Children with idiopathic short stature have significantly different gut microbiota than their normal height siblings: a case-control study.

Objectives: To investigate the role of gut microbiota (GM) in pathogenesis of idiopathic short stature (ISS) by comparing GM of ISS children to their normal-height siblings. Methods: This case-control study, conducted at the Schneider Children's Medical Center's Institute for Endocrinology and Diabetes between 4/2018-11/2020, involved 30 pairs of healthy pre-pubertal siblings aged 3-10 years, each comprising one sibling with ISS and one with normal height. Outcome measures from fecal analysis of both siblings included GM composition analyzed by 16S rRNA sequencing, fecal metabolomics, and monitoring the growth of germ-free (GF) mice after fecal transplantation. Results: Fecal analysis of ISS children identified higher predicted levels of genes encoding enzymes for pyrimidine, purine, flavin, coenzyme B, and thiamine biosynthesis, lower levels of several amino acids, and a significantly higher prevalence of the phylum Euryarchaeota compared to their normal-height siblings (p<0.001). ISS children with higher levels of Methanobrevibacter, the dominant species in the archaeal gut community, were significantly shorter in stature than those with lower levels (p=0.022). Mice receiving fecal transplants from ISS children did not experience stunted growth, probably due to the eradication of Methanobrevibacter caused by exposure to oxygen during fecal collection. Discussion: Our findings suggest that different characteristics in the GM may explain variations in linear growth. The varying levels of Methanobrevibacter demonstrated within the ISS group reflect the multifactorial nature of ISS and the potential ability of the GM to partially explain growth variations. The targeting of specific microbiota could provide personalized therapies to improve growth in children with ISS.

A Whey-Based Diet Can Ameliorate the Effects of LPS-Induced Growth Attenuation in Young Rats.

Chronic inflammation in childhood is associated with impaired growth. In the current study, a lipopolysaccharide (LPS) model of inflammation in young rats was used to study the efficacy of whey-based as compared to soy-based diets to ameliorate growth attenuation. Young rats were injected with LPS and fed normal chow or diets containing whey or soy as the sole protein source during treatment, or during the recovery period in a separate set of experiments. The body and spleen weight, food consumption, humerus length, and EGP height and structure were evaluated. Inflammatory markers in the spleen and markers of differentiation in the EGP were assessed using qPCR. The LPS led to a significant increase in the spleen weight and a decrease in the EGP height. Whey, but not soy, protected the animals from both effects. In the recovery model, whey led to increased EGP height at both 3 and 16 d post treatment. The most affected region in the EGP was the hypertrophic zone (HZ), which was significantly shortened by the LPS treatment but enlarged by whey. In conclusion, LPS affected the spleen weight and EGP height and had a specific effect on the HZ. Nutrition with whey protein appeared to protect the rats from the LPS-induced growth attenuation.

Associations of Obesity with Linear Growth and Puberty.

BACKGROUND: The prevalence of obesity in childhood has increased dramatically in recent decades with increased risk of developing cardiometabolic and other comorbidities. Childhood adiposity may also influence processes of growth and puberty. SUMMARY: Growth patterns of obesity during childhood have been shown to be associated with increased linear growth in early childhood, leading to accelerated epiphyseal growth plate (EGP) maturation. Several hormones secreted by the adipose tissue may affect linear growth in the context of obesity, both via the growth hormone IGF-1 axis and via a direct effect on the EGP. The observation that children with obesity tend to mature earlier than lean children has led to the assumption that the degree of body fatness may trigger the neuroendocrine events that lead to pubertal onset. The most probable link between obesity and puberty is leptin and its interaction with the kisspeptin system, which is an important regulator of puberty. However, peripheral action of adipose tissue could also be involved in changes in the onset of puberty. In addition, nutritional factors, epigenetics, and endocrine-disrupting chemicals are potential mediators linking pubertal onset to obesity. In this review, we focused on interactions of obesity with linear growth and pubertal processes, based on basic research and clinical data in humans. KEY MESSAGE: Children with obesity are subject to accelerated linear growth with risk of impaired adult height and early puberty, with its psychological consequences. The data highlight another important objective in combatting childhood obesity, for the prevention of abnormal growth and pubertal patterns.

Different Effects of Soy and Whey on Linear Bone Growth and Growth Pattern in Young Male Sprague-Dawley Rats.

