کورتیکوتروپین (ACTH) - ایمونوگلوبولینهای راکتیو در نوجوانان در رابطه با رفتار ضد اجتماعی و پاسخ سطح کورتیزول ناشی از استرس. مطالعه مسیرهای پیاده روی

. Introduction Antisocial behavior often develops during childhood and adolescence and can manifest itself in delinquency. Research into neurobiological mechanisms involved in antisocial behavior has focused on the hypothalamic–pituitary–adrenal (HPA) axis, an important regulator of the physiological stress response. Its dysgregulation has been linked to externalizing problems (van Goozen et al., 2007, Alink et al., 2008 and Ruttle et al., 2011). While some studies have reported associations of aggression and antisocial behavior with low cortisol levels (McBurnett et al., 2000, Shoal et al., 2003, Poustka et al., 2010 and Haltigan et al., 2011), others have found no association (Klimes-Dougan et al., 2001), or higher cortisol levels in adolescents with externalizing behavior problems (McBurnett et al., 2005 and van Bokhoven et al., 2005). The inconsistent findings and weak associations between cortisol and antisocial behavior increase the need for investigating other factors of HPA axis functioning that could influence antisocial behavior. Corticotropin (ACTH)-reactive immunoglobulins (IgG) are one such factor to explore. High levels of ACTH IgG were found in prisoners and males with conduct disorder (Fetissov et al., 2006), suggesting their association with antisocial behavior, maybe through interfering with the HPA axis and altering stress-related behavior. It is plausible that ACTH IgG influence cortisol release upon stress by modulating ACTH action. This in turn could result in stress-induced behavior changes like impulsivity and aggression (Haller and Kruk, 2006). A similar model has previously been proposed for IgG against α-MSH, an ACTH-derived peptide (Sinno et al., 2009). According to this model, neuropeptide-reactive IgG may play a role in peptide transportation, protecting peptides from degradation by peptidases. An increase in affinity of such IgG would result in neutralization, decreasing peptide transportation and biological activity (Sinno et al., 2009). Both unbound (‘free’) IgG and IgG in immune complexes circulate in the blood. To be able to distinguish between the roles of free and total IgG, it may be informative to look at both pools separately (Gustaw et al., 2008 and Deloumeau et al., 2010). The previous study of ACTH IgG and antisocial behavior included males with severe conduct problems (n = 20), prisoners (n = 20), and healthy volunteers (n = 22) ( Fetissov et al., 2006). It was the first study to demonstrate elevated ACTH IgG levels in aggressive and violent subjects compared to healthy controls. In this study, antisocial behavior of the controls was not measured and likely the prisoners were at the extreme end of the antisocial behavior spectrum. The study could not adjust for potential covariates (e.g. age, socioeconomic status). Our study is the first to investigate associations between ACTH IgG and both antisocial behavior and HPA axis response in a large sample of adolescents from the general population, making it possible to adjust for covariates. Most studies on antisocial behavior and low cortisol levels have focused on males ( McBurnett et al., 2000, Shoal et al., 2003, Popma et al., 2006 and Poustka et al., 2010), but a similar pattern has been reported in girls ( Pajer et al., 2001). Both HPA axis and the immune system are differentially regulated in males and females ( Gaillard and Spinedi, 1998). Knowing this, we were interested in exploring sex differences in our study. Thus, the aim of this study was to validate the finding that ACTH IgG levels are associated with antisocial behavior in a large sample of male adolescents, and to investigate if such associations could also be found in females. Furthermore, we hypothesized that ACTH IgG are associated with HPA axis activity. We tested this by linking serum ACTH IgG levels to cortisol responses during a social stress test. We further characterized ACTH IgG properties by studying their affinity kinetics in adolescents with high or low ACTH IgG levels.

