol diurnal rhythm is not reliable in stimulated saliva. Moreover, the effect of saliva stimulation has to be taken into account for every marker individually (Fig. 2, Ref. 22).

Testosterone and the brain: from cognition to autism.

Sex and gender matter in all aspects of life. Humans exhibit sexual dimorphism in anatomy, physiology, but also pathology. Many of the differences are due to sex chromosomes and, thus, genetics, other due to endocrine factors such as sex hormones, some are of social origin. Over the past decades, huge number of scientific studies have revealed striking sex differences of the human brain with remarkable behavioral and cognitive consequences. Prenatal and postnatal testosterone influence brain structures and functions, respectively. Cognitive sex differences include especially certain spatial and language tasks, but they also affect many other aspects of the neurotypical brain. Sex differences of the brain are also relevant for the pathogenesis of neuropsychiatric disorders such as autism spectrum disorders, which are much more prevalent in the male population. Structural dimorphism in the human brain was well-described, but recent controversies now question its importance. On the other hand, solid evidence exists regarding gender differences in several brain functions. This review tries to summarize the current understanding of the complexity of the effects of testosterone on brain with special focus on their role in the known sex differences in healthy individuals and people in the autism spectrum.

Specificity of gut microbiota in children with autism spectrum disorder in Slovakia and its correlation with astrocytes activity marker and specific behavioural patterns.

Recent research suggests the involvement of bidirectional gut-brain axis in autism spectrum disorder (ASD). The aim of this study was to establish the role of changed gut microbiota in behavioural and gastrointestinal manifestations, but also in astrocyte activation in children with ASD. Distinct faecal microbiota in children with ASD was found to be more heterogeneous compared to that in neurotypical children. Different bacterial abundance and correlation with behavioural and GI manifestations revealed several bacterial genera possibly important for ASD. Microbial-neuronal cross talk could be accomplished through S100B, released by glial cells, activated by low grade inflammation. More complex studies are required to understand ASD pathogenesis.

Does the 2nd and 4th digit ratio reflect prenatal androgen exposure?

OBJECTIVES:  The aim of our study was to describe the effect of prenatal testosterone exposure on 2D:4D in both sexes, and to determine whether this effect is mediated via the androgen receptor. In addition, the sex differences in lengths of 2D, 4D, and 2D:4D ratio were analyzed. BACKGROUND:  Clinical studies suggest a negative correlation between prenatal testosterone exposure and ratio of the lengths of the second and fourth digits (2D:4D). However, less is known about the underlying molecular mechanisms. METHODS:  Pregnant rats were treated with olive oil, testosterone, flutamide or testosterone with flutamide daily from the fourteenth day of pregnancy until delivery. The finger lengths of adult offspring were measured using both, digital scanning of the paws and µCT analysis of the phalanges. RESULTS:  None of the aforementioned methods revealed any effect of testosterone on 2D:4D. µCT measurements showed that prenatal hyperandrogenism in both sexes leads to shorter 2D compared to controls. Moreover, the testosterone treatment in males resulted in the shortening of 4D when compared to controls. CONCLUSION:  Prenatal hyperandrogenism leads to shorter lengths of 2D and 4D; however, it does not affect 2D:4D ratio. Whether other steroid hormones and/or testosterone metabolites affect the 2D:4D ratio requires further investigation (Tab. 5, Fig. 3, Ref. 32).

Metabolic and renal effects of dietary advanced glycation end products in pregnant rats - a pilot study.

Thermally processed food contains advanced glycation end products (AGEs) including N(epsilon)-(carboxymethyl)lysine (CML). Higher AGEs or circulating CML were shown to be associated with pregnancy complications such as preeclampsia and gestational diabetes. It is unclear whether this association is causal. The aim of our study was to analyze the effects of dietary CML and CML-containing thermally processed food on metabolism in pregnant rats. Animals were fed with standard or with AGE-rich diet from gestation day 1. Third group received standard diet and CML via gavage. On gestation day 18, blood pressure was measured, urine and blood were collected and the oral glucose tolerance test was performed. Plasma AGEs were slightly higher in pregnant rats fed with the AGE-rich diet (p=0.09). A non-significant trend towards higher CML in plasma was found in the CML group (p=0.06). No significant differences between groups were revealed in glucose metabolism or markers of renal functions like proteinuria and creatinine clearance. In conclusion, this study does not support the hypothesis that dietary AGEs such as CML might induce harmful metabolic changes or contribute to the pathogenesis of pregnancy complications. The short duration of the rodent gestation warrants further studies analyzing long-term effects of AGEs/CML in preconception nutrition.

