Haroon I. Sheikh

G-CSF-mediated inhibition of JNK is a key mechanism for Lactobacillus rhamnosus-induced suppression of TNF production in macrophages.

Lactobacillus rhamnosus is a human commensal with known immunomodulatory properties. To date the mechanism of these immunomodulatory effects is not well understood. To unravel the immunomodulatory signalling mechanism, we investigated the effects of two strains of L. rhamnosus, L. rhamnosus GG and GR‐1, in modulating production of tumour necrosis factor‐α (TNF) in human monocytic cell line THP‐1 and mouse macrophages. Live L. rhamnosus GG and GR‐1 or their spent culture supernatant induced minuscule amounts of TNF production but large quantities of granulocyte‐colony stimulating factor (G‐CSF) in macrophages compared with those induced by pathogenic Escherichia coli GR‐12 and Enterococcus faecalis. By using neutralizing antibodies and G‐CSF receptor knockout mice, we demonstrated that G‐CSF secreted from L. rhamnosus GG‐ and GR‐1‐exposed macrophages suppressed TNF production induced by E. coli‐ or lipopolysaccharide‐activated macrophages through a paracrine route. The suppression of TNF production by G‐CSF was mediated through activation of STAT3 and subsequent inhibition of c‐Jun‐N‐terminal kinases (JNKs). The inhibition of JNK activation required STAT3α‐mediated de novo protein synthesis. This demonstrates a novel role of G‐CSF in L. rhamnosus‐triggered anti‐inflammatory effects and its mechanism in the suppression of TNF production in macrophages.

The dopamine D2 receptor gene and depressive and anxious symptoms in childhood: associations and evidence for gene–environment correlation and gene–environment interaction

Objectives Research implicates the A1 allele of the dopamine D2 receptor gene (DRD2) Taq1A polymorphism in the development of depression and anxiety. Furthermore, recent papers suggest that children with A1 allele of this gene may receive less positive parenting, and that the effects of this gene on child symptoms may be moderated by parenting. We sought to replicate and extend these findings using behavioral measures in a nonclinical sample of young children. Methods In a sample of 473 preschool-aged children and their mothers, structured clinical interview measures and maternal reports of child symptoms were collected, and standardized observations of parent–child interactions were conducted. Results An association was detected between the DRD2 A1 allele and symptoms of depression and anxiety indexed using interview and parent report methods. As found in previous reports, children with the DRD2 A1 allele received less supportive parenting and displayed higher levels of negative emotionality during parent–child interactions. Tests of mediation and moderation were conducted. Conclusion We found associations between the DRD2 A1 allele and early-emerging anxious and depressive symptoms in a community sample of preschool-aged children, and evidence of a gene–environment correlation and moderation of the main effect of child genotype on child symptoms by parenting.

Assessing stress reactivity indexed via salivary cortisol in preschool-aged children

Identifying a stressor paradigm that elicits mean increases in salivary cortisol in young children has proven elusive, possibly due to characteristics of the paradigms used and how and when cortisol is sampled. We therefore examined the validity of a standardized task (adapted from Lewis and Ramsay, 2002) and procedures developed to assess cortisol reactivity in 215 preschool-aged children. Children participated in a standardized stress task during a home visit, which was videorecorded for future coding. Salivary cortisol samples were obtained at baseline and 10, 20, 30, 40, and 50 min post-stress. In support of the validity of the task, significant increases in cortisol levels from baseline were found, followed by a significant decline, and a quadratic function provided a good fit to the data. Children also showed a significant increase in negative emotions and a decrease in positive emotions over the course of the stress task. Results indicate that the task successfully elicited the hypothesized cortisol response in 3-year-old children.

The Role of Brain-Derived Neurotrophic Factor Genotype, Parental Depression, and Relationship Discord in Predicting Early-Emerging Negative Emotionality

The brain-derived neurotrophic factor (BDNF) gene is a plausible candidate for early-emerging negative emotionality (NE), and evidence suggests that the effects of this gene may be especially salient in the context of familial risk for child maladjustment. We therefore examined whether the single-nucleotide polymorphism producing a valine-to-methionine substitution at codon 66 (val66met) of the BDNF gene was associated with childhood NE, in the context of parental depression and relationship discord. A sample of 413 three-year-old children was assessed for NE using standardized laboratory measures. The children’s parents completed clinical interviews as well as a measure of marital satisfaction. Children with at least one BDNF methionine (met) allele exhibited elevated NE when a parent had a history of depressive disorder or when relationship discord was reported by a parent. In contrast, this allele was associated with especially low NE when parental depression was absent and when the parental relationship was not discordant. Our findings suggest that the BDNF met allele confers increased child sensitivity to both positive and negative familial influences.

Corticotropin-releasing hormone system polymorphisms are associated with children's cortisol reactivity.

The hypothalamic-pituitary-adrenal (HPA) axis underlies both adaptive and maladaptive responses to stress and may be an important marker of childhood vulnerability to psychopathology, although little is known about genetic variants that influence cortisol reactivity. We therefore examined associations between corticotrophin-releasing hormone (CRH) system gene (CRH, CRHR1 and CRHBP) variants and cortisol reactivity in preschoolers. A community sample of 409 three-year-old children completed a standardized stress task to elicit HPA axis activation. Salivary samples were obtained at the baseline and at 10-min intervals post-stress for a total of six samples. Salivary cortisol was measured using standard ELISA (enzyme-linked immunosorbent assay) protocols and cortisol reactivity was operationalized by calculating cortisol change scores ([baseline]-[peak cortisol post-stressor]). A single nucleotide polymorphism (SNP) marker panel containing 18 SNPs was used to tag the full-length CRH (4 SNPs), CRHR1 (7 SNPs) and CRHBP (7 SNPs) genes. Significant main effects on childrens cortisol reactivity (all ps<0.05) were found for loci on CRHR1 and CRHBP. Haplotypes of the CRHR1 linkage region were also associated with cortisol reactivity (all ps<0.01). Additionally, we found multiple interactions between tag-SNPs in all three gene-coding regions predicting cortisol reactivity (all ps<0.05). Individual differences in childrens cortisol reactivity are related to genetic variation in CRH system gene-coding regions. Our results have important implications for future research on the role of HPA axis function in the development of disorders such as anxiety and depression.

