Circulating levels of hormones, lipids, and immune mediators in post-traumatic stress disorder - a 3-month follow-up study.

A number of peripheral blood analytes have been proposed as potential biomarkers of post-traumatic stress disorder (PTSD). Few studies have investigated whether observed changes in biomarkers persist over time. The aim of this study was to investigate the association of combat-related chronic PTSD with a wide array of putative PTSD biomarkers and to determine reliability of the measurements, i.e., correlations over time. Croatian combat veterans with chronic PTSD (n = 69) and age-matched healthy controls (n = 32), all men, were assessed at two time points separated by 3 months. Serum levels of lipids, cortisol, dehydroepiandrosterone-sulfate (DHEA-S), prolactin, and C-reactive protein were determined. Multiplex assay was used for the simultaneous assessment of 13 analytes in sera: cytokines [interferon-γ, interleukin (IL)-1ß, IL-2, IL-4, IL-6, TNF-α], adhesion molecules (sPECAM-1, sICAM-1), chemokines (IL-8 and MIP-1α), sCD40L, nerve growth factor, and leptin. Group differences and changes over time were tested by parametric or non-parametric tests, including repeated measures analysis of covariance. Reliability estimates [intraclass correlation coefficient (ICC) and kappa] were also calculated. Robust associations of PTSD with higher levels of DHEA-S [F(1,75) = 8.14, p = 0.006)] and lower levels of prolactin [F(1,75) = 5.40, p = 0.023] were found. Measurements showed good to excellent reproducibility (DHEA-S, ICC = 0.50; prolactin, ICC = 0.79). Serum lipids did not differ between groups but significant increase of LDL-C after 3 months was observed in the PTSD group (t = 6.87, p < 0.001). IL-8 was lower in the PTSD group (t = 4.37, p < 0.001) but assessments showed poor reproducibility (ICC = -0.08). Stable DHEA-S and prolactin changes highlight their potential to be reliable markers of PTSD. Change in lipid profiles after 3 months suggests that PTSD patients may be more prone to hyperlipidemia. High intra-individual variability in some variables emphasizes the importance of longitudinal studies in investigations of PTSD biomarkers.

Telomere shortening and immune activity in war veterans with posttraumatic stress disorder.

BACKGROUND: There is increasing evidence that chronic stress accelerates telomere erosion in leukocytes/peripheral blood mononuclear cells (PBMCs). However, functional changes associated with telomere shortening are poorly understood. We hypothesized that war veterans with PTSD would have shorter telomeres in PBMCs and that these cells might exhibit changes in measures of immune reactivity such as proliferation, cytokine production and expression of regulators of immune responses. METHODS: We measured relative telomere length and basal telomerase activity in PBMCs of 62 individuals (PTSD patients (N=30); age-matched healthy controls (N=17), elderly volunteers (N=15)). In parallel, we have assessed proliferation of activated T cells, interferon (IFN)-γ, interleukin (IL)-2, IL-4, tumor necrosis factor (TNF)-α and IL-6 cytokine production and expression of programmed death 1 (PD-1) receptor and its ligand PD-L1 on activated T cells. RESULTS: Middle-aged war veterans with current PTSD had shorter PBMC telomere length than their age-matched healthy controls while the elderly had the shortest telomeres. There was no difference in telomerase activity between PTSD patients and healthy controls while telomerase activity was significantly lower in the elderly. While the elderly group exhibited robust changes in immune activity such as increased production of proinflammatory cytokines (TNF-α, IL-6) and reduced proliferation of all T cells, the PTSD group showed reduced proliferative response of CD8(+) T cells to high concentrations of mitogen and reduced spontaneous production of IL-2 and IFN-γ. CONCLUSIONS: This study adds to the accumulating evidence that psychological trauma and chronic stress are associated with accelerated telomere attrition. However, changes in immune function associated with stress-related telomere shortening are not well understood. Although much less pronounced in PTSD patients than in elderly persons, reduced proliferative responses of T cells accompanied by shorter telomeres might be a sign of early immunosenescence. Together with reduced production of Th1 cytokines, observed immune changes may contribute to health risks associated with PTSD.

Patients with posttraumatic stress disorder exhibit an altered phenotype of regulatory T cells.

