Sabina Rabatić

Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry

Acute RSV infection in infancy may produce some asthma‐like symptoms and may be followed by a recurrent wheeze later in childhood. It has been proposed that RSV infection stimulates type‐2 cytokine responses, resembling those found in atopy and asthma. Peripheral blood cells were obtained from RSV‐infected infants (n = 30) and healthy controls (n = 10). After in vitro restimulation of the cells, intracellular IL‐4 and interferon‐gamma (IFN‐γ) were measured by flow cytometry. The cells from RSV‐infected infants produced more IL‐4 and less IFN‐γ than those from healthy controls. IL‐4 production was more frequent in CD8 than in CD4 cells, and the bias toward IL‐4 production was greatest in infants with mild infections, whereas IFN‐γ production increased with disease severity. Our conclusions are that RSV infection is associated with IL‐4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine‐producing cells.

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.

Increased Toll-like receptor 4 expression in infants with respiratory syncytial virus bronchiolitis

The fusion protein of the respiratory syncytial virus (RSV) binds to the pattern recognition receptors, TLR4 and CD14, and initiates innate immunity response to the virus. The aim of the study was to investigate the expression of TLR4 on peripheral blood lymphocytes and monocytes in peripheral blood of infants in both acute and convalescent phase of RSV bronchiolitis (n = 26). In addition, TNF‐α expression in lipopolysaccharide‐stimulated monocytes was also assessed. The results showed TLR4 to be expressed predominantly by monocytes in both sick infants and controls. During the acute phase of infection monocytes up‐regulated TLR4 in eight infants, which returned to the levels recorded in controls 4–6 weeks from infection. There was no difference in the percentage of TNF‐α secreting monocytes. Of the clinical parameters tested, minimal oxygen saturation was found to correlate negatively with this expression in the group of infants with increased TLR4. Additional studies are under way to correlate this finding with the outcome of the immune response to RSV.

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.

Met-enkephalin modulates stress-induced alterations of the immune response in mice.

Overnight restraint stress of mice decreased ConA-driven lymphocyte proliferation, plaque-forming cell response to sheep red blood cells (SRBC), and NK activity in the spleen, but the phagocytic activity was enhanced. Injection of methionine-enkephalin (MENK), 10 mg/kg, i.p., 30 min before restraint, abolished these changes (except for the NK activity) and attenuated the stress-induced elevation of glucocorticoids. However, MENK itself affected the immune responses like stress: It decreased NK activity and the PFC response and enhanced phagocytic activity. Contrary to results with stress, MENK had no effect on cell proliferation. The opioid-receptor antagonist naloxone given before restraint reversed the stress-induced enhancement of phagocytosis and the decrease of T-cell proliferation. Alterations of the immune responses induced by restraint stress seem to be mediated by at least two mechanisms: activation of the hypothalamus-pituitary-adrenal (HPA) axis and the secretion of opioid peptides. MENK injected before stress may interfere with either or both mechanisms. T or B lymphocytes seem to be affected by the activation of the HPA axis, and phagocytes by a direct opioid action, whereas NK cells seem to be under the influence of another control mechanism.

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.

Expression of Lymphocytes FcεRII/CD23 in Allergic Children Undergoing Hyposensitization

Owing to the proposed role of Fc epsilon RII/CD23 in allergic diseases, we analyzed the expression of this receptor on peripheral blood lymphocytes (pan-B, pan-T and CD4+ or CD8+ T cells) and its autoproteolytic product sCD23 in serum. This was done in 10 asthmatic children allergic to Dermatophagoides pteronyssinus (Dpt) before and 6 weeks after hyposensitization. FACS analysis of double, direct immunofluorescence staining of the whole blood revealed an elevated percentage of Fc epsilon RII/CD23+ lymphocytes in allergic children (10.29 +/- 5.0), a significantly higher percentage than in nonallergic children (5.7 +/- 2.4, p < 0.05). The majority of Fc epsilon RII/CD23+ were on B cells. A significant positive correlation between the percentages of CD23+ lymphocytes and serum IgE levels was found (Spearman rank = 0.63, p < 0.05). The percentage of CD20+CD23+ lymphocytes significantly decreased after 6 weeks of hyposensitization (6.2 +/- 3.6, p < 0.05), while the percentage of CD20+ lymphocytes remained unchanged. Similarly, hyposensitization was followed by a reduction of total serum IgE levels, but Dpt-specific IgG4 and IgE remained unchanged.