Enumeration of haemagglutinin-specific CD8+ T cells after influenza vaccination using MHC class I peptide tetramers.

With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-gamma+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.

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.

Effect of cysteinyl leukotriene receptor antagonist on CD11b and CD23 expression in asthmatic children.

BACKGROUND: Cysteinyl leukotrienes are potent pro-inflammatory mediators that contribute to the pathophysiologic features observed in allergic asthma. Inhibitors of leukotriene receptors represent novel therapy in asthma treatment. In addition to the protection from early asthmatic responses, these drugs have recently been shown to protect from late airway responses too. METHODS: We studied the effect of treatment with an oral antagonist of cysteinyl leukotriene receptors on the increased expression of the low-affinity IgE receptor, CD23, on B cells, and of its ligands, CD11b and CD11c, on CD4(+) T cells and monocytes in peripheral blood of patients with allergic asthma. In this uncontrolled open-label study, 14 children with allergic asthma received montelukast, a cysteinyl leukotrine receptor antagonist, for a period of 6 weeks after demonstrating forced expiratory volume in 1 s (FEV(1)) of less than 80% of the predicted value. Samples of peripheral heparinized blood and sera were obtained before and after therapy completion. Three-colour immunofluorescence analysis was performed, and expression of CD11b and CD11c on CD4(+) T lymphocytes and monocytes as well as the expression of CD21 and CD23 on B cells were determined (n=12). Peripheral blood eosinophil count, changes in FEV(1) and peak expiratory flow rate (PEFR), asthma exacerbations, and as-needed use of beta-agonist were also monitored. RESULTS: Montelukast improved FEV(1) and PEFR, and decreased peripheral eosinophil counts in all study patients. There was no significant change in the expression of CD21 and CD23 on B cells. The expression of CD11c on CD4(+) T cells and of both CD11b and CD11c on monocytes remained similar to the pretreatment expression. However, the percentage of CD11b(+)CD4(+) T lymphocytes significantly decreased after treatment with montelukast. This was accompanied by a significant decrease in the levels of total IgE. CONCLUSION: The capacity of 6-week montelukast therapy to reduce the percentage of CD11b CD4(+) T cells might be a mechanism leading to the immune response modulation on this T cell subset interaction with CD23-expressing B cells and subsequent down-regulation of IgE synthesis.

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.

Characteristics of Puumala and Dobrava infections in Croatia.

In this study, two different hantaviruses, Puumala virus (PUUV) and Dobrava virus (DOBV), were demonstrated for the first time to coexist and cause hemorrhagic fever with renal syndrome (HFRS) in Croatia. Phylogenetic analysis showed some differences among the nucleotide sequences of PUUV originating from Dinara mountain, which was more closely related to Austrian PUUV than other Croatian PUUV from Mala Kapela mountain. More consistency was found among the Croatian DOBV. HFRS was verified in 85 of 201 suspected cases recorded in 1995 during the largest HFRS outbreak in Croatia. Most of these cases were soldiers. With the exception of the coastal region and islands, all of Croatia was found to be an area endemic for HFRS. A statistically significantly higher proportion of DOBV-infected patients had acute renal failure, visual disturbance, severe thrombocytopenia, and elevated levels of nonsegmented leukocytes, creatine, and total bilirubin. The prevalence of gastrointestinal and electrocardiography disorders also was greater in DOBV-infected patients. Interestingly, significantly more PUUV-infected patients had elevated systolic blood pressure on admission to the hospital. Further prospective studies are necessary to shed more light on differences in HFRS severity associated with PUU and DOB viruses.

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 (i.e., 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 (i.e., 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.

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, alpha2-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.

[Characteristics of immunoreactivity in neonates and young children. Review of the literature]. / Posebnosti imunoreaktivnosti u novorodencadi i male djece. Pregled literature.

