A modified and cost-effective method for hair cortisol analysis.

Hair cortisol may hold potential as a biomarker for assessment of chronic psychological stress. We report a modified and cost-effective method to prepare hair samples for cortisol assay. Hair samples were ground using an inexpensive ball grinder - ULTRA-TURRAX tube drive. Cortisol was extracted from the powder under various defined conditions. The data showed that the optimal conditions for this method include cortisol extraction at room temperature and evaporation using a stream of room air. These findings should allow more widespread research using economical technology to validate the utility of hair cortisol as a biomarker for assessing chronic stress status.

Hair Cortisol Concentrations Are Associated with Hair Growth Rate.

There is a growing interest in hair cortisol concentrations as a valuable biomarker for the assessment of metabolic diseases and chronic psychological stress. Fifty-three volunteers were recruited, and hair segments proximal to the scalp were collected from each individual. A cost-effective ball mill was used for the preparation of hair samples, and ELISA was performed to analyze cortisol concentrations. Results indicate that the frequency of hair washing affects the hair cortisol concentration. The group that washed their hair every day had significantly lower cortisol concentrations than the group that washed it less often. However, no significant differences were detected between cosmetic-treated and nontreated hair samples. The study also shows that hair cortisol concentrations in the first 3 cm of hair segments proximal to the scalp corresponded to average hair growth rate based on 1 cm/month. Thus, hair cortisol concentrations of segments 3 cm proximal to the scalp may represent cumulative stress exposure over the previous 3 months. These findings will allow more widespread research to validate the utility of hair cortisol as a potential biomarker to assess chronic stress.

Gene polymorphisms of stress hormone and cytokine receptors associate with immunomodulatory profile and psychological measurement.

OBJECTIVE: We sought to identify whether stable single nucleotide polymorphisms (SNPs) of various endocrine and immune molecules could be used as biomarkers associated with specific immune alterations and chronic stress measures in normal humans. METHODS: A total of 207 volunteer participants answered stress questionnaire and gave peripheral blood cells for identification of SNPs in genes coding for glucocorticoid receptor (GR), beta 2 adrenergic receptor (B2AR), interferon-gamma receptors (IFNGR1, IFNGR2), and interleukin-4 receptor (IL4R). Immunoregulatory profiles were measured by flow cytometry and genotyping assays were performed by allelic discrimination real-time PCR. RESULTS: Several significant differences were revealed in associations between stress marker and immune indicators based on SNP categories. For instance, Th1 levels of the minor alleles of GR TthIIII (AA) and IFNGR2 Q64R (Arg/Arg) groups were positively associated with chronic stress (PSS) (p = 0.024 and 0.005, respectively) compared with wild type (WT) and negatively associated with PSS in the heterozygous genotypes of GR BclI and IL4R Ile50Val (p = 0.040 and p = 0.052, respectively). Treg levels of the minor alleles of BclI (GG) and IFNGR1 T-56C (CC) groups were positively associated with PSS (p = 0.045 and p = 0.010, respectively) and negatively associated in the minor allele (Val/Val) of IL4R Ile50Va and the heterozygous genotype of IL4R Q576R (p = 0.041 and p = 0.017, respectively) compared to WT. CONCLUSION: The data support the notion that gene polymorphisms from various components of the psychoneuroendocrine-immune network may be useful as biomarkers to categorize individual stress-associated immune responses.

Effects of strenuous exercise on Th1/Th2 gene expression from human peripheral blood mononuclear cells of marathon participants.

Physical stressors, such as strenuous exercise, can have numerous effects on the human body including the immune system. The aim of this study was to evaluate the gene expression profile of Th1/Th2 cytokines and related transcription factor genes in order to investigate possible immune imbalances before and after a marathon. Blood samples were collected from 16 normal volunteers 24-48 h before and one week after completing a marathon race. Gene expression of Th1 and Th2 related cytokines from human peripheral blood mononuclear cells (PBMC) was analyzed using Human Th1-Th2-Th3 RT(2) Profiler PCR Array and qRT-PCR that measured the transcript levels of 84 genes related to T cell activation. We found that PBMC express a characteristic Th2-like gene profile one week post-marathon compared to pre-marathon. The majority of genes up-regulated one week post-marathon such as IL-4, GATA3, and CCR4 were Th2 associated. For Th1-related genes, CXCR3 and IRF1 were up-regulated one week post-marathon. There was a trend of down-regulation of two Th1 related genes, T-bet and STAT1. Th3-related gene expression patterns did not change in the study. The ratios of both IFN-γ/IL-4 and T-bet/GATA3 gene expressions were significantly lower one week after marathon. These findings suggest that a Th1/Th2 immune imbalance persisted at least 1 week after completion of a marathon which offers a mechanistic rationale for the increased risk of upper respiratory tract infections often reported after strenuous exercise.