Vitamin D in autistic children and healthy controls.

Insufficient levels of vitamin D have been demonstrated by many authors as a risk factor for autistic patients, however, the causality has not been reliably elucidated. In the present study, levels of calcidiol were determined in group of autistic children and compared with age matched healthy children as controls. Suboptimal levels of calcidiol in more than 60 % of both autistic patients as well as of control group were found. No significant differences in vitamin D between autistic children and healthy controls were observed.

Erythrocyte deformability in children with autism spectrum disorder: correlation with clinical features.

Biomechanical properties of erythrocytes play an important role in health and disease. Deformability represents intrinsic property of erythrocytes to undergo deformation that is crucial for their passage through the narrow capillaries. The erythrocyte damage can lead to compromised tissue perfusion and consequently play a role in the pathogenesis of various diseases including neurological ones. Data available in databases indicate that erythrocytes in autism spectrum disorder (ASD) are altered. This may affect the clinical symptoms of ASD. The aim of our study was to determine erythrocyte deformability in 54 children with ASD and correlate it with clinical symptoms. We found significant negative correlation between erythrocyte deformability and score in C domain of the Autism Diagnostic Interview-Revised (ADI-R) diagnostic tool describing the measure of restrictive, repetitive, and stereotyped behaviors and interests, mainly observable in C1 and C2, but not in C3 and C4 subdomains. This supports the findings of other authors and suggest that behavioral domain C comprises of more subcategories with different underlying etiology. Our results also indicate that abnormalities in erythrocyte deformability may be involved in ASD pathomechanisms and contribute to its clinical manifestation. Further research is necessary to bring more data and identify erythrocyte deformability as prognostic biomarker in ASD.

Plasma levels of glial cell marker S100B in children with autism.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with increasing incidence. Recent evidences suggest glial cells involvement in autism pathophysiology. S100B is a calcium binding protein, mainly found in astrocytes and therefore used as a marker of their activity. In our study, children with autism had higher plasma concentrations of S100B compared to non-autistic controls. No association of S100B plasma levels with behavioral symptoms (ADI-R and ADOS-2 scales) was found. Plasma S100B concentration significantly correlated with urine serotonin, suggesting their interconnection. Correlation of plasma S100B levels with stool calprotectin concentrations was found, suggesting not only brain astrocytes, but also enteric glial cells may take part in autism pathogenesis. Based on our findings, S100B seems to have a potential to be used as a biomarker of human neurodevelopmental disorders, but more investigations are needed to clarify its exact role in pathomechanism of autism.

Projection length stimulated by oxytocin is modulated by the inhibition of calcium signaling in U-87MG cells.

Neuropeptide oxytocin contributes to the regulation of glial cell morphology. The precise mechanisms, however, are not yet fully understood. In the present study, we have investigated whether an oxytocin-induced increase of intracellular calcium is required for cell extension in astrocyte-like U-87MG cells. Oxytocin (1 µM) significantly increased the length of the cell projections measured by the green-fluorescent protein labeled microtubule-associated protein after 48 h. The knockdown of oxytocin receptors (OXTR) in U-87MG cells prevented the elongation of the projections. Incubation of U-87MG cells in the presence of oxytocin, resulted in a significant increase of intracellular calcium, specifically blocked by the OXTR antagonist L-371,257. Both quercetin, which is a phosphoinositide 3-kinase inhibitor, and the phospholipase C inhibitor U-73122 reduced oxytocin-induced elevation of intracellular calcium concentration. Conversely, neither diltiazem, an L-type voltage-gated calcium channel blocker nor tetracaine, which is a blocker of the ryanodine receptors, showed an effect on intracellular calcium levels. Treatment of cells with quercetin, U-73122 and the voltage-gated calcium channel blockers cilnidipine, ω-agatoxin and mibefradil prevented the elongation of projections stimulated by oxytocin. Oxytocin treatment resulted in a significant increase in gene and protein expression of the scaffolding protein SHANK3. Our results clearly show that activation of OXTRs contributes to the elongation of cell projections in astrocyte-like U-87MG cells and that this effect is mediated by an extracellular calcium influx accompanied by an increase in scaffolding proteins expression.

Vitamin D, neurosteroids and autism.

