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.

Beta-cell secretory function and CD25 + lymphocyte subsets in the early stage of type 1 diabetes mellitus.

Cellular immunologic tests have not been used for diagnostic purposes in individuals at risk for autoimmune insulitis or in patients with partial beta-cell destruction because of a lack of studies that show their predictive value. In this study we initially evaluated 43 patients with recent-onset Type 1 diabetes (disease duration

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.

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.

Decision-tree approach to the immunophenotype-based prognosis of the B-cell chronic lymphocytic leukemia.

Use of a nonlinear prediction method, such as machine learning, is a valuable choice in predicting progression rate of disease when applied to the highly variable and correlated biological data such as those in patients with chronic lymphocytic leukemia (CLL). In this work, decision-tree approach to cell phenotype-based prognosis of CLL was adopted. The panel of 33 (32 different phenotypic features and serum concentration of sCD23) parameters was simultaneously presented to the C4.5 decision tree which extracted the most informative of them and subsequently performed classification of CLL patients against the modified Rai staging system. It has been shown that substantial correlation between the percentage of expression of the CD23 molecule on CD19+ B-cells, the level of sCD23, the percentage of CD45RA+, and the absolute number of CD4CD45RA+RO+ T-cells and the clinical stages, exists. The prediction vector, composed of their concatenated values, was able to correctly associate 83% of the cases in the low-risk group (Rai stage 0), 100% of the cases in the intermediate-risk group (Rai stage I and II), and 89% of the cases in the high-risk group (Rai stage III and IV) of CLL patients. Predictivity of this vector was 100%, 95%, and 89%, respectively. In conclusion, from the described analysis, it may be inferred that two processes play important roles in the progression rate of CLL: 1.deregulated function of the CD23 gene in B-cells accompanied by the appearance of its cleaved product sCD23 in the sera; and 2. functionally impaired and imbalanced CD4 T-cell subpopulations found in the peripheral blood of CLL patients.

Prognostic significance of cell cycle parameters in infiltrative ductal breast carcinoma.

Flow-cytometric DNA analysis was performed retrospectively from paraffin-embedded blocks in 158 consecutive ductal infiltrative breast carcinoma patients grades I-III. Normal breast tissue was used as control. Tumor proliferative activity, cell ploidy, and DNA index (DI) were related to age of patients, histological grade of tumor, tumor size, axillary lymph node status, estrogen and progesterone receptor status, menopausal status,TNM clinical classification, and survival. There was a significant association between DNA aneuploidy and a high cellular proliferative activity, increased DI, poor differentiation of tumor, primary tumor size, number of positive lymph nodes, and postmenopausal state. Increased proportion of cells in S-phase was associated with positive lymph node status and higher number of positive lymph nodes. The cell cycle parameters had no prognostic value either for overall survival of disease-free survival of the patients.

Phenotypic analysis of receptor-ligand pairs on B-cells in B-chronic lymphocytic leukemia.

Whole-blood three-color immunofluorescence analysis was used to investigate the role of CD5/CD72 and CD21/CD23 receptor-ligand pair formation on B-chronic lymphocytic leukemia (B-CLL) cells as well as sCD23 and bcl-2 oncoprotein expression in disease progression and activity and total tumor mass in B-cell chronic leukemia (B-CLL) patients. Thirty-four patients with B-CLL and 19 controls were included in the study. The majority of B-cells in B-CLL patients coexpressed CD5 and CD72 as well as the CD23 antigen. Unlike B-cells in B-CLL patients, B-cells in all healthy controls tested had high expression of CD21 antigen. We identified two groups of B-CLL patients according to high (n = 20) or low levels (n = 14) of CD21 expression on CD19+CD23+ B-cells. Only in the patients with high CD21 expression, were sCD23 levels positively correlated with factors known to have prognostic significance in B-CLL (Rai stage and TTM) and could, therefore, be used as a prognostic parameter for these B-CLL patients. Bcl-2 oncoprotein expression did not differ between these patient groups. We presumed that in patients with a lower expression of CD21 antigen, the contribution of the CD21 molecule to homotypic adhesion was lacking. Further studies are necessary to determine the possible association of higher expression of the CD21 antigen with disease progression and the aggressive character of the B-CLL.

Prognostic parameters for survival of patients with malignant mesenchymal tumors of the uterus.

