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.

Decreased Toll-like receptor 8 expression and lower TNF-α synthesis in infants with acute RSV infection.

BACKGROUND: Toll-like receptors (TLRs) are part of the innate immune system, able to recognize pathogen-associated molecular patterns and activate immune system upon pathogen challenge. Respiratory syncytial virus (RSV) is a RNA virus particularly detrimental in infancy. It could cause severe lower respiratory tract disease and recurrent infections related to inadequate development of anti-viral immunity. The reason could be inadequate multiple TLRs engagement, including TLR8 in recognition of single-stranded viral RNA and diminished synthesis of inflammatory mediators due to a lower expression. METHODS: Intracellular TLR8 expression in peripheral blood monocytes from RSV-infected infants was profiled and compared to healthy adults and age matched controls. Whether the observed difference in TLR8 expression is a transitory effect, infants in convalescent phase (4-6 weeks later) were retested. Specific TLR8-mediated TNF-α production in monocytes during an acute and convalescent phase was analyzed. RESULTS: RSV-infected and healthy infants had lower percentage of TLR8-expressing monocytes than healthy adults whereas decreased of TLR8 protein levels were detected only for RSV-infected infant group. Lower protein levels of TLR8 in monocytes from RSV-infected infants, compared to healthy infants, negatively correlated with respiratory frequency and resulted in lower TNF-α synthesis upon a specific TLR8 stimulation. In the convalescent phase, levels of TLR8 increased, accompanied by increased TNF-α synthesis compared to acute infection. CONCLUSIONS: Lower TLR8 expression observed in monocytes, during an acute RSV infection, might have a dampening impact on early anti-viral cytokine production necessary to control RSV replication, and subsequently initiate an adaptive Th1 type immune response leading to severe disease in infected infants.

Expression of chemokine receptor CX3CR1 in infants with respiratory syncytial virus bronchiolitis.

Respiratory syncytial virus (RSV) glycoprotein G mimics fractalkine, a CX(3)C chemokine, which mediates chemotaxis of leukocytes expressing its receptor, CX(3)CR1. The aim of this study was to examine the relationship between RSV infection and expression of perforin and IFN-gamma in CX(3)CR1-expressing peripheral blood CD8(+) T cells. Samples were collected from infants with RSV bronchiolitis, both in the acute and convalescence phase (n = 12), and from their age- and sex-matched healthy controls (n = 15). Perforin expression and IFN-gamma secretion in CX(3)CR1(+) CD8(+) T cells were assessed by four-color flow cytometry. The NF-kappaB p50 and p65 subunit levels were also determined as markers of RSV-induced inflammation. Study results showed perforin and CX(3)CR1 expression to be significantly lower in the convalescent phase of infected infants than in healthy controls. There was no significant difference in IFN-gamma secretion and NF-kappaB binding activity between two time-points in RSV-infected infants, or when compared with healthy controls. Infants with prolonged wheezing had lower acute-phase CX(3)CR1 levels in peripheral blood. These data indicate existence of an event persisting after acute RSV infection that is able to modulate effector functions of cytotoxic T cells, and also link disease severity with CX(3)CR1 expression.

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.

[Regulatory T cells]. / Regulacijski T-limfociti.

Regulatory T-cells are a subset of T cells that have beene extensively studied in modern immunology. They are important for the maintenance of peripheral tolerance, and have an important role in various clinical conditions such as allergy, autoimmune disorders, tumors, infections, and in transplant medicine. Basically, this population has a suppressive effect on the neighboring immune cells, thus contributing to the local modulation and control of immune response. There are two main populations of regulatory T cells - natural regulatory T cells, which form a distinct cellular lineage, develop in thymus and perform their modulatory action through direct intercellular contact, along with the secreted cytokines; and inducible regulatory T cells, which develop in the periphery after contact with the antigen that is presented on the antigen presenting cell, and their primary mode of action is through the interleukin 10 (IL-10) and transforming growth factor beta (TGF-alpha) cytokines. Natural regulatory T cells are activated through T cell receptor after contact with specific antigen and inhibit proliferation of other T cells in an antigen independent manner. One of the major difficulties in the research of regulatory T cells is the lack of specific molecular markers that would identify these cells. Natural regulatory T cells constitutively express surface molecule CD25, but many other surface and intracellular molecules (HLA-DR, CD122, CD45RO, CD62, CTLA-4, GITR, PD-1, Notch, FOXP3, etc.) are being investigated for further phenotypic characterization of these cells. Because regulatory T cells have an important role in establishing peripheral tolerance, their importance is manifested in a number of clinical conditions. In the IPEX syndrome (immunodysregulation, polyendocrinopathy and enteropathy, X-linked), which is caused by mutation in Foxp3 gene that influences the development and function of regulatory T cells, patients develop severe autoimmune reactions that involve autoimmune endocrine disorders (type 1 diabetes, thyroiditis), respiratory and nutritive allergy, eczema and severe infections. In different types of allergy (pollen allergy, dust mite, nutritive allergens, contact hypersensitivity, etc.) and autoimmune diseases (such as rheumatoid arthritis, multiple sclerosis and type 1 diabetes) a lower number or decreased functional capability of regulatory T cells have been described. In inflammatory conditions and infections, this cell population has an important task in restricting immune response and protecting the host from excessive damage. This ability of regulatory T cells can be used by some pathogens (Epstein Barr virus, Mycobacterium tuberculosis, Leishmania major, etc.) and tumor cells to avoid host response and therefore contribute to the development of some pathological conditions. The knowledge gained on the phenotype and function of regulatory T cells could be useful in many medical conditions. In allergy, autoimmune diseases and in transplant procedures in medicine it would be desirable to increase their function, thus to partially suppress the immune system activity. On the other hand, in some infections and tumors, it would be preferable to decrease the activity of regulatory T cells and boost the function of effector T cells. Regulatory T cells comprise a very active field of immunology, therefore monitoring and modulating of their activity is of great potential significance in a broad spectrum of clinical conditions. By developing and standardizing methods for their monitoring, it would be possible to follow additional parameters of certain clinical conditions and possibly utilize them in therapy.