The aim of this investigation was to determine the better protein for supporting optimal linear growth, as the exact composition and benefits of specific dietary proteins in supporting linear growth is unknown. In the current study, we compared the effect of soy and whey proteins, both proteins contain all essential amino acids and are considered the best proteins in their categories. Young male rats were subjected to multiple feeding protocols using iso-energetic diets containing soy or whey as the sole protein source. The rats were allowed to eat ad libitum for 11, 24, or 74 days in the first set of experiments, and the soy group was pair-fed to the whey group in the second set. The differences in weight gain, food consumption, and humeri length of the soy group that were greater at the beginning of the ad libitum experiments lessened over time. Pair-fed experiments revealed that the increased weight and humeri length resulted from the differences in food consumption. However, other parameters were protein specific. Bone quality, which was better in the soy group at 24 days, was matched by the whey group and even surpassed that of the soy group in the long-term experiment, with a significantly greater bone mineral density, cortical thickness, and growth plate. Although in the short term the levels of insulin like growth factor (IGF)-I were similar between the groups, IGF-I increased with age in the whey group, and the levels at the long-term experiment were significantly higher compared to the soy group. Furthermore, using the pair fed setup made it clear that when the difference in food consumption were no longer playing part, whey was more efficient in increasing IGF-I. There were no indications of metabolic sequelae. Although the use of soy is gaining in popularity as a sustainable protein, our findings indicate a better effect of whey on linear growth by leading to slower growth with better-organized epiphyseal growth plates and bone quality.

Anxiety and Cognition in Cre- Collagen Type II Sirt1 K/O Male Mice.

Introduction: Using transgenic collagen type II-specific Sirt1 knockout (CKO) mice we studied the role of Sirt1 in nutritional induced catch up growth (CUG) and we found that these mice have a less organized growth plate and reduced efficiency of CUG. In addition, we noted that they weigh more than control (CTL) mice. Studying the reason for the increased weigh, we found differences in activity and brain function. Methods: Several tests for behavior and activity were used: open field; elevated plus maze, Morris water maze, and home cage running wheels. The level of Glu- osteocalcin, known to connect bone and brain function, was measured by Elisa; brain Sirt1 was analyzed by western blot. Results: We found that CKO mice had increased anxiety, with less spatial memory, learning capabilities and reduced activity in their home cages. No significant differences were found between CKO and CTL mice in Glu- osteocalcin levels; nor in the level of brain SIRT1. Discussion/Conclusion: Using transgenic collagen type II-specific Sirt1 knockout (CKO) mice we found a close connection between linear growth and brain function. Using a collagen type II derived system we affected a central regulatory mechanism leading to hypo activity, increased anxiety, and slower learning, without affecting circadian period. As children with idiopathic short stature are more likely to have lower IQ, with substantial deficits in working memory than healthy controls, the results of the current study suggest that SIRT1 may be the underlying factor connecting growth and brain function.

Cartilage -specific knockout of Sirt1 significantly reduces bone quality and catch-up growth efficiency.

BACKGROUND: Spontaneous catch-up (CU) growth occurs when a growth-restricting factor is resolved. However, its efficiency is sometimes inadequate and growth deficits remain permanent. The therapeutic toolbox for short stature is currently very limited, thus, finding new regulatory pathways is important for the development of novel means of treatment. Our previous studies using a nutrition-induced CU growth model showed that the level of sirtuin-1 (Sirt1) was significantly increased in food-restricted animals and decreased during CU growth. AIM: This study sought to investigate the role of Sirt1 in modulating the response of the epiphyseal growth plate (EGP) to nutritional manipulation. METHOD: Collagen type II-specific Sirt1 knockout (CKO) mice were tested for response to our CU growth model consisting of a period of food restriction followed by re-feeding. RESULTS: The transgenic CKO mice weighed more than the control (CTL) mice, their EGP was higher and less organized, specifically at the resting and proliferative zones, leading to shorter bones. Ablation of Sirt1 in the chondrocytes was found to have a dramatic effect on bone mineralization on micro-CT analysis. The CKO mice were less responsive to the nutritional manipulation, and their CU growth was less efficient. They remained shorter than the CTL mice who corrected the food restriction-induced growth deficit during the re-feeding period. CONCLUSIONS: Sirt1 appears to be important for normal regulation of the EGP. In its absence, the EGP is less organized and CU growth is less efficient. These results suggest that SIRT1 may serve as a novel therapeutic target for short stature.