Vitamin D had been for a long time investigated for its effects on bone metabolism. Recently has been observed that the incidence of some neurodevelopmental disorders (including autism) increases hand in hand with vitamin D deficiency. Indeed, vitamin D was reported to modulate the biosynthesis of neurotransmitters and neurotrophic factors; moreover, its receptor was found in the central nervous system. Vitamin D deficiency was therefore assessed as a risk factor for autism, however the biological mechanism has not yet been revealed. In our review we focused on potential connections among vitamin D, steroids and autism. Potential mechanisms of vitamin D action are also discussed.

Sex differences in the effect of prenatal testosterone exposure on steroid hormone production in adult rats.

Maternal hyperandrogenism during pregnancy might have metabolic and endocrine consequences on the offspring as shown for the polycystic ovary syndrome. Despite numerous experiments, the impact of prenatal hyperandrogenic environment on postnatal sex steroid milieu is not yet clear. In this study, we investigated the effect of prenatal testosterone excess on postnatal concentrations of luteinizing hormone, corticosterone and steroid hormones including testosterone, pregnenolone, progesterone, estradiol and 7beta-hydroxy-epiandrosterone in the offspring of both sexes. Pregnant rats were injected daily with either testosterone propionate or vehicle from gestational day 14 until parturition. The hormones were evaluated in plasma of the adult offspring. As expected, females had lower testosterone and higher pregnenolone, progesterone and estradiol in comparison to males. In addition, corticosterone was higher in females than in males, and it was further elevated by prenatal testosterone treatment. In males, prenatal testosterone exposure resulted in higher 7beta-hydroxy-epiandrosterone in comparison to control group. None of the other analyzed hormones were affected by prenatal testosterone. In conclusion, our results did not show major effects on sex hormone production or luteinizing hormone release in adult rats resulting from testosterone excess during their fetal development. However, maternal hyperandrogenism seems to partially affect steroid biosynthesis in sex-specific manner.

How do we love? Romantic love style in men is related to lower testosterone levels.

Testosterone has been widely investigated in associations with many aspects of social interactions, emotions and behavior. No research has been conducted on its contribution to the variability of love styles in human. The aim of this paper was to uncover the possible relationship between not only the actual plasma testosterone levels, but also the prenatal testosterone level (expressed as 2D:4D ratio) and the sensitivity of androgen receptor and love typology in young healthy men. There are six love styles which are primary including Eros (passionate romantic love), Ludus (playful) and Storge (friendly) and secondary love consisting of Mania (obsessive), Pragma (practical realistic) and Agape (altruistic). Our results pointed out that low testosterone concentrations are associated with higher score for Eros, Ludus, Pragma, Mania love style. No significant association was proved for other tested parameters of androgenicity (2D:4D, sensitivity of androgen receptor) and love style after correction was applied. Different attitudes and behavior in relationships do have a biological foundation related to endogenous testosterone levels in plasma. Future studies should address questions about the family and social background of participants to differentiate here between moral rules or/and social-conventional rules.

Fecal calprotectin levels correlate with main domains of the autism diagnostic interview-revised (ADI-R) in a sample of individuals with autism spectrum disorders from Slovakia.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impaired social interaction and communication, as well as repetitive behavior and restricted interests. There is convincing evidence that the intestinal inflammation is involved in etiology of ASD. Increased levels of inflammatory markers were shown to be associated with more aberrant behaviors and communication of subjects with ASD. Calprotectin in the feces is produced by activated neutrophils and epithelial cells of the gut mucosa, and its levels reflect local inflammation of the gastrointestinal tract. Concentration of fecal calprotectin was determined by ELISA method in 87 individuals with ASD and 51 controls, of that 29 siblings of children with ASD and 22 non-related controls. In non-relatives significantly lower values of fecal calprotectin were observed than in both subjects with ASD and their siblings. In the group with ASD significant correlations of fecal calprotectin with all domains of the ADI-R diagnostic tool were found: qualitative abnormalities in reciprocal social interaction and communication, restrictive and repetitive patterns of behavior. Results suggest that low grade intestinal inflammation may be one of factors implicated in the pathophysiology of ASD.

Testosterone in relation to behavioral problems in pre-pubertal boys with autism spectrum disorders.