Associations between cytokine receptor polymorphisms and variability in laboratory immune parameters in normal humans.

In every study involving human immune parameters, large inter-subject variability occurs which can make interpretation of results difficult. The aim of this study was to evaluate whether genetic variants in cytokine receptors could associate with variability in laboratory immune measures. A total of 207 normal volunteers were recruited in this study. Immunoregulatory profiles were measured by flow cytometry and genotyping assays were performed by allelic discrimination real-time PCR. Immunoregulatory profiles were categorized according to various single nucleotide polymorphisms (SNPs) of cytokine receptors including T-56C and G-611A of IFN-γ receptor 1 (IFNGR1); Q64R of IFNGR2; and Ile50Val, Q576R and S503P of IL4R. Results reveal that Th1 levels were significantly higher in the heterozygous of the IFNGR1 T-56C polymorphism (minor allele) compared to wild-type (WT, major allele) (p = 0.006). For the Q576R of IL4R, Th1/Th2 ratio was significantly lower for the homozygous SNP (Arg/Arg) compared to the WT (Gln/Gln) (p = 0.035). In addition, the significant interaction effects of demographic characteristics on SNP-immune parameter associations were reported as well. We conclude that cytokine receptor polymorphisms might associate with variability in laboratory immune measures. Approach of SNP analysis of cytokine receptors can be useful in categorizing baseline immune responses to more accurately evaluate clinical immune data.

Immunomodulatory effects of dexamethasone on gene expression of cytokine and stress hormone receptors in peripheral blood mononuclear cells.

Glucocorticoid (GC) such as cortisol in humans is a major stress hormone and can influence immunomodulation through various mechanisms including impact on regulatory T-cell (RTC) elements and changes in Th1/Th2 cytokine balance. In this study, we sought to determine the immunomodulatory effects of GC equivalent dexamethasone (DEX) at various concentrations (10(-7), 10(-8), and 10(-9) M) on gene expression of RTC, cytokine receptors and stress hormone receptors from normal human peripheral blood mononuclear cells (PBMC) in an in vitro stress model in 24 h (acute stress) vs 11-day (chronic stress) cultures. Results revealed that the mRNA of forkhead box P3 (FoxP3) was significantly decreased at 24 h with 10(-7) and 10(-8) M DEX. After 11 days, FoxP3 expression in the 10(-8) M DEX cultures had returned to baseline but was still down-regulated with 10(-7) M DEX. GC receptor (GR) mRNA decreased and ß2 adrenergic receptor (ß 2AR) mRNA increased significantly after 24 h exposure to DEX. These changes had returned to baseline in the 11-day cultures. The IFN-γR/IL-4R ratio, an alternative marker for Th1/Th2 balance, was significantly increased at 24 h and decreased after 11-day cultures with 10(-7) and 10(-8) M DEX compared to control cultures. These findings further contribute to our understanding of the mechanisms associated with the effects of acute vs. chronic stress on normal immune balance.

Prevalence and cardiometabolic associations of the glucocorticoid receptor gene polymorphisms N363S and BclI in obese and non-obese black and white Mississippians.

OBJECTIVE: Polymorphisms (SNP) in the glucocorticoid receptor (GR) gene can alter sensitivity to glucocorticoids. Previous studies of the N363S and BclI SNP in the GR gene have shown a metabolic syndrome phenotype in mostly non-African populations. The obesity phenotype of African Americans (AA) seems to be more severe than that of Caucasians. DESIGN: We aimed to assess the prevalence of N363S and BclI in obese and non-obese Caucasian (n=26) and African (n=23) Mississippians (age: 23-63 years) to investigate associations with body composition (body mass index/BMI, waist-to-hip ratio), metabolic parameters (salivary cortisol, fasting glucose and insulin, hemoglobin A1C, fructosamine, HOMA-IR index), and psychological stress perception (blood pressure/BP, perceived stress scale/PSS). RESULTS: All subjects were homozygous for wildtype N363N. BclI polymorphism genotype frequencies among the 23 AA were: homozygous CC (57%), GG (4%), and heterozygous CG (39%), and among the 26 white women: homozygous CC (35%), GG (19%), and heterozygous CG (46%). Linear and logistic regression analyses including a parsimonious model identified BMI as a statistically significant parameter between the two ethnic groups (BMI was 3.13 kg/m2 higher in AA). Within the AA group, BMI, waist-to-hip ratio, log (HOMA-IR), PSS scores, BP, and hyperlipidemia showed no statistically significant relationships for the BclI polymorphism. PSS scores were 15.2 for AA vs. 14.7 for white women (normal mean: 14.7 vs. 12.8). CONCLUSION: Black Mississippians have a higher BMI than whites, which may be related to the presence of the BclI polymorphism and increased glucocorticoid sensitivity. Although more blacks (52%) than whites (38%) had elevated BP, PSS scores in both groups suggest that a high BMI is not regarded as abnormal or stressful. This might negatively impact behavior change regarding lifestyle modifications with increased physical activity and healthier food choices. Larger studies, particularly in African populations, are needed to better define metabolic and psychological characteristics in relation to the N363S and BclI GR gene polymorphisms.