The association between adipocytokines and glycemic control in women with gestational diabetes mellitus.

Objective: To evaluate the relationship between adipocytokines and glycemic control.Study design: Prospective observational trial of gestations with gestational diabetes mellitus (GDM). Fasting glucose (FG), insulin, adiponectin, leptin, chemerin, retinol-binding protein 4 (RBP-4), osteocalcin, and resistin were measured. HomeOstasis model assessment of insulin resistance (HOMA-IR) and QUantitative insulin sensitivity ChecK Index (QUICKI) were calculated. Women who required medications for glycemic control were compared to women using nutritional therapy only.Results: Overall, 75 women were included -26 (34.7%) required medications to achieve good glycemic control. Factors associated with poor control are as follows: low resistin (aOR 0.84), HOMA-IR (aOR 1.96), QUICKI (aOR 0.62), first trimester FG (aOR 1.43), and maternal age (aOR 1.26). HOMA-IR and QUICKI performed highest for prediction. Resistin, first trimester FG, maternal age, and QUICKI had an AUC of 0.878, sensitivity and specificity of 87.5% for the prediction of the need for medications.Conclusions: Low resistin is associated with poor control. A model utilizing maternal age, first trimester fasting glucose, and first visit QUICKI yields good predictability.

Growth hormone therapy in children with idiopathic short stature - the effect on appetite and appetite-regulating hormones: a pilot study.

AIM: To investigate the effect of growth hormone (GH) therapy on appetite-regulating hormones and to examine the association between these hormones and the response to GH, body composition, and resting energy expenditure (REE). METHODS: Nine pre-pubertal children with idiopathic short stature underwent a standard meal test before and 4 months following initiation of GH treatment. Ghrelin, GLP-1, leptin, and insulin levels were measured; area under the curve (AUC) was calculated. Height, weight, body composition, REE, and insulin-like growth factor levels were recorded at baseline and after 4 and 12 months. RESULTS: Following 4 months of GH therapy, food intake increased, with increased height-standard deviation score (SDS), weight-SDS, and REE (p < .05). Significant changes in appetite-regulating hormones included a decrease in postprandial AUC ghrelin levels (p = .045) and fasting GLP-1 (p = .038), and an increase in fasting insulin (p = .043). Ghrelin levels before GH treatment were positively correlated with the changes in weight-SDS (fasting: r = .667, p = .05; AUC: r = .788, p = .012) and REE (fasting: r = .866, p = .005; AUC: r = .847, p = .008) following 4 months of GH therapy. Ghrelin AUC at 4 months was positively correlated with the changes in height-SDS (r = .741, p = .022) and fat-free-mass (r = .890, p = .001) at 12 months of GH treatment. CONCLUSIONS: The reduction in ghrelin and GLP-1 following GH treatment suggests a role for GH in appetite regulation. Fasting and meal-AUC ghrelin levels may serve as biomarkers for predicting short-term (4 months) changes in weight and longer term (12 months) changes in height following GH treatment. The mechanisms linking GH with changes in appetite-regulating hormones remain to be elucidated. ABBREVIATIONS: SDS: standard deviation score; REE: resting energy expenditure; SMT: standard meal test; AUC: area under the curve; ISS: idiopathic short stature; SGA: small for gestational age; FFM: fat-free-mass; FM: fat mass; EER: estimated energy requirements; DRI: dietary reference intakes; IQR: inter-quartile range.

Leptin stimulates aromatase in the growth plate: limiting catch-up growth efficiency.