BACKGROUND: 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. METHODS: Flow cytometric cell cycle analysis (proliferative activity, DNA ploidy, and DNA index) was performed on archival paraffin embedded blocks from 80 patients with malignant mesenchymal uterine neoplasms (endometrial stromal sarcomas, malignant smooth muscle tumors, and malignant Müllerian mixed tumors). The Cox proportional hazards regression model was used to assess relative effects of the following factors on patient survival: clinical stage, mode of therapy, DNA+proliferative activity, DNA index, histologic type, cellularity, degree of atypia, mitotic activity, and depth of myometrial invasion. RESULTS: There were 9 low grade stromal sarcomas, 17 high grade stromal sarcomas, 8 smooth muscle neoplasms with uncertain malignant potential, 23 leiomyosarcomas, and 16 homologous and 7 heterologous malignant Müllerian mixed tumors. In univariate analysis for stromal sarcomas, statistical significance was found for DNA ploidy+proliferative activity (P < 0.001), histologic type (P = 0.005), and DNA index (P < 0.001). In multivariate analysis, DNA index appeared to be the only significant parameter influencing patient survival (P = 0.005). In univariate analysis for malignant smooth muscle neoplasms, statistical significance was detected for mitotic activity (P = 0.049) and International Federation of Gynecology and Obstetrics classification (P = 0.021), but in multivariate analysis, clinical stage appeared to be the only significant parameter influencing patient survival (P = 0.032). In univariate analysis for malignant Müllerian mixed tumors, statistical significance was found for the depth of myometrial invasion (P = 0.039), DNA index (P = 0.037), and clinical stage (P = 0.013), but in multivariate analysis, only the depth of myometrial invasion (P = 0.036) and clinical stage (P = 0.025) were of statistical significance. CONCLUSIONS: The most powerful prognostic indicator for stromal sarcomas was the DNA index, for malignant smooth muscle neoplasms it was the clinical stage, and for malignant Müllerian mixed tumors it was the depth of myometrial invasion.

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.

Antibody-dependent cellular cytotoxicity, ingestion and digestion in Dermatophagoides pteronyssinus-sensitive asthmatic children.

Dermatophagoides pteronyssinus (DP) is, for unknown reasons, the commonest cause of asthma attacks in children suffering from reaginic bronchial asthma. The underlying immune disorder is also unclear. The authors analyzed phagocytosis (ingestion), digestion and antibody-dependent cellular cytotoxicity (ADCC) of peripheral blood leukocytes in 20 asthmatic children hypersensitive to DP, aged 2 to 14 years. The tests were performed while the children were entirely asymptomatic and under no therapy. The aim was to determine the possible difference in comparison to healthy children and to assess the correlation of these results with the total serum IgE level, DP-specific IgE and duration of the disease. Ingestion in asthmatics did not differ significantly from that in controls, while digestion and ADCC were significantly (P < 0.01) lower in asthmatics. This phenomenon could contribute to their difficulties in the elimination of allergens, immune complexes and microbial, particularly viral antigens, making them more susceptible to allergic reaction and infections. No significant correlation to the total serum IgE level, DP-specific IgE and duration of the disease was found.

Significance of aneuploidy in non-Hodgkin lymphomas.

The incidence of aneuploidy was determined by flow cytometric analysis in paraffin-embedded tumor samples of 125 patients with non-Hodgkin lymphoma (NHL). There were 48 low-grade (LG) and 77 high-grade (HG) tumors. Aneuploidy was found in 34 (27%) NHL, 15 (31%) LG, and 19 (25%) HG tumors. The analysis of patient survival showed a significantly better survival of patients with LG than HG NHL, but the presence of aneuploidy did not influence the survival on the level of all patients, patients with LG or patients with HG NHL. In patients with LG NHL a tendency of diploid vs. aneuploid patients to survive longer was observed, but only at P = 0.056 significance level.

Tumor cell cycle in patients with stage I endometrial carcinoma.

Flow cytometric cell cycle analysis was performed on paraffin-embedded blocks from 49 patients with stage I endometrial carcinoma. Care was taken to separate tumor tissue from normal tissue in each specimen; normal tissue was used as a control for each individual specimen. DNA index, proliferative activity, and cell DNA aneuploidy were correlated with known parameters of tumor malignancy. Increased DNA index corresponded well with the DNA aneuploid tumors, poor tumor differentiation (G3), myometrial invasion of more than one-third, more malignant histologic type of tumor, and low concentration of estrogen (< or = 10 fmole/mg) and progesterone (< or = 25 fmole/mg) receptors. Similar results were obtained for tumor cell proliferative activity (percentage of cells in S + G2/M phases) and for DNA aneuploid tumors. Since more than 90% of patients with stage I endometrial carcinoma survived the 5-year postoperation period, analyzed parameters could not be checked for survival-related prognostic significance. However, our data indicate that cell cycle analysis may be instrumental for objective ranking of several known prognostic parameters.