Newborns and infants are particularly susceptible to infections, and this appears to be due to deficient immuneresponsiveness. The survey of the pertaining literature (95 references) reveals the particularities of newborns' and infants' immune responses. After the brief survey of basic immunological terms, innate (natural) and acquired (specific), systemic and local (regional) immune responses are described. Characteristics of cellular [leukocytes, polymorphonuclears, neutrophils, eosinophils, mast cells, monocytes, macrophages, natural killer (NK-) cells] and humoral (complement system, fibronectin, C-reactive protein) components of innate immunity are surveyed. Follows the analysis of cellular and humoral participants in specific immune responses: antigen presenting cells (macrophages, dendritic cells), B cells (immunoglobulins), T cells (TCR-1 T cells, TCR-2 T cells). Finally, the characteristics of local immunity are described. The presented overview of the immune responses reveals a partial immune system's immaturity (maturational deficiency) in newborns and infants.

Expression of CD23 antigen and its ligands in children with intrinsic and extrinsic asthma.

The role of the low-affinity IgE receptor CD23 in immune reactions has been further emphasized by recent discoveries of novel surface ligands for CD23: CD21, CD11b, and CD11c. We previously observed the difference between the expression of CD23 and CD21 antigens in children suffering from extrinsic asthma when compared to healthy controls. In the present study, we investigated the expression of CD23 and its ligand CD21 on CD20 B cells in 44 asthmatic children (23 allergic and 21 nonallergic) using three-color immunofluorescence analysis. In addition, the expression of two other ligands for CD23, CD11b, and CD11c, on T cells (CD3+), a subpopulation of T cells (CD4+ and CD8+), natural killer cells (CD56+), and monocytes (CD14+) was tested by two-color immunofluorescence analysis in 12 allergic and 14 nonallergic children. We found that children with extrinsic asthma had higher levels of CD23+ B cells than those with intrinsic asthma. No difference was observed in the percentage of either CD23+CD21+ or CD23-CD21+ B cells. The CD11b antigen was expressed on each tested population, but only on CD4+ T cells was CD11b significantly increased in children with extrinsic asthma. CD11c was expressed mainly on monocytes, and no difference was observed between tested groups. The increased percentage of CD11b antigen on CD4+ T cells and the increased percentage of CD23 antigen on B cells in children with extrinsic asthma provide further evidence of the immunologic differences between intrinsic and extrinsic asthma.

Decrease in CD23+ B lymphocytes and clinical outcome in asthmatic patients receiving specific rush immunotherapy.

Rush immunotherapy (RIT) has been documented as useful in the treatment of patients with allergic bronchial asthma. To investigate the mechanisms of its action, we studied changes in the serum levels of total IgE, allergen-specific IgE and IgG4, and expression of CD23 on peripheral blood B cells in patients receiving RIT. Twenty patients with perennial bronchial asthma were evaluated before the beginning of RIT, as well as 6 weeks and 6 months later. Compared to pretreatment values, the level of Der-p-specific IgG4 and IgE significantly increased after 6 weeks and 6 months of RIT, while the total serum IgE remained unchanged. Furthermore, after 6 months of RIT, the percentage of CD23+B cells and its CD23 receptor density significantly decreased. Since the symptom score improved and the need for medication decreased, we evaluated RIT as a useful procedure. After 6 months, 30% of patients did not have an asthma attack, with no medication in the last month, while 10% of them were asthma free for the last 3 months. No significant correlation between the clinical improvement, and in vitro changes was found. Furthermore, the observed in vitro changes were not significantly different in patients who responded with clinical improvement, compared to those with unchanged intensity of asthma. In conclusion, during specific RIT we found a significant increase in Der-p-specific IgE and IgG4 antibodies, as well as a moderate decrease in CD23+ B cells and its CD23 receptor density. These findings suggest a change in the lymphokine profile of patients receiving specific immunotherapy, and that the inhibition of IL-4-induced B cell stimulation may be hypothesized as the most important mechanism.

Psychological condition hormone levels in war trauma.

Psychological and hormonal responses to various degrees of war-related traumatic experience were analysed in 91 subjects. Their psychological responses (psychosomatic, personality traits, etc.) were evaluated by the COR-NEX2 test. Based on test results, the subjects were classified into three groups: G1 = normal, G2 = moderate, and G3 = severe response. The distribution of subjects in the three groups was related to the intensity and duration of stress that they had been exposed to. Serum levels of cortisol, prolactin, beta-endorphin, thyroxin and triiodothyronine were analysed in all subjects. The levels of cortisol and prolactin were significantly decreased in subjects expressing a severe psychological response, while the level of prolactin correlated with COR-NEX2 test scores. Although relations to other intervening variables are to be investigated, our results indicated that endocrine changes, following trauma, were not random, but rather related to stress-induced psychological responses, and not to trauma per se.