Catch-up growth (CUG) in childhood is defined as periods of growth acceleration, after the resolution of growth attenuation causes, bringing the children back to their original growth trajectory. Sometimes, however, CUG is incomplete, leading to permanent growth deficit and short stature. The aim of this study was to investigate the mechanisms that limit nutritional-CUG. Specifically, we focused on the crosstalk between leptin, increased by re-feeding, and sex hormones, which increase with age. In vivo studies were performed in young male Sprague Dawley rats fed ad libitum or subjected to 10/36 days of 40% food restriction followed by 90-120 days of re-feeding. In vitro studies were performed on ATDC5 cells. Analyses of mRNA and protein levels were done using qPCR and Western blot, respectively. CUG was complete in body weight and humerus length in animals that were food-restricted for 10 days but not for those food-restricted for 36 days. In vitro studies showed that leptin significantly increased aromatase gene expression and protein level as well as the expression of estrogen and leptin receptors in a dose- and time-dependent manner. The effect of leptin on aromatase was direct and was mediated through the MAPK/Erk, STAT3 and PI3K pathways. The crosstalk between leptin and aromatase in the growth plate suggests that re-feeding during puberty may lead to increased estrogen level and activity, and consequently, irreversible premature epiphyseal growth plate closure. These results may have important implications for the development of novel treatment strategies for short stature in children.

Food restriction followed by refeeding with a casein- or whey-based diet differentially affects the gut microbiota of pre-pubertal male rats.

Researchers are gaining an increasing understanding of host-gut microbiota interactions, but studies of the role of gut microbiota in linear growth are scarce. The aim of this study was to investigate the effect of food restriction and refeeding with different diets on gut microbiota composition in fast-growing rats. Young male Sprague-Dawley rats were fed regular rat chow ad libitum (control group) or subjected to 40% food restriction for 36 days followed by continued restriction or ad libitum refeeding for 24 days. Three different diets were used for refeeding: regular vegetarian protein chow or chow in which the sole source of protein was casein or whey. In the control group, the composition of the microbiota remained stable. Food restriction for 60 days led to a significant change in the gut microbiota at the phylum level, with a reduction in the abundance of Firmicutes and an increase in Bacteroidetes and Proteobacteria. Rats refed with the vegetarian protein diet had a different microbiota composition than rats refed the casein- or whey-based diet. Similarities in the bacterial population were found between rats refed vegetarian protein or a whey-based diet and control rats, and between rats refed a casein-based diet and rats on continued restriction. There was a significant strong correlation between the gut microbiota and growth parameters: humerus length, epiphyseal growth plate height, and levels of insulin-like growth factor 1 and leptin. In conclusion, the type of protein in the diet significantly affects the gut microbiota and, thereby, may affect animal's health.

The unique clinical spectrum of maturity onset diabetes of the young type 3.

Phenotypic variability in maturity-onset diabetes of the young (MODY) makes screening criteria for genomic analysis challenging. We describe the clinical spectrum in a large pedigree with HNF1A-MODY; as generations progressed, the course and outcome became poorer. Although uncommon, pancreatic autoantibodies and diabetes ketoacidosis should not exclude the diagnosis of MODY.

Beta Palmitate Improves Bone Length and Quality during Catch-Up Growth in Young Rats.

Palmitic acid (PA) is the most abundant saturated fatty acid in human milk, where it is heavily concentrated in the sn-2-position (termed beta palmitate, BPA) and as such is conserved in all women, regardless of their diet or ethnicity, indicating its physiological and metabolic importance. We hypothesized that BPA improves the efficiency of nutrition-induced catch up growth as compared to sn-1,3 PA, which is present in vegetable oil. Pre-pubertal male rats were subjected to a 17 days food restriction followed by re-feeding for nine days with 1,3 PA or BPA-containing diets. We measured bone length, epiphyseal growth plate height (EGP, histology), bone quality (micro-CT and 3-point bending assay), and gene expression (Affymetrix). The BPA-containing diet improved most growth parameters: humeri length and EGP height were greater in the BPA-fed animals. Further analysis of the EGP revealed that the hypertrophic zone was significantly higher in the BPA group. In addition, Affymetrix analysis revealed that the diet affected the expression of several genes in the liver and EGP. Despite the very subtle difference between the diets and the short re-feeding period, we found a small but significant improvement in most growth parameters in the BPA-fed rats. This pre-clinical study may have important implications, especially for children with growth disorders and children with special nutritional needs.

The reliability, concurrent validity and association with salivary oxytocin of the self-report version of the Inventory of Callous-Unemotional Traits in adolescents with conduct disorder.