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.

[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.

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.

The increased type-1 and type-2 chemokine levels in children with acute RSV infection alter the development of adaptive immune responses.

Severe RSV infections and frequent recurrence could be related to the altered polarization of type-2/type-1 T cells. This increases the importance of determining distinctive chemokines and chemokine receptor profiles on memory T cells. We analyzed systemic adaptive T cell response in the acute (n=17) and convalescent phase (n=7) of RSV-infected children, in the acute (n=11) and convalescent phase (n=6) of children with other viral respiratory infections (adenovirus and influenza virus), and in healthy children (n=18). Expression of CCR4 and CXCR3 on effector-memory (TEM) and central-memory (TCM) T cells was compared between tested groups. Serum concentrations of specific chemokines were determined. High CXCL10 levels were detected in acutely infected children regardless of virus pathogen, whereas increased CCL17 production was RSV-specific. Higher percentages of CCR4+ CD4 TEM cells in acute RSV infection were accompanied with higher percentages of CXCR3+ CD8 TEM cells, whereas the development of long-lived memory CXCR3+ CD4 and CD8 TCM cells seems to be compromised, as only children with other viral infections had higher percentages in the convalescent phase. Presence of type-2 and type-1 adaptive antiviral immune response, together with insufficient development of long-lived type-1 T cell memory, could play an important role in RSV pathogenesis and reinfection.

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.

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.

Effect of house dust mite immunotherapy on transforming growth factor beta1-producing T cells in asthmatic children.

BACKGROUND: Recent evidence suggests that regulatory T cells (Treg cells) and immunosuppressive cytokines, such as transforming growth factor BETA1 (TGF-BETA1) and interleukin 10 (IL-10), may have a role in clinically effective allergen specific immunotherapy (SIT). OBJECTIVE: To evaluate the effect of SIT on the induction of Treg cells in house dust mite-allergic children and on the expression of specific Treg cell markers (cytotoxic T-lymphocyte-associated protein 4 [CTLA-4], IL-10, and TGF-BETA1). METHODS: In this uncontrolled open-label study, the percentage of peripheral blood CD4+ Treg cells (CD69 CD45RO+CTLA-4+ and CD3+CD4+CD25+FOXP3+) and the expression of molecules associated with their functions (CTLA-4, TGF-BETA1, and IL-10) were analyzed using flow cytometry in 16 children allergic to house dust mites before and at 3 and 12 months of subcutaneous SIT. Clinical variables, such as symptom score, medication requirements, forced expiratory volume in 1 second, peak expiratory flow rate, and serum IgE levels, were also determined. Ten healthy children were included as controls. RESULTS: All the clinical variables improved during immunotherapy. The percentage of CD4+CD25+CD69-CD45RO+ Treg cells remained unchanged. The percentage of CTLA-4+ -expressing Treg cells transiently increased after 3 months of immunotherapy, whereas the percentage of FOXP3+ Treg cells did not change after 1 year of immunotherapy. Levels of IL-10+ cells transiently decreased after 3 months of immunotherapy. Four children who required inhaled fluticasone propionate administration for significant symptom worsening had no statistically significant increase in TGF-BETA1-secreting T cells at 12 months of SIT, in contrast to 12 children without inhaled corticosteroid treatment. CONCLUSIONS: The increase in TGF-BETA1-positive T cells only in children without significant symptom worsening requiring inhaled corticosteroid treatment limits the usefulness of TGF-BETA1 in monitoring response to allergen immunotherapy.

Double trouble: hemorrhagic fever with renal syndrome and leptospirosis.

The clinical picture of hemorrhagic fever with renal syndrome can closely mimic that of unicteric leptospirosis and vice versa. This is the first description of dual infection with Dobrava virus and leptospira and alteration of immune parameters in a Croatian soldier.

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.