BACKGROUND: Regulatory T cells (Tregs) play a key role in immune homeostasis in vivo. Tregs have a critical role in preventing the development of autoimmune diseases and defects in Treg function are implicated in various autoimmune disorders. Individuals with posttraumatic stress disorder (PTSD) have higher prevalence of autoimmune disorders than the general population. We hypothesized that war veterans with PTSD would exhibit a decreased number and/or altered phenotype of Tregs. METHODS: We analyzed peripheral blood mononuclear cells (PBMCs) of patients with PTSD (N = 21) (mean age = 45.9) and age-matched healthy controls (N = 23) (mean age = 45.7) to determine the proportion of Tregs and their phenotype according to the expression of CD127 and HLA-DR markers which describe the differentiation stages of Tregs. In addition, we analyzed the expression of membrane ectoenzyme CD39 on Tregs of the study groups, an important component of the suppressive machinery of Tregs. RESULTS: We found no differences in the proportion of Tregs between PTSD patients and controls, but PTSD patients had a higher percentage of CD127(-)HLA-DR(-) Tregs and a lower percentage of CD127(lo)HLA-DR(+) Tregs compared to controls. There was no difference in expression of CD39 on Tregs of the study groups. CONCLUSIONS: Although the proportions of Tregs in PTSD patients were unchanged, we found that they exhibit a different phenotype of Tregs that might be less suppressive. Impaired differentiation and function of Tregs is likely involved in disruption of immune homeostasis in PTSD.

Determination of leucocyte subsets in human saliva by flow cytometry.

OBJECTIVE: Validation of a flow cytometry-based method for the determination of major leucocyte subsets [polymorphonuclear (PMN) cells, monocytes, T cells and B cells] in paraffin-stimulated whole human saliva. DESIGN: Salivary leucocyte subsets were determined by four-colour flow cytometry in eight healthy volunteers on three consecutive days. Comparison of leucocyte subsets between saliva and whole blood was also performed. Day-to-day variability and intraclass correlation coefficients (ICC) were determined as indicators of assay reliability. RESULTS: It was observed that PMN cells were the predominant cells in the saliva. Percentages of mononuclear cells ranged from 0.3% to 7.2%, with monocytes composing the highest percentage, followed by T cells and B cells. Regardless of high intra-individual day-to-day variability, proportions of leucocyte subsets did not significantly change over three measurements, and high ICCs were calculated for T cells and monocytes. CONCLUSION: Flow cytometry can be used as non-invasive and reproducible method for the analysis of leucocyte subsets in human saliva. Further investigation of pathological and other conditions that have the potential to influence salivary leucocyte subsets is warranted.

Repeated assessments of endocrine- and immune-related changes in posttraumatic stress disorder.

OBJECTIVE: It is assumed that stress-related changes in the endocrine and immune systems are key mediators involved in the development of diseases associated with posttraumatic stress disorder (PTSD). Evidence suggests that those changes might be related to the duration of PTSD. The aim of our study was to investigate the differences in selected endocrine- and immune-related variables between PTSD patients and control subjects, and whether these differences persist over time. METHODS: We assessed 39 Croatian war veterans with PTSD and 25 healthy volunteers (civilians without traumatic experience), all men, at two time points separated by 5.6 years (median; interquartile range: 5.4-6.3). Cortisol and prolactin levels were measured by radioimmunoassays while interleukin-6 and tumor necrosis factor-α were determined by enzyme-linked immunosorbent assays. Immune function was assessed by in vitro natural killer cell cytotoxicity (NKCC). Lymphocyte counts, immunophenotype and intracellular glucocorticoid receptor expression in various lymphocyte subsets were determined by three-color flow cytometry. RESULTS: At the first assessment, moderate to large effect size estimates of differences between patients and controls were observed for most of the measured variables. Only prolactin levels and lymphocyte counts remained significantly elevated in PTSD patients at the second assessment with low to moderate effect size estimates of differences between patients and controls in other variables. CONCLUSION: Observed endocrine- and immune-related changes in PTSD over time may depend on the duration of the allostatic load posed by the disorder and its impact on interactions between the endocrine and immune systems involved in stress response.

Exaggerated platelet reactivity to physiological agonists in war veterans with posttraumatic stress disorder.

