Mirjana Grubišić-Ilić

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.

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.

Epidemiological study of suicide in the Republic of Croatia – comparison of war and post-war periods and areas directly and indirectly affected by war

We examined the differences in the suicide characteristics between areas directly and indirectly affected by war activities and in war and post-war periods according to the following variables: suicide rate, sex, age and method of suicide. Analysis was done on 5349 suicides committed in the period 1993-1998 (war and post-war years). The suicide rates in the Republic of Croatia oscillated in the pre-war, war and post-war periods (1985-2000) but without significant differences. In the areas directly affected by war, the suicide rate was significantly lower than in other areas during the study period 1993-1998 (chi-square = 10.3245; P = 0.0017). The number of suicides in both sexes declined in the areas directly affected by war-more in men than in women; the difference between sexes was statistically significant (chi-square = 3.6697; P = 0.055). Middle- and old-aged people were the population with high suicide risk in both areas (t = 1.76; P = 0.078). There were significant differences in the methods of suicides between war and non-war areas (chi-square = 108.8473; P = 0.001). Firearms or explosive devices were the methods used more significantly for suicides in the areas directly affected by war than in other areas, whereas hanging was more frequently used in the areas indirectly affected by war.

Outcome of influenza vaccination in combat-related post-traumatic stress disorder (PTSD) patients

Post‐traumatic stress disorder (PTSD) is an anxiety disorder that can occur after exposure to extreme traumatic experience such as war trauma, and is accompanied by fear, helplessness or horror. Exposure to trauma can result in immune dysregulation and influence susceptibility to infectious disease as well as vaccine efficacy. The aim of the study was to determine the relation of psychological stress and the immune response to influenza vaccination in combat‐related PTSD patients (n = 28). Detection of anti‐viral antibody titre was performed by inhibition of haemagglutination assay. Ex vivo tetramer staining of CD8+ T lymphocytes was used to monitor T cells specific for human leucocyte antigen (HLA)‐A*0201‐restricted influenza A haemagglutinin antigens before and after vaccination. Twenty patients showed a fourfold antibody titre increase to one or both influenza A viral strains, and 18 of them showed the same response for both influenza B viral strains. Ten of 15 healthy controls showed a fourfold rise in antibody titre to both influenza A viral strains and eight of them showed the same response for both influenza B viral strains. HLA‐A*0201+ PTSD patients (n = 10) showed a significant increase of influenza‐specific CD8 T cells after vaccination. Although those PTSD patients had a lower number of influenza‐specific CD8+ T cells before vaccination compared to HLA‐A*0201+ healthy controls (n = 6), there was no difference in influenza A antibody titre between PTSD patients and control subjects before vaccination. The generated humoral and cellular immune response in PTSD patients argues against the hypothesis that combat‐related PTSD in war veterans might affect protection following influenza vaccination.