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.

T-cell subsets in asthmatic children.

Using monoclonal antibodies authors determined the percentage of CD2(+)-, CD4(+)- and CD8(+)-lymphocytes in the peripheral blood of 45 asthmatic children. In 38 subjects asthma was reaginic, while in 7 it was nonreaginic. The aim of the study was to detect an abnormality which might contribute to the pathogenesis of the disease. A correlation test was done to assess the association of the above-mentioned percentages with the serum IgE level and the duration of the illness. The percentage of CD2(+)-lymphocytes and CD4+ lymphocytes does not differ significantly among the patient groups themselves nor in relation to healthy children. The percentage of CD8(+)-lymphocytes in asthmatic children, particularly in the group with reaginic asthma, is significantly lower (P < 0.01) than in healthy children. A significant negative correlation (P < 0.05) was found between the percentage of CD8(+)-cells and the serum IgE level in asthmatics. There was no significant correlation between the CD2(+)-lymphocyte and CD(4+)-lymphocyte percentages and the serum IgE level, nor between any of examined percentages and the duration of the illness. The authors conclude that, in children with reaginic asthma, a primarily lower percentage of CD8(+)-lymphocytes, which bear a suppressor function, may be one of the causes for over-production of IgE and is therefore an important factor in the pathogenesis of the illness.

[Phagocytic activity in rheumatoid arthritis]. / Fagocitna aktivnost u reumatoidnom artritisu.

The phagocytic activity of leukocytes was studied in 41 patients with active rheumatoid arthritis, 24 of whom were treated with gold compounds and the remaining 17 with non-steroidal anti-inflammatory drugs. The control group comprised 137 patients with no signs of inflammatory rheumatic diseases. The mean age of patients was 54.8 years (range: 32-75) and that of the controls, 53.5 years (range: 32-75). Spontaneous mobility (P < 0.001), phagocytic activity (P < 0.001) and antibody-dependent cytotoxicity (P < 0.001) of leukocytes were significantly lower in patients with rheumatoid arthritis compared to the control group. No significant differences were observed between patients treated with gold compounds and those receiving nonsteroidal anti-inflammatory drugs with respect to mean spontaneous mobility (U = 198, P = 0.44), phagocytic activity (U = 185, P = 0.31) and antibody-dependent cytotoxicity (U = 183, P = 0.29) of leukocytes. Among patients treated with gold compounds phagocytic activity was significantly lower in those receiving TauredonR compared to those receiving AuropanR (U = 33.5, P = 0.05). The antibody-dependent cytotoxicity was comparable in patients receiving Tauredon and Auropan, respectively (U = 58.5, P = 0.48).

[Reference values in immunodiagnosis: the approach using the Senieur protocol]. / Referentne vrijednosti u imunodijagnostici: pristup prema Senieur protokolu.

In an attempt to standardize the normal values for immunological tests it is necessary to establish strict admission criteria. The EURAGE Concerted Action Programme on Ageing of the European Community described the SENIEUR protocol with criteria for immunological studies in man, based on clinical and laboratory information. Our results on humoral and cellular components of immunity in dependence of age, according to SENIEUR protocol admission criteria are presented.

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.

T-lymphocyte subpopulations in children's atopic asthma.

A group of children with atopic asthma (42) and the control group of normal children (30) were examined in order to determine the level of suppressor and helper T lymphocyte subpopulations in each group by using monoclonal antibodies. The asthmatic children were divided into two subgroups: one group received no therapy (30) and the other was treated with specific hyposensitization (12). Suppressor T lymphocytes were significantly lower in the subgroup of non-treated asthmatic children (p less than 0.01) and significantly higher (p less than 0.01) in the subgroup of children treated with immunotherapy. The stimulation index using mitogens was higher in the group of asthmatic children. The results suggest that the reduction and functional damage of suppressor T lymphocytes may have an influence on the pathogenesis of asthma and that immunotherapy may be beneficial in the treatment of atopic asthma in children.