Variability in laboratory immune parameters is associated with stress hormone receptor polymorphisms.

OBJECTIVES: Interpretation of laboratory immune data in healthy human subjects is often challenging due to wide inter-subject variability. Since endocrine and immune mediators have been mutually interlinked, a potential explanation for the significant variability seen in immune data even when controlled for technical variability and demographics is differences in the binding affinity of ligand with hormone receptors on the surface of immune cells, which can be associated with single nucleotide polymorphisms (SNP). METHODS: We categorized immunoregulatory cellular profiles from PBMC of 207 healthy volunteers according to glucocorticoid receptor (GR: Bcl1, TthIIII, and A3669G) and ß2-adrenergic receptor (ß2AR: Gly16Arg and Gln27Glu) polymorphisms. Subjects were genotyped for each SNP, and Th1, Th2, Th1/Th2 ratio, regulatory T cell (T(reg)), Tr1, and Th3 cell numbers were assessed. Immune parameters in the SNP groups were compared to the wild type (WT). RESULTS: Significant differences were observed in Th2 and the Th1/Th2 ratio for the ß2AR SNP Gly16Arg. Th1, the Th1/Th2 ratio, and Tr1 differed significantly by SNP of Gln27Glu. In addition, the effect of age on Th2 and the effect of the body mass index on the Th1/Th2 ratio significantly differed across subtypes of the Gly16Arg SNP. Significant differences based on allergic status and gender were also seen for T(reg), Th1, and Th2 across Gly16Arg, Gln27Glu, and TthIIII SNP. CONCLUSIONS: These data suggest that SNP from various components of the stress-immune network may be useful for subgrouping of immune responses to more accurately categorize psychoneuroimmunological components of stress risk in individual subjects. This approach may have significant research and clinical potential.

Low gene expression of bone morphogenetic protein 7 in brainstem astrocytes in major depression.

The noradrenergic locus coeruleus (LC) is the principal source of brain norepinephrine, a neurotransmitter thought to play a major role in the pathology of major depressive disorder (MDD) and in the therapeutic action of many antidepressant drugs. The goal of this study was to identify potential mediators of brain noradrenergic dysfunction in MDD. Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-ß superfamily, is a critical mediator of noradrenergic neuron differentiation during development and has neurotrophic and neuroprotective effects on mature catecholaminergic neurons. Real-time PCR of reversed transcribed RNA isolated from homogenates of LC tissue from 12 matched pairs of MDD subjects and psychiatrically normal control subjects revealed low levels of BMP7 gene expression in MDD. No differences in gene expression levels of other members of the BMP family were observed in the LC, and BMP7 gene expression was normal in the prefrontal cortex and amygdala in MDD subjects. Laser capture microdissection of noradrenergic neurons, astrocytes, and oligodendrocytes from the LC revealed that BMP7 gene expression was highest in LC astrocytes relative to the other cell types, and that the MDD-associated reduction in BMP7 gene expression was limited to astrocytes. Rats exposed to chronic social defeat exhibited a similar reduction in BMP7 gene expression in the LC. BMP7 has unique developmental and trophic actions on catecholamine neurons and these findings suggest that reduced astrocyte support for pontine LC neurons may contribute to pathology of brain noradrenergic neurons in MDD.

Effects of acute stress-induced immunomodulation on TH1/TH2 cytokine and catecholamine receptor expression in human peripheral blood cells.