Child dopamine active transporter 1 genotype and parenting: evidence for evocative gene-environment correlations.

The dopamine active transporter 1 (DAT1) gene is implicated in psychopathology risk. Although the processes by which this gene exerts its effects on risk are poorly understood, a small body of research suggests that the DAT1 gene influences early emerging negative emotionality, a marker of childrens psychopathology risk. As child negative emotionality evokes negative parenting practices, the DAT1 gene may also play a role in gene-environment correlations. To test this model, children (N = 365) were genotyped for the DAT1 gene and participated in standardized parent-child interaction tasks with their primary caregiver. The DAT1 gene 9-repeat variant was associated with child negative affect expressed toward the parent during parent-child interactions, and parents of children with a 9-repeat allele exhibited more hostility and lower guidance/engagement than parents of children without a 9-repeat allele. These gene-environment associations were partially mediated by child negative affect toward the parent. The findings implicate a specific polymorphism in eliciting negative parenting, suggesting that evocative associations play a role in elevating childrens risk for emotional trajectories toward psychopathology risk.

Genotyping the BDNF rs6265 (val66met) polymorphism by one- step amplified refractory mutation system PCR

Background The brain derived neutrophic factor (BDNF), a 27 kD polypeptide, is one of the most widely expressed neurotrophins in the brain, regulating neural development and plasticity. The BDNF gene contains a functional single-nucleotide polymorphism (rs6265), which results in a valine to methionine substitution (val66met), leading to reduced mature BDNF expression. This polymorphism has been widely implicated in a host of psychiatric disorders and is a focus of many ongoing psychiatric genetic studies. Objective To develop an efficient and rapid method to detect the val66met polymorphism in a one-step PCR reaction. Method and results We have designed four PCR primers that amplify the BDNF gene region containing rs6265. The specificity of the four primers in a single PCR reaction amplifies two allele-specific amplicons (253 and 201 bp) and the entire region (401 bp) as an internal control, which are easily distinguished on a polyacrylamide gel. The effectiveness and efficiency of the results are validated by traditional NlaIII restriction enzyme digestion, sequencing of resulting bands and confirmation on 308 genomic DNA samples. Conclusion This new method describes a rapid, sensitive, cost effective and high throughput genotyping of the BDNF val66met polymorphism, ideal for large-scale genotyping studies.

Hair cortisol concentrations in higher- and lower-stress mother–daughter dyads: A pilot study of associations and moderators

Hair cortisol concentrations (HCC) are receiving increased attention as a novel biomarker of psychophysiological responses to chronic stress, with potential relevance for psychopathology risk research. We examined the validity of HCC as a marker of psychosocial stress in mother (M(age)  = 37.87 years)-daughter (M(age)  = 7.62 years) dyads characterized by higher (n = 30) or lower (n = 30) maternal chronic stress. Additionally, we examined whether early care moderated similarity of HCC levels within dyads. Higher-stress mothers had significantly lower HCC compared to lower-stress mothers, consistent with other research showing that chronic stress leads to blunted HPA axis activity over time. Further, HCC in daughters were significantly and positively associated with previously assessed salivary cortisol stress reactivity. Finally, mother-daughter HCC associations were significantly moderated by negative parenting styles, such that associations became stronger as quality of parenting decreased. Findings overall indicate that HCC may be a useful marker of cortisol responses to chronic stress.

Parenting and Child DRD4 Genotype Interact to Predict Children’s Early Emerging Effortful Control

Effortful control (EC), or the trait-like capacity to regulate dominant responses, has important implications for childrens development. Although genetic factors and parenting likely influence EC, few studies have examined whether they interact to predict its development. This study examined whether the DRD4 exon III variable number tandem repeat polymorphism moderated the relation between parenting and childrens EC. Three hundred and eighty-two 3-year-olds and primary caregivers completed behavioral tasks assessing childrens EC and parenting. Childrens DRD4 genotypes moderated the relation between parenting and EC: Children with at least one 7-repeat allele displayed lower EC in the context of negative parenting than children without this allele. These findings suggest opportunities for modifying early risk for low EC.

The Serotonin Transporter Promoter Polymorphism and Childhood Positive and Negative Emotionality

Association studies of the serotonin transporter promoter polymorphism (5-HTTLPR) and negative emotionality (NE) are inconclusive. However, emerging evidence suggests that the association between this polymorphism and NE may be influenced by levels of another temperament trait, positive emotionality (PE). Therefore, this study examined whether the association between the 5-HTTLPR and NE was moderated by PE. A community sample of 413 three-year-old children completed a standardized battery of laboratory tasks designed to tap temperamental emotionality. Children were also genotyped for the 5-HTTLPR. No direct association between 5-HTTLPR genotype and NE was found. However, the interaction of child PE and NE predicted 5-HTTLPR genotype. Furthermore, children with a short allele who were also low in PE had significantly greater NE than children without a short allele or children with high PE. Our findings suggest that the short allele of the 5-HTTLPR is associated with NE only in the context of low PE. Inconsistent links between NE and this gene in previous research may stem from the failure to consider other temperament traits that moderate associations.