The present study evaluated the self-report version of the Inventory of Callous-Unemotional Traits (ICU-SR) in terms of reliability, concurrent validity, and correlation with salivary oxytocin levels, a potential biomarker of CU traits. 67 socially at-risk male adolescents (mean 16.2 years) completed the ICU-SR, ICU teacher-version (ICU-TR), Strengths and Difficulties Questionnaire, and their medical files were coded for previous antisocial acts using Brown-Goodwin Lifetime Aggression Scale. Salivary samples were assayed for oxytocin. The reliability of ICU-SR was lower (α = 0.71) than ICU-TR (α = 0.86). ICU-SR mean score was significantly lower than ICU-TR (M = 25.29, SD = 8.02; M = 33.14, SD = 9.47). ICU-TR but not ICU-SR, significantly correlated with history of antisocial acts (r = 0.40). Two-way analysis of variance showed a significant effect of conduct disorder and oxytocin on ICU-TR but not ICU-SR [F(1,59) = 6.53; F(1,59) = 6.08], and a significant interaction only for ICU-TR [F(1,59) = 2.89]. Subjective self-reports of CU traits may be less reliable and valid than teachers' reports.

Which dietary components modulate longitudinal growth?

PURPOSE OF REVIEW: Linear growth in children is sensitive to nutritional status; the growth of the human skeleton requires many different nutritional factors for energy and building blocks: proteins, lipids, carbohydrates and micronutrients. However, what are the specific nutritional factors that are required for proper growth and what is the composition that will be most beneficial is still not known. RECENT FINDINGS: Recent findings indicate that macro and micronutrients are required as building blocks and as cofactors for important enzymes. In addition, they stimulate linear growth by acting as regulatory factors and also affect gut microbiome. Some interesting studies regarding the effect of proteins and amino acids are presented. SUMMARY: Most studies investigated the effect of replacing a single micronutrient that was deficient; however, in real life, deficiency of one nutritional element is commonly associated with other deficiencies. Therefore, it is a reasonable clinical approach, both in developing and developed countries, to use a mixture of both macro and micronutrients to support growth. How much of each of the components and what is the best composition are still open questions that require more research.

Effect of Nutrition on Statural Growth
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In children, proper growth and development are often regarded as a surrogate marker for good health. A complex system controls the initiation, rate, and cessation of growth, and thus gives a wonderful example of the interactions between genetics, epigenetics, and environmental factors (especially stress and nutrition). Malnutrition is considered a leading cause of growth attenuation in children. This review summarizes our current knowledge regarding the mechanisms linking nutrition and skeletal growth, including systemic factors, such as insulin, growth hormone, insulin-like growth factor-1, fibroblast growth factor-21, etc., and local mechanisms, including mTOR, miRNAs, and epigenetics. Studying the molecular mechanisms regulating skeletal growth may lead to the establishment of better nutritional and therapeutic regimens for more effective linear growth in children with malnutrition and growth abnormalities. 
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How Milk and Its Proteins Affect Growth, Bone Health, and Weight
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Milk has long been recognized to constitute a complete, well-balanced source of the nutrients and energy required to ensure the proper postnatal growth and development of infants. A growing body of evidence suggests the positive effects of dairy products and particularly of milk proteins on linear growth also in older children, both healthy or during recovery from malnutrition. This evidence led the way to the performance of extensive research aimed to delineate the components of milk and the mechanisms acting to make milk so effective. The present review summarizes the current knowledge regarding the influence of milk and its proteins on linear growth in healthy and malnourished children, focusing also on other important aspects of healthy growth, including bone health, weight status, and body composition.
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Quantitative proteomics of rat livers shows that unrestricted feeding is stressful for proteostasis with implications on life span.

Studies in young mammals on the molecular effects of food restriction leading to prolong adult life are scares. Here, we used high-throughput quantitative proteomic analysis of whole rat livers to address the molecular basis for growth arrest and the apparent life-prolonging phenotype of the food restriction regimen. Over 1800 common proteins were significantly quantified in livers of ad libitum, restriction- and re-fed rats, which summed up into 92% of the total protein mass of the cells. Compared to restriction, ad libitum cells contained significantly less mitochondrial catabolic enzymes and more cytosolic and ER HSP90 and HSP70 chaperones, which are hallmarks of heat- and chemically-stressed tissues. Following re-feeding, levels of HSPs nearly reached ad libitum levels. The quantitative and qualitative protein values indicated that the restriction regimen was a least stressful condition that used minimal amounts of HSP-chaperones to maintain optimal protein homeostasis and sustain optimal life span. In contrast, the elevated levels of HSP-chaperones in ad libitum tissues were characteristic of a chronic stress, which in the long term could lead to early aging and shorter life span.