An association between traumatic stress and cardiovascular disease (CVD) is supported by various epidemiological studies. Platelet activation and binding of activated platelets to leukocytes contributes to the pathophysiology of CVD. Evidence of hyperactive sympathetic nervous system, altered expression of platelet α(2)-adrenoreceptors (α(2)AR), and altered platelet adenylate cyclase activity in patients with posttraumatic stress disorder (PTSD) suggest that platelet reactivity in PTSD may be altered as well. We tested whether platelet reactivity to increasing doses of adenosine-diphosphate (ADP), epinephrine (EPI), or their combination differs between war veterans with PTSD (n=15) and healthy controls (n=12). For this purpose, citrated whole blood was incubated with increasing concentrations of ADP (0.1, 1, 10 µM), EPI alone (10 nM, 100 nM, 1000 nM), or EPI (10 nM, 100 nM, 1000 nM) in combination with 0.1 µM ADP. A subset of samples was also incubated with 10 µM yohimbine (YOH), α(2)AR antagonist, to distinguish receptor-specific effects. Platelet CD62P expression and formation of platelet-leukocyte aggregates (PLA) [platelet-monocyte (P-Mo), -lymphocyte (P-Ly), and -neutrophil (P-Ne) aggregates] were measured using three-color flow cytometry. Platelet reactivity was higher in war veterans with PTSD when compared to controls, as determined by greater CD62P expression and formation of PLA in response to ADP alone or in combination with EPI. Platelet reactivity also correlated with the severity of PTSD symptoms. Preliminary experiments with YOH indicate that stress-associated EPI elevations may contribute to platelet activation through a α(2)AR-dependent mechanism. The enhanced platelet reactivity observed in our study may be the underlying mechanism contributing to the development of CVD in PTSD patients.

Natural killer cell cytotoxicity and lymphocyte perforin expression in veterans with posttraumatic stress disorder.

OBJECTIVE: To examine the effect of posttraumatic stress disorder (PTSD) on the measures of immune function and the hypothalamic-pituitary-adrenal axis components, and to determine whether additional life stressors affect measured variables. METHODS: We simultaneously examined the natural killer cell cytotoxicity (NKCC), perforin and glucocorticoid receptor (GCR) expression in natural killer (NK) and cytotoxic T (CD8) cells, as well as serum cortisol concentration in a group of Croatian war veterans with chronic, combat-related PTSD (n=29) and a group of healthy, age-matched men (n=13). PTSD patients were divided into two subgroups: compensation-seeking (n=15) and retired or compensation non-seeking (n=14) subjects. The former includes those involved in the process of getting disability-based army retirement as an additional life stressor. RESULTS: NKCC was decreased in both PTSD groups when compared to controls. Impairment of NKCC could not be attributed to the perforin expression as perforin was not decreased in comparison to controls. Moreover, the increased level of perforin was recorded in NK cells of retired PTSD subjects. Both PTSD groups shared an increased relative quantity of GCR in lymphocytes, whereas no difference between the groups in the baseline levels of serum cortisol was observed. CONCLUSIONS: Diminished NKCC was not accompanied by perforin insufficiency in PTSD subjects, and other causes should be examined. An additional life stressor does not contribute considerably to either immune or endocrine system related changes.

Changes in immune and endocrine systems in posttraumatic stress disorder - prospective study.

INTRODUCTION AND AIM: Posttraumatic stress disorder (PTSD) is associated with increased medical morbidity, particularly from the autoimmune and cardiovascular diseases. Changes in the endocrine and immune system are key mediators in this process. The aim of our study was to investigate weather hormones (cortisol and prolactin), proinflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α)), components of HPA-axis (lymphocyte expression of glucocorticoid receptor (GR)), immune function (natural killer cell cytotoxicity) and peripheral blood percentages of various lymphocyte subpopulations (T cells, helper T cells, cytotoxic T cells, B cells and natural killer cells) change in patients with posttraumatic stress disorder over time.

[Psychoneuroimmunology--regulation of immunity at the systemic level]. / Psihoneuroimunologija--regulacija imunosti na razini organizma kao cjeline.

Innate and acquired immune reactions are controlled by their intrinsic regulatory mechanisms, ie. by an array of cytokines that mediate communication among cells of the immune system itself and with other cells and tissues, e. g. in areas of inflammation. In addition, the immune system is also subjected to systemic regulation by the vegetative and endocrine systems since immune cells express receptors for neurotransmitters and hormones. Neuroendocrine signals may enhance or suppress the immune reaction, accelerate or slow it, but do not affect specificity. Various stressful factors, including the psychosocial ones, affect immunity. In turn, cytokines generated by the immune system influence hormonal secretion and central nervous system, producing specific behavioral changes (the "sickness behavior") accompanying infectious and inflammatory diseases. That includes somnolence, loss of apetite, depression or anxiety and decrease of cognitive abilities, attention and memory. Local immune systems in skin and mucosa are also subjected to systemic neuroendocrine regulation and possess intrinsic neuroregulatory networks as well. These mechanisms render skin and respiratory and digestive tracts responsive to various forms of stress. Examples are neurodermitis, asthma and ulcerative colitis. In children, the immune and the neuroendocrine systems are still developing, particularly in fetal, neonatal and early infant periods, and exposure to stressful experiences at that time may result in late consequences in the form of deficient immunity or greater risks for allergic or autoimmune reactions. Recognition of the participation of neuroendocrine mechanisms in regulation of immunity helps us understand alterations and disturbances of immune reactions under the influence of stressful factors but so far has not produced reliable therapeutic implications. Psychosocial interventions involving the child and its family may be useful.