AIMS: There is evidence that psychological stress can modulate immune functions. It has been hypothesized that acute stressors can affect both immune balance (including Th1 and Th2 cytokines) and expression of stress hormone receptors. This study investigated the impact of an acute stressor on gene expressions of glucocorticoid receptor (GR), and ß2-adrenergic receptor (ß2AR) in leukocytes. The effect on T regulatory cells (Treg), regulatory cytokines IL-10 and TGF-ß, Th1 and Th2 cytokines and their receptors IFN-γR and IL-4R was also studied. METHOD: Fourteen normal volunteers completed an acute laboratory stressor, and blood samples were collected before, immediately after, and 1, 2, 6 and 24 h after completion of the tasks. Cytokine production and Treg were determined by flow cytometry. Gene expressions of receptors were analyzed by real-time PCR. RESULTS: IFN-γ was increased immediately and 1 h after stressor (p<0.05, respectively) and upregulation of IFN-γR mRNA was noted at 2, 6 and 24 h (p<0.01, respectively). IL-10 was decreased at 2 h (p<0.01). There were no significant changes in post-task IL-4R, Treg, or TGF-ß. ß2AR mRNA was increased at 2, 6 and 24 h (p<0.01, respectively). On the other hand, no significant alterations were observed in GR expression. CONCLUSION: An acute stressor increased Th1 cytokine production and its receptor expression. ß2AR but not GR was significantly increased after an acute stressor, which supports the hypothesis that catecholamine-mediated signal pathways in communication with the central nervous and immune systems play a fundamental role in acute stress-mediated immune alterations.

Stress and allergic diseases.

Allergy describes a constellation of clinical diseases that affect up to 30% of the world's population. It is characterized by production of allergen-specific IgE, which binds to mast cells and initiates a cascade of molecular and cellular events that affect the respiratory tract (rhinitis and asthma), skin (dermatitis, urticaria), and multiple systems (anaphylaxis) in response to a variety of allergens including pollens, mold spores, animal danders, insect stings, foods, and drugs. The underlying pathophysiology involves immunoregulatory dysfunctions similar to those noted in highly stressed populations. The relationships in terms of potential for intervention are discussed.

Immunomodulatory effects of in vitro stress hormones on FoxP3, Th1/Th2 cytokine and costimulatory molecule mRNA expression in human peripheral blood mononuclear cells.

BACKGROUND: Stress influences immune function through mechanisms including an impact on regulatory elements. We have previously demonstrated that glucocorticoids (GCs) and catecholamines can influence immunomodulation including changes in Th1/Th2 cytokine production. These immunoregulatory imbalances are associated with elevated cortisol in vivo and in the presence of GCs in vitro. METHODS: We examined the effects of dexamethasone (DEX) and epinephrine (EPI) on the balance of regulatory T cells (Tregs), Th1/Th2 cytokine gene expression by peripheral blood mononuclear cells (PBMCs) from 16 normal subjects after 24-hour and 11-day cultures with tetanus toxoid. We wished to determine whether the immunomodulatory effects of stress hormones involved differences in costimulatory signal pathways including CD28, CTLA-4 (CD152), and CD80/86. RESULTS: Our results revealed that FoxP3 mRNA expression (representing Tregs) was decreased in PBMCs cultured with DEX relative to the control within 24 h (short term). DEX decreased the Th1/Th2 cytokine mRNA balance after 11 days (long term). CD28 and CD80 mRNA were decreased in short-term incubation with DEX whereas the effect of inhibition by DEX on CTLA-4 mRNA was detected only in long-term cultures. CONCLUSIONS: These results suggest that the Treg (FoxP3 mRNA) and CD28/CD80 costimulatory signal pathway may be a target of stress hormones in acute stress while the CTLA-4/CD80-86 pathway may be more susceptible in chronic stress.

Dopamine receptor gene expression in human amygdaloid nuclei: elevated D4 receptor mRNA in major depression.

Previous findings from this laboratory demonstrating changes in dopamine (DA) transporter and D2 receptors in the amygdaloid complex of subjects with major depression indicate that disruption of dopamine neurotransmission to the amygdala may contribute to behavioral symptoms associated with depression. Quantitative real-time RT-PCR was used to investigate the regional distribution of gene expression of DA receptors in the human amygdala. In addition, relative levels of mRNA of DA receptors in the basal amygdaloid nucleus were measured postmortem in subjects with major depression and normal control subjects. All five subtypes of DA receptor mRNA were detected in all amygdaloid subnuclei, although D1, D2, and D4 receptor mRNAs were more abundant than D3 and D5 mRNAs by an order of magnitude. The highest level of D1 mRNA was found in the central nucleus, whereas D2 mRNA was the most abundant in the basal nucleus. Levels of D4 mRNA were highest in the basal and central nuclei. In the basal nucleus, amounts of D4, but not D1 or D2, mRNAs were significantly higher in subjects with major depression as compared to control subjects. These findings demonstrate that the D1, D2 and D4 receptors are the major subtypes of DA receptors in the human amygdala. Elevated DA receptor gene expression in depressive subjects further implicates altered dopaminergic transmission in the amygdala in depression.