Introduction Antisocial behavior often develops during childhood and adolescence and can manifest itself in delinquency. Research into neurobiological mechanisms involved in antisocial behavior has focused on the hypothalamic–pituitary–adrenal (HPA) axis, an important regulator of the physiological stress response. Its dysgregulation has been linked to externalizing problems (van Goozen et al., 2007, Alink et al., 2008 and Ruttle et al., 2011). While some studies have reported associations of aggression and antisocial behavior with low cortisol levels (McBurnett et al., 2000, Shoal et al., 2003, Poustka et al., 2010 and Haltigan et al., 2011), others have found no association (Klimes-Dougan et al., 2001), or higher cortisol levels in adolescents with externalizing behavior problems (McBurnett et al., 2005 and van Bokhoven et al., 2005). The inconsistent findings and weak associations between cortisol and antisocial behavior increase the need for investigating other factors of HPA axis functioning that could influence antisocial behavior. Corticotropin (ACTH)-reactive immunoglobulins (IgG) are one such factor to explore. High levels of ACTH IgG were found in prisoners and males with conduct disorder (Fetissov et al., 2006), suggesting their association with antisocial behavior, maybe through interfering with the HPA axis and altering stress-related behavior. It is plausible that ACTH IgG influence cortisol release upon stress by modulating ACTH action. This in turn could result in stress-induced behavior changes like impulsivity and aggression (Haller and Kruk, 2006). A similar model has previously been proposed for IgG against α-MSH, an ACTH-derived peptide (Sinno et al., 2009). According to this model, neuropeptide-reactive IgG may play a role in peptide transportation, protecting peptides from degradation by peptidases. An increase in affinity of such IgG would result in neutralization, decreasing peptide transportation and biological activity (Sinno et al., 2009). Both unbound (‘free’) IgG and IgG in immune complexes circulate in the blood. To be able to distinguish between the roles of free and total IgG, it may be informative to look at both pools separately (Gustaw et al., 2008 and Deloumeau et al., 2010). The previous study of ACTH IgG and antisocial behavior included males with severe conduct problems (n = 20), prisoners (n = 20), and healthy volunteers (n = 22) ( Fetissov et al., 2006). It was the first study to demonstrate elevated ACTH IgG levels in aggressive and violent subjects compared to healthy controls. In this study, antisocial behavior of the controls was not measured and likely the prisoners were at the extreme end of the antisocial behavior spectrum. The study could not adjust for potential covariates (e.g. age, socioeconomic status). Our study is the first to investigate associations between ACTH IgG and both antisocial behavior and HPA axis response in a large sample of adolescents from the general population, making it possible to adjust for covariates. Most studies on antisocial behavior and low cortisol levels have focused on males ( McBurnett et al., 2000, Shoal et al., 2003, Popma et al., 2006 and Poustka et al., 2010), but a similar pattern has been reported in girls ( Pajer et al., 2001). Both HPA axis and the immune system are differentially regulated in males and females ( Gaillard and Spinedi, 1998). Knowing this, we were interested in exploring sex differences in our study. Thus, the aim of this study was to validate the finding that ACTH IgG levels are associated with antisocial behavior in a large sample of male adolescents, and to investigate if such associations could also be found in females. Furthermore, we hypothesized that ACTH IgG are associated with HPA axis activity. We tested this by linking serum ACTH IgG levels to cortisol responses during a social stress test. We further characterized ACTH IgG properties by studying their affinity kinetics in adolescents with high or low ACTH IgG levels.