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impairment in social communication and presence of stereotyped/restricted behaviors. Children with ASD very often demonstrate co-morbid psychiatric problems, problems known to be affected by testosterone in neurotypical populations. However, there are few reports investigating relationships between testosterone and psychiatric conditions in children with ASD. The aim of this study was to determine the relationship between plasmatic levels of testosterone and behavioral/emotional problems in pre-pubertal boys with ASD. The study sample consisted of 31 pre-pubertal boys (ages 3-10) with ASD. Parents completed the Nisonger Child Behavior Rating Form (NCBRF) to assess specific behavioral/emotional problems as observed in the previous 2 months. Plasmatic testosterone levels were determined in boys according to standardized procedures. It was found that there were positive correlations between testosterone levels and the conduct problems subscale (p=0.034, rs=0.382) of NCBRF and also between testosterone levels and the hyperactive subscale (p=0.025, rs=0.402) of NCBRF. Findings in this study are in line with research conducted in the neurotypical population. This is the first large study investigating testosterone and emotional/behavioral problems in ASD and warrants further research in this field in order to clarify the etiopathogenesis of psychiatric co-morbidities and improve their treatment.

Bio-behavioral model of aggression in autism spectrum disorders-pilot study.

Children with autism spectrum disorders (ASD) have a high rate of irritability and aggressive symptoms which have significant impact on their lives, families and society. The etiology of aggression in humans is likely complex and includes both biological and behavioral causes. Biological approaches have focused on hormones and neurotransmitters that are hypothesized to contribute to the etiology and clinical manifestation of aggressive behavior in humans. Testosterone is a male sex hormone and some studies suggest that it can play a role in the complex etiology of aggressive behavior. Two specific subtypes of aggression have been identified: explosive and non-explosive. Explosive aggression is accompanied by a raged affect and is usually more dangerous and not immediately responsive to behavioral treatment. We propose that individuals with ASD and explosive aggression will have higher androgen activity and higher arousal than neurotypical children and children with ASD without explosive aggression. We employed a unique method for aggression assessment- functional behavioral analysis- to obtain objective and quantitative measures of aggression and arousal signs. In our pilot study, we proposed to determine bio-behavioral model of explosive aggression in children with ASD which will predict which children will be most responsive to antiandrogen therapy and behavioral therapy (Tab. 1, Fig. 1, Ref. 31).

Testosterone and estradiol are not affected in male and female patients with obstructive sleep apnea treated with continuous positive airway pressure.

BACKGROUND: Continuous positive airway pressure (CPAP) improves symptoms in patients with obstructive sleep apnea syndrome (OSAS). It is currently unclear, whether CPAP also alters endocrine parameters such as sex hormone levels. In a previous study, we have found no changes in sex hormones in patients with OSAS after one night with CPAP. AIM: The aim of this study was to prove long-term effects of CPAP on sex hormone concentrations in patients with OSAS. METHODS: Twenty-two women and 67 men with severe OSAS (respiratory distress index > 30/h) were enrolled in the study. Fasting blood venous samples were taken before CPAP therapy and after 1 and 6 months of CPAP treatment. Testosterone and estradiol were measured in all samples using commercially available ELISA kits. RESULTS: No effects of long-term CPAP treatment were found on testosterone or estradiol levels in OSAS patients of either gender. CONCLUSIONS: The results are in line with previous smaller studies. However, our study is larger and longer than previously published studies. In addition, this is the first study analyzing the effects of CPAP on testosterone and estradiol and in both genders. Positive effects of CPAP on sexual functions reported in other studies might, thus, be mediated by other than endocrine effects.

Increased plasma levels of the high mobility group box 1 protein (HMGB1) are associated with a higher score of gastrointestinal dysfunction in individuals with autism.

Autism is a disorder of neural development characterized by impairments in communication, social interaction, restricted interests and repetitive behavior. The etiology of autism is poorly understood, the evidence indicates that inflammation may play a key role. In autism a high prevalence of gastrointestinal disturbances is reported, that are linked to a low-grade chronic inflammation of the intestinal mucosa. High mobility group box 1 protein (HMGB1) is an intranuclear protein that can be passively released from necrotic cells or actively secreted under inflammatory conditions as alarmin or late proinflammatory cytokine. The objective of this study was to measure plasma levels of HMGB1 in individuals with autism and to analyze their association with gastrointestinal symptoms. The study involved 31 subjects with low-functioning autistic disorder aged 2-22 years and 16 healthy controls. Plasma HMGB1 levels were significantly higher in individuals with autism than in controls (13.8+/-11.7 ng/ml vs. 7.90+/-4.0 ng/ml, p<0.02). In subjects with plasma HMGB1 levels higher than 11 ng/ml severe forms of GI disorders were more prevalent (83.3 %) than in subjects with lower levels (38.9 %, p<0.04). Results of the study support the involvement of the systemic low-grade inflammation in the pathomechanisms of autism and its possible association with GI symptoms.