Skeletal effect of casein and whey protein intake during catch-up growth in young male Sprague-Dawley rats.

The aim of the present study was to determine whether the type of protein ingested influences the efficiency of catch-up (CU) growth and bone quality in fast-growing male rats. Young male Sprague-Dawley rats were either fed ad libitum (controls) or subjected to 36 d of 40 % food restriction followed by 24 or 40 d of re-feeding with either standard rat chow or iso-energetic, iso-protein diets containing milk proteins - casein or whey. In terms of body weight, CU growth was incomplete in all study groups. Despite their similar food consumption, casein-re-fed rats had a significantly higher body weight and longer humerus than whey-re-fed rats in the long term. The height of the epiphyseal growth plate (EGP) in both casein and whey groups was greater than that of rats re-fed normal chow. Microcomputed tomography yielded significant differences in bone microstructure between the casein and whey groups, with the casein-re-fed animals having greater cortical thickness in both the short and long term in addition to a higher trabecular bone fraction in the short term, although this difference disappeared in the long term. Mechanical testing confirmed the greater bone strength in rats re-fed casein. Bone quality during CU growth significantly depends on the type of protein ingested. The higher EGP in the casein- and whey-re-fed rats suggests a better growth potential with milk-based diets. These results suggest that whey may lead to slower bone growth with reduced weight gain and, as such, may serve to circumvent long-term complications of CU growth.

Growth attenuation is associated with histone deacetylase 10-induced autophagy in the liver.

Our previous data suggested that the histone deacetylase (HDAC) SIRT1 is involved in mediating the effect of nutrition on growth. The aim of the present research was to study the mechanism by which additional HDACs may be involved in nutrition-induced linear growth. The in vivo studies were performed in young male Sprague-Dawley rats that were either fed ad libitum (AL) or subjected to 10days of 40% food restriction (RES) and then refed (CU). For in vitro studies, Huh7 hepatoma cells were used. Food restriction led to significant reduction in liver weight, concomitant with increased autophagy (i.e., a decrease in the level of P62 and an increase in the expression level of Ambra1 and Atg16L2 genes in the RES group). At the same time, we found that the level of HDAC10 was significantly increased. Overexpression of HDAC10 in Huh7 hepatoma cells led to reduced cell viability and increased autophagy as shown by increased conversion of LC3-I to LC3-II. An increase in the level of HDAC10 was also obtained when mTOR was inhibited by Rapamycin. siRNA directed against HDAC10 abolished the effect of Rapamycin on cell viability and Ambra1 and Atg16L2 increased expression. These results suggest that increased levels of HDAC10 may mediate the effect of malnutrition on growth attenuation and autophagy. Deciphering the role of epigenetic regulation in the nutrition-growth connection may pave the way for the development of new forms of treatment for children with growth disorders.

Salivary oxytocin in adolescents with conduct problems and callous-unemotional traits.

Callous-unemotional (CU) traits correlate with the severity and prognosis of conduct disorder in youth. The neuropeptide oxytocin (OT) has been linked to prosocial behaviors, including empathy and collaboration with others. This study discusses a possible role for OT in the biology of delinquent behavior. We hypothesized that in delinquent youth OT secretion will correlate with the severity of conduct problems and specifically with the level of CU traits. The study group included 67 male adolescents (mean age 16.2 years) undergoing residential treatment, previously assessed by an open clinical interview and history for the psychiatric diagnosis. Staff based Inventory of Callous-Unemotional traits for psychopathy and Strength and Difficulties Questionnaire were administered, and patients' medical and social personal files were systematically coded for previous history of antisocial acts using the Brown-Goodwin Questionnaire. Salivary OT was assayed by ELISA. Salivary OT levels were inversely correlated with conduct problems severity on Strength and Difficulties Questionnaire (r = -0.27; p ≤ 0.01). Recorded history of antisocial acts did not correlate with current OT levels. Odds ratio (OR) for significant CU traits among subjects with conduct problems was increased in low-OT (OR = 14, p ≤ 0.05) but not in high-OT subjects (OR = 6, p ≥ 0.05). Children with conduct problems and low levels of salivary OT are at risk for significant CU traits. These results suggest a possible role for salivary OT as a biomarker for CU traits and conduct problems severity.