. Results 3.1. Descriptives Girls had higher mean levels of free and total ACTH IgG than boys (Table 1). ACTH IgG levels did not differ between GSST participants and non-participants, they also did not differ between OC-users and non-OC-users. The cortisol response to the GSST was blunted in OC-users, as shown earlier (Bouma et al., 2009). Free and total ACTH IgG levels correlated positively with each other (girls ρ = 0.34; boys ρ = 0.42) and with age (girls ρ = 0.17 (total); boys ρ = 0.09 (free) −0.22 (total)), with PD (ρ = 0.08 (free) −0.12 (total)) in boys and with SES (ρ = −0.16 (total) and irregular menstruation (ρ = 0.16, free) in girls (supplementary Table 1). Table 1. Descriptives. Boys Girls N Mean SD N Mean SD Age (years) 573 16.2 0.64 657 16.2 0.68 Physical development 554 0.01 0.97 635 0.00 0.99 Socioeconomic status 566 0.11 0.79 651 0.10 0.76 Antisocial behavior (ASBQ) 554 0.29** 0.33 641 0.16 0.22 Smoking (≥1 cigarette/week) 158 (28.5%) 204 (31.8%) ACTH immunoglobulins (OD) Free IgG 573 0.59* 0.44 657 0.67b 0.50 Total IgG 573 2.14** 0.42 657 2.24b 0.39 HPA axis activity (nmol/l) AUCincr 270 82.1* 105.5 192 (no OC) 94 (OC) 70.4a 19.0 115.1 41.4 Recovery 277 1.8* 2.1 294 1.2 b 2.3 Abbreviations: ASBQ, Antisocial Behavior Questionnaire; ACTH, corticotropin; OD, optical density; HPA axis, hypothalamic–pituitary–adrenal axis; AUCincr, area under the curve during social stress test with respect to the starting value; OC, oral contraceptives; SD, standard deviation. a p < 0.001 difference between girls taking OC and girls not taking OC. b No difference between girls taking OC and girls not taking OC. * p < 0.01 difference between boys and girls. ** p < 0.001 difference between boys and girls. Table options 3.2. ACTH IgG and antisocial behavior In the model for antisocial behavior, the interaction between sex and free ACTH IgG was significant (β = 0.17; p = 0.02), therefore we stratified by gender. In boys, high total ACTH IgG levels were associated with a higher score for antisocial behavior, and this association was stronger at higher levels of free ACTH IgG ( Table 2). The model improved significantly (−2 loglikelihood difference (LLH) = 5.1; 1 df; p = 0.02) when the interaction between free and total ACTH IgG was included. In girls, high levels of free ACTH IgG were associated with a lower antisocial behavior score. The model did not improve after adding the interaction between free and total ACTH IgG (−2 LLH difference = 0.3; 1 df; p = 0.60). The level of total ACTH IgG was not related to antisocial behavior in girls. To explore relations with varying antisocial behaviors we carried out logistic regression analyses with groups of items of the ASBQ. The strongest relationships were with destruction of property, involvement in physical fights and theft (supplementary Table 2). Table 2. Associations between ACTH immunoglobulin (IgG) and antisocial behavior. Boys and girls Boys Girls β 95% CI p β 95% CI p β 95% CI p Free ACTH IgG −0.02 −0.19; 0.09 0.50 −0.64 −1.36; 0.08 0.08 −0.20 −0.39; −0.01 0.04 Total ACTH IgG 0.04 −0.18; 0.68 0.26 1.05 0.03; 2.07 0.04 0.33 −0.29; 0.94 0.30 Free × total ACTH IgG 1.03 0.13; 1.93 0.03 Covariates in the model: age, socioeconomic status. Abbreviations: ACTH, corticotropin; CI, confidence interval. Bold values: p<0.05. Table options 3.3. ACTH IgG and stress-induced cortisol response In the model for AUCincr, the interaction between sex and free ACTH IgG was significant (β = −0.94; p < 0.01), and in the model for recovery, the interaction between sex and total ACTH IgG was significant (β = −1.62; p < 0.01), hence we stratified by gender. Levels of free ACTH IgG were negatively associated with AUCincr in boys (β = −0.67; p < 0.01, Table 3). Total ACTH IgG level was associated with higher recovery of the cortisol response in girls (β = 0.84; p = 0.05, Table 3). The associations between ACTH IgG and cortisol responses did not change after adding menstrual cycle stage as covariate. Unadjusted cortisol responses in boys and girls with low (first quartile) or high (third quartile) free and total ACTH IgG levels are depicted in Fig. 1. There was a trend toward a negative association between antisocial behavior and AUCincr (β = −0.13, 95% CI: −0.73; 0.01, p = 0.08) in the total sample. The interaction between sex and antisocial behavior was not significant. The association with Recovery was not significant (β = −0.01, 95% CI: −0.09; 0.07, p = 0.81). Table 3. Associations between ACTH immunoglobulin (IgG) and HPA axis activity during social stress. AUCincr Recovery β 95% CI p β 95% CI p Boys and girls Total ACTH IgG −0.09 −1.12; 0.94 0.86 −0.06 −0.63; 0.51 0.84 Free ACTH IgG −0.04 −0.45; 0.17 0.37 0.02 −0.15; 0.18 0.86 Boys Total ACTH IgG 0.07 −1.27; 1.40 0.88 −0.65 −1.42; 0.13 0.10 Free ACTH IgG −0.67 −1.10; −0.24 <0.01 −0.04 −0.30; 0.21 0.78 Girls Total ACTH IgG −0.38 −1.01; 1.15 0.63 0.85 −0.00; 1.70 0.05 Free ACTH IgG 0.31 −0.11; 0.74 0.15 0.05 −0.17; 0.28 0.63 Covariates in the model: age, socioeconomic status, physical development, smoking, OC use (girls). Abbreviations: ACTH, corticotropin; AUCincr, area under the curve during social stress test with respect to the starting value; Recovery, recovery after social stress test; CI, confidence interval; OC, oral contraceptives. Bold values: p<0.05. Table options Cortisol levels during the social stress test in boys and girls with high and ... Figure 1. Cortisol levels during the social stress test in boys and girls with high and low free or total ACTH immunoglobulin (IgG) levels. Subjects with low ACTH IgG levels were defined as those in the first quartile, high ACTH IgG levels as those in the third quartile. Values are shown with standard. Abbreviation: ACTH, corticotropin Figure options 3.4. Affinity kinetics There were no significant differences in age, PD and SES between boys or girls selected for high and low total ACTH IgG levels. ACTH-reactive IgM and IgA did not differ significantly between groups (Fig. 2b and c). Total IgG and IgM concentrations were higher in girls with high ACTH IgG levels (Fig. 2d and e). Total IgA and ACTH peptide concentration did not differ between groups (Fig. 2f and h). As expected based on the positive association with total ACTH IgG in boys but not in girls, in the selected samples antisocial behavior scores were higher in boys with high total ACTH IgG levels (Fig. 2g). Free ACTH IgG levels were higher in girls with high total ACTH IgG levels (Fig. 2i). The association rate constant (Ka) was lower in boys and girls with high compared to low total ACTH IgG (Fig. 2j). Dissociation rate constant (Kd) and affinity constant at equilibrium (KD) did not differ between groups (Fig. 2k and l). The model fit for affinity kinetics after applying the Langmuir (1:1) binding model had an average Chi2 of 317 (SD = 162). ACTH-reactive immunoglobulins (Ig), serum total Ig concentrations and affinity ... Figure 2. ACTH-reactive immunoglobulins (Ig), serum total Ig concentrations and affinity measures in selected samples with high or low total ACTH IgG. Comparison between boys and girls with high or low ACTH IgG levels: (a) total ACTH IgG levels, these were used for selection; (b) ACTH-reactive IgM levels; (c) ACTH-reactive IgA levels; (d) total IgG levels; (e) total IgM levels; (f) total IgA levels; (g) antisocial behavior scores; (h) ACTH peptide levels; (i) free ACTH IgG levels; (j) Ka of ACTH-reactive IgG; (k) Kd of ACTH-reactive IgG; and (l) KD of ACTH-reactive IgG. *p < 0.05, **p < 0.01. Abbreviations: ACTH, corticotropin; OD, optical density; Ka, association rate constant; Kd, dissociation rate constant; KD, dissociation constant at equilibrium.