Ante Sabioncello

Flow cytometric determination of glucocorticoid receptor (GCR) expression in lymphocyte subpopulations: lower quantity of GCR in patients with post-traumatic stress disorder (PTSD).

Assessment of the intracellular glucocorticoid receptor (GCR) level may be useful in monitoring functional disturbances of the hypothalamic‐pituitary‐adrenocortical axis or effects of prolonged steroid therapy. Cytosolic ligand binding assays have recently been supplemented by flow cytometric determination of receptor expression in individual cells. A method based on multiparametric analysis of whole blood by simultaneous labelling of intracellular GCRs and surface markers of lymphocyte subsets is described. We examined 25 healthy male volunteers and 35 age‐ and sex‐matched post‐traumatic stress disorder (PTSD) patients within 8 years from traumatic event. PTSD patients had a lower relative quantity of GCR in all lymphocyte populations tested as compared with healthy volunteers. NK cells of both groups showed higher expression of GCR than other lymphocyte subsets. In PTSD patients, the expression of GCR in B lymphocytes was also higher than in T cell. Although serum cortisol level was lower in PTSD patients, there was no correlation between cortisol level and GCR expression. Multiparameter flow cytometric determination of GCR expression in lymphocyte subpopulations may provide a useful tool for monitoring immunoregulatory action of glucocorticoids.

Prognostic Parameters for Survival of Patients with Malignant Mesenchymal Tumors of the Uterus

Malignant mesenchymal uterine neoplasms are the most aggressive type of primary uterine tumors, with most patients dying within a few years of diagnosis. Thus, it would be very important to define prognostic factors for predicting the malignancy potential of at least some of their subtypes.

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.

Immune, endocrine, and psychological responses in civilians displaced by war.

Objectives The objectives of this study were to assess the influence of trauma caused by forced expulsion from home in a war-ravaged region on the psychological, hormonal, and immune responses in displaced persons and to analyze the relationships between psychometric, hormonal, and immunologic variables. Methods Participants were 20 displaced and 14 control women. Psychosomatic response was evaluated using the COR-NEX2 test. Serum concentrations of cortisol, prolactin, endorphin, thyroxine, and triiodothyronine were measured by radioimmunoassay. Immunophenotyping and lymphocyte proliferation were determined by flow cytometry, and phagocyte functions (ie, ingestion and antibody-dependent cytotoxicity) against 51Cr-labeled sheep red blood cells were assessed through radioactivity uptake and release, respectively. Results In comparison with control women, displaced women had higher COR-NEX2 test scores; higher serum cortisol, prolactin, and endorphin levels; an increase in activated phenotype within all three measured cell populations (ie, B, T, and natural killer cells); as well as an enhanced proportion of proliferating lymphocytes in freshly isolated samples. However, the phytohemagglutinin-stimulated proliferative response, estimated as the stimulation index, was lower in displaced women. A complex pattern of relations between psychological, hormonal, and immune responses was observed. Conclusions Chronic psychological stress elicited multiple, predominantly stimulatory influences on immune functions.

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.

Role of peripheral blood mononuclear cell (PBMC) phenotype changes in the pathogenesis of haemorrhagic fever with renal syndrome (HFRS)

Hantaviruses cause an important human illness, HFRS. Blood samples from 22 HFRS‐positive, six seronegative patients and 15 healthy controls were examined in 1995, during the largest HFRS epidemic in Croatia. Results of double‐ and triple‐colour immunofluorescence analysis showed an increased percentage of cytotoxic T cells (CD3+CD8+) in seropositive patients compared with seronegatives and healthy controls. The majority of seropositive HFRS patients expressed activation and memory antigens on T and B lymphocytes. The percentage of CD23+ and CD21+ B lymphocytes was lower in seropositive patients. HFRS patients had elevated levels of sCD23 and five had elevated total IgE. The increased expression of both early and late T cell activation antigens, e.g. CD25, CD71 and HLA‐DR, memory cells and sCD23 positively correlated with biochemical parameters (AST, ALT, urea, α2‐globulin) during the acute phase of HFRS. The phenotypic changes observed, especially early and late T cell activation markers, as well as memory cells, could be useful parameters in the evaluation of HFRS course, and prognostic factors of HFRS severity. Additional attention should be paid to liver involvement in the pathogenesis of HFRS.

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.

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)-1β, 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.

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.