Circulating lymphocyte subsets, natural killer cell cytotoxicity, and components of hypothalamic-pituitary-adrenal axis in Croatian war veterans with posttraumatic stress disorder: cross-sectional study.

AIM: To determine peripheral blood lymphocyte subsets--T cells, helper T cells, cytotoxic T cells, B cells, and natural killer cells, natural killer cell cytotoxicity, serum cortisol concentration, and lymphocyte glucocorticoid receptor expression in Croatian combat veterans diagnosed with chronic posttraumatic stress disorder (PTSD); and to examine the relationship between the assessed parameters and the time passed since the traumatic experience. METHODS: Well-characterized group of 38 PTSD patients was compared to a group of 24 healthy civilians. Simultaneous determination of lymphocyte subsets and the expression of intracellular glucocorticoid receptor was performed using three-color flow cytometry. Natural killer cell cytotoxicity was measured by (51)Cr-release assay and the serum cortisol concentration was determined by radioimmunoassay. RESULTS: We found higher lymphocyte counts in PTSD patients than in healthy controls (2294.7+/-678.0/microL vs 1817.2+/-637.0/microL, P=0.007) and a positive correlation between lymphocyte glucocorticoid receptor expression and the number of years that passed from the traumatic experience (r(s)=0.43, P=0.008). Lymphocyte glucocorticoid receptor expression positively correlated with serum cortisol concentration both in PTSD patients (r=0.46, P=0.006) and healthy controls (r=0.46, P=0.035). CONCLUSION: This study confirmed that the immune system was affected in the course of chronic PTSD. Our findings also indicated that the hypothalamic-pituitary-adrenal axis profile in PTSD was associated with the duration of the disorder. Due to the lack of power, greater sample sizes are needed to confirm the results of this study.

Baseline level of platelet-leukocyte aggregates, platelet CD63 expression, and soluble P-selectin concentration in patients with posttraumatic stress disorder: a pilot study.

Platelets may have an important role in the development of cardiovascular disease (CVD) as a result of chronic stress. We conducted a pilot study to evaluate the effect of posttraumatic stress disorder (PTSD) on baseline platelet activation. Platelet-leukocyte aggregates (PLA) and CD63 expression were measured by flow cytometry, and soluble (s)P-selectin concentration was determined in sera of 20 Croatian male combat veterans with PTSD and 20 healthy civilians. Groups were matched in sex, age, body mass index (BMI) and traditional CVD risk factors. Our data showed no differences in measured parameters. Other platelet activation markers should be determined and a larger sample size used in future studies.

Effectiveness of novel PGM-containing incomplete Seppic adjuvants in rabbits.

Peptidoglycan monomer (PGM) is adjuvant active molecule in experimental mice, although its adjuvanticity is much lower in comparison to potent adjuvants. The novel adjuvant formulations were developed by incorporation of PGM into Montanide ISA 206 and Montanide ISA 720 adjuvants, with the aim to enhance its adjuvanticity by protecting it from the fast degradation and metabolic clearance. Adjuvanticity of the novel adjuvant formulations was tested in rabbits for induction of protein-specific antibodies. Both novel adjuvants ISA206(PGM) and ISA720(PGM) were significantly stronger than Montanide adjuvants themselves, and also significantly more potent than Complete Freund Adjuvant. Montanide ISA 720 was shown as much better carrier of PGM, since the novel ISA720(PGM) adjuvant was significantly stronger adjuvant than the ISA206(PGM).

[MHC tetramers: tracking specific immunity]. / MHC-tetrameri: na tragu specificnoj imunosti.