Papers published by members of the Croatian Academy of Medical Sciences, registered in international bibliographic data bases in the period 1986-1990.

Scientific productivity of the CAMS members was analyzed according to the number of papers published in periodicals covered by international Medline and Science Citation Index data bases from 1986 to 1990. Results showed the mean scientific productivity per CAMS member, and the relationship between scientific productivity and age, field of medicine or respective biomedical discipline. The pattern of scientific productivity was also found to change, when analyzed according to Medline or SCI data bases in separate.

Multiple changes of immunologic parameters in prisoners of war. Assessments after release from a camp in Manjaca, Bosnia.

OBJECTIVE: To assess immune reactivity in men just released from a war prisoner camp. PARTICIPANTS: Random sample of 29 men from a group of 764 liberated detainees in war prisoner camp in Bosnia, 15 matched healthy control subjects, and pre-war historical control subjects. MAIN OUTCOME MEASURES: Report on immune reactivity parameters, such as lymphocyte immunophenotypes, natural killer cell and phagocyte function, serum cytokines, and hormones. RESULTS: Compared with control subjects, detainees had significantly lowered red blood cell count, hemoglobin mass concentration, hematocrit, total serum proteins, and albumin level, while the percentage and count of monocytes and non-segmented neutrophils were increased. Flow cytometry revealed a significant increase in percentage of activated lymphocytes, activated T lymphocytes, Tc/s lymphocytes, B lymphocytes, and total HLA-DR lymphocytes. The absolute counts of activated lymphocytes and activated T lymphocytes were also significantly increased. The percentages of naive Th/i lymphocytes and the ratio of CD4:CD8 lymphocytes were decreased. The in vitro natural killer cell cytotoxic activity and phagocytic functions of ingestion and digestion were significantly depressed. Serum interferon, serum cortisol, and prolactin were also significantly lowered. Serum tumor necrosis factor was increased. CONCLUSIONS: Alterations in the main parameters of the immune system and depression of important immune effector functions may have resulted from the psychological stress, physical deprivation, and malnutrition experienced by these war camp prisoners during their detainment.

Characterization and partial purification of the Croatian national standard Dermatophagoides pteronyssinus allergen extract.

Lyophilized Dermatophagoides pteronyssinus (Der p) allergen extract (AE) and partially purified Der p extract (PAE) were prepared and characterized. Partial purification of AE was performed by gel filtration on Sephadex G-100 and Sephacryl S-300. Crossed immunoelectrophoresis (CIE) disclosed the same precipitating lines in AE and PAE preparations. The relative potencies of AE and PAE were determined and compared with the WHO International Standard for Der p by the RAST inhibition method. The potencies were 6.5 x 10(5) IU and 1.5 x 10(6) IU, respectively. Biologic standardization by quantitative skin testing was performed with AE (20 selected patients) and PAE (12 patients). Median Ch was calculated by linear regression analysis (log-log model). One ampoule of AE contained 65,300 BU and 1 ml (vial) of PAE contained 166,000 BU. Der p AE could serve as a croatian national standard for further production of Der p allergenic extracts.

Expression of lymphocytes Fc epsilon RII/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.

Influence of circadian light-dark alternations on macrophages and lymphocytes of CBA mouse.

The influence of circadian 12 h light-12 h dark alternations on CBA mouse macrophages and lymphocytes was determined using tests for macrophage spreading and ingestion ability or flow cytometry immunophenotyping of blood, lymph node, and spleen lymphocytes. The animals were tested every 4 h around the clock. Collected macrophages were incubated in vitro for 3 or 18 h. Monoclonal antibodies permitted detection of T-lymphocytes, suppressor-cytotoxic T-lymphocytes, helper-inducer T-lymphocytes, or B-lymphocytes. Two types of analyses were performed: First, the difference between the same intervals of the 12 h light or dark period was determined. The macrophage ingestion was significantly lower at the beginning and higher at the end of the dark period. We have also found a significant increase in blood T-lymphocytes of helper-inducer T-lymphocyte percentages and of the T helper-inducer: T suppressor-cytotoxic ratio during the dark period. Second, the ultradian variation during the 12 h light or dark period was determined. The variability was significant both for macrophage spreading and ingestion. Multiple significant variations of lymph node, spleen, or blood lymphocyte percentages were also observed. All of these data indicate that daily alteration of the lighting regimen significantly influences mouse peritoneal macrophage functions and various lymphocyte subsets.

Immunological reactivity in psoriatic arthritis.

Immunologic reactivity of patients with psoriatic arthritis and the influence of the activity of psoriatic arthritis on immunologic reactivity were analysed. The group of psoriatic arthritis patients, aged 17 to 65 years, consisted of 36 males and 13 females with confirmed diagnosis. 51 age and sex-matched volunteers served as the control group (32 males and 19 females aged 21 to 62 years). The following parameters of immunologic reactivity were analysed: number of leucocytes, percentage of the lymphocytes in peripheral blood, number and percentage of T lymphocytes with positive CD3, CD4 and CD8, number and percentage of B lymphocytes, number and percentage of B lymphocytes with surface IgG, IgA and IgM, number and percentage of large granular lymphocytes (LGL), lymphocytic transformation by phytohaemagglutinin and phagocytic index. The authors found statistically significant reduction of the number and percentage of CD4 T lymphocytes, number and percentage of B lymphocytes with surface IgG as well as significantly decreased lymphocyte transformation and phagocytic activity of peripheral granulocytes in patients with psoriatic arthritis in comparison to control group.

Function of granulocytes in B-chronic lymphatic leukaemia.

To study the function of granulocytes in patients with B-cell chronic lymphatic leukaemia (B-CLL), granulocytes were separated from peripheral blood of 48 patients (mean age: 69 years) and 35 apparently healthy age-matched volunteers. Spontaneous mobility, ingestion, digestion and antibody dependent cellular cytotoxicity (ADCC) of granulocytes were assessed. Decreased spontaneous mobility was found in granulocytes from patients with B-CLL but between the two groups no detectable differences were encountered in the other parameters tested. No alterations of granulocytes functions were found to be correlated with clinical stages of B-CLL. If granulocytes functions were compared in treated (chlorambucil, steroids) and untreated patients, a significant decrease in digestion was found in treated patients.

Polymorphonuclear leucocyte and natural killer cell functions in mature and premature newborns.

In this study the phagocytic and natural killer cell (NK) functions in 17 premature and 30 mature newborns are compared. The ability of polymorphonuclear phagocytes (PMNs) to ingest, digest and lyse (antibody-dependent cell-mediated cytotoxicity, ADCC) opsonized sheep red blood cells and NK activity were tested. Examinations were performed in cord and venous blood within 6 h or 3-4 days after delivery. Results of examinations were compared with normal values for the group of healthy 4- to 15-year-old children. To assess the influence of the newborn's maturity and age on the tested PMNs and NK functions, the following comparisons were made. (1) Cord vs. peripheral venous blood: only ADCC was higher in peripheral than in cord blood. (2) Mature newborn cells obtained either 6 h or 3-4 days after delivery: ingestion and ADCC were lower and NK activity was higher 3-4 days after delivery. (3) Premature vs. mature newborn cells tested 3-4 days after delivery: ingestion and ADCC were higher while NK activity was lower in premature newborns. (4) Premature newborns tested at 3-4 days vs. mature newborns tested within 6 h after birth: ingestion was lower in the prematures while digestion, ADCC and NK activity were similar. (5) Cells from all newborns tested vs. those of healthy older children: results depend on the interval after birth when newborns were tested. Thus, within the first 6 h after delivery, mature newborns had higher ingestion and ADCC capacity but lower digestion and NK activity. Later, 3-4 days after birth, ingestion, ADCC and NK activity were lower in mature newborns. In the prematures at that interval NK activity was lower. (6) There was a positive correlation between gestational age and NK activity of newborns.