In an adaptive immune response, antigen is recognized by two distinct sets of highly variable receptor molecules: (1) immunoglobulins, that serve as antigen receptors on B cells and (2) the antigen-specific receptors on T cells. T cells play important role in the control of infection and in the development of protective immunity. These cells can also mediate anti-tumor effects and, in case of autoimmune syndromes, contribute to the development and pathology of disease. The specificity of T cells is determined by T cell receptors (TCR). Understanding of the success of immune responses requires the direct measurement of antigen-specific T lymphocytes. Cell with major histocompatibility complex (MHC) class I molecules are able to present antigens to antigen-specific CD8+ cytotoxic T lymphocytes. MHC class I molecules present small peptides (epitopes) processed from intracellular antigens such as viruses and intracellular bacteria. MHC class I molecules in humans are designated as human leukocyte antigen (HLA) class I and divided into HLA-A, -B and -C. CD8+ T cells recognize MHC class I molecules and after activation produce proteins that destroy infected cells. MHC class II molecules receive their peptides mainly from extracellular and soluble antigens and present them to the CD4+ T helper cells. A recently described technique that can be used in flow cytometry enables us to quantify ex vivo antigen-specific T cells by binding of soluble tetramer MHC-peptide complexes attached to fluorochrome. Quantitative analyses of antigen-specific T cell populations provide important information on the natural course of immune responses. The interaction of T cell receptors on T lymphocytes with tetrameric MHC-peptide complexes mimics the situation on the cell surface, and allows for reliable binding. Tetramers consist of four biotinylated HLA-peptide epitope complexes bound to streptavidin conjugated with fluorescent dye. Tetramer technology has sensitivity of detection as little as 0.02% of total cytotoxic T cell pool or T helper cell pool (i.e. approximately 1 in 50.000 lymphocytes). The combination of this technology with intracellular cytokine staining methods opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production). MHC tetramers class I have yielded useful insights into in vivo dynamic and function of antigen-specific CD8+ T cells in viral infections, parasitic infections, cancer, autoimmune disease and transplantation. This knowledge is of special interest for immunotherapy, diagnostic monitoring of T cell mediated immunity, and the development of new vaccination strategies. There is some possibility for cell therapy with antigen-specific CD8+ T cells for various diseases including cancer and viral infections. Targeted immunotherapy of selective deletion of auto--or alloreactive T cells with MHC tetramers may be important for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs. The utility of this technique for the immunotherapy in vivo needs to be confirmed and modified in further research. Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of cellular immune system.

Immune parameters in hemorrhagic fever with renal syndrome during the incubation and acute disease: case report.

We describe immune parameters in a Croatian soldier who presented with mild flu-like symptoms and interstitial inflammatory infiltrate in the lungs on an X-ray during the incubation phase of hemorrhagic fever with renal syndrome (HFRS). Enzyme-linked immunosorbent assay (ELISA) IgM and polymerase chain reaction (PCR) were negative. Two weeks later, he developed HFRS caused by the Puumala virus. We performed two-color immunofluorescence cytometry with monoclonal antibodies identifying the activation markers on T cells. Serum samples were also examined by enzyme immunoassay (EIA) for the presence of interleukins IL-2 and IL-6 and their soluble receptors (sR). The analysis of early and late activation markers during the period of incubation revealed a small increase in the percentage of helper (CD4+CD25+) T cells and no significant increase in total activated (HLA-DR+TCR+) and cytotoxic (CD8+CD71+) T cells as compared with healthy controls. In the serum, only the concentration of soluble IL-6 receptor was increased. However, when the patient developed HFRS, all activation markers on T cells increased. Concentrations of sIL-2Ralpha and IL-6 remained increased two and six days after HFRS onset, respectively, whereas sIL-6R increased six days after HFRS onset. IL-2 concentration did not change. Our case indicates that rapid, modern diagnostic tools are necessary in the diagnosis of infectious diseases and their differential diagnosis. Immunological tests, which provide information on the patient immune status and especially on early changes in immune parameters, may contribute to the improvement of the diagnosis, prognosis, and therapy of HFRS.

Influence of adjuvant-active peptidoglycan monomer on specific T cell responses in mice.

Peptidoglycan monomer (PGM) originating from Brevibacterium divaricatum is a non-toxic, non-pyrogenic, water-soluble immunostimulator. It potentiates humoral immune response to ovalbumin (OVA) in mice upregulating both immunoglobulin (IgG) 1 and IgG2a antibody subclasses. This study concerns the influence of PGM on T cell activation and cytokine networks in response to OVA. OVA-specific proliferative response as well as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) secretion in lymph node cell cultures of immunised mice were studied. Due to pharmacokinetic properties of PGM, namely its fast metabolism and excretion, special emphasis was on choosing the appropriate time for lymph node removal and duration of cell cultivation for each cytokine. PGM treatment in addition to OVA resulted in an increase of lymph node cellularity, stimulation of OVA-specific IFN-gamma and IL-4 production as well as of OVA-specific proliferative response. Results demonstrate that PGM stimulated both Th1 and Th2 subpopulations.