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.

Age-related changes in functions of peripheral blood phagocytes.

Ingestion, digestion and antibody-dependent cell-mediated cytotoxicity (ADCC) of opsonized sheep red blood cells (SRBC), as effector functions of peripheral blood phagocytes, were studied in newborns, children, mature and aged adults. All tested functions changed non-synchronously during the lifetime. The ingestion was maximal in newborns, digestion in children and ADCC in mature adults. The ingestion was minimal in aged, but digestion was minimal both in newborns and aged. Such changes of phagocytes' functions could possibly contribute to differences in immune reactions of the age-groups studied. The study indicates the need for establishing age-adjusted normal values for major granulocyte and monocyte effector functions.

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.

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.

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

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.

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).

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.

Leucocyte migration inhibition test and tuberculin hypersensitivity. "Escape" and some other factors influencing the clinical use.

Tuberculin hypersensitivity was evaluated in 71 Mantoux-positive and 58 Mantoux-negative persons by the leucocyte migration inhibition test. The capillary tube technique discriminated these two groups, but variability of results and overlapping of the groups were considerable. The shorter the incubation period, the better discrimination between the groups. Only after short incubation (2 h) was the correlation between the intensities of Mantoux reactions and migration inhibitions evident. The observed variability in migration areas, similar in the Mantoux-positives and controls, was the result of technical causes and variations in cells' ability to migrate. Further, in Mantoux-positive persons the migration progression (per min) increased during the first 2 h, but slowed down later on, more in controls than in chambers with antigen. Smaller migration areas in controls (early intervals) tended to produce lower migration indices than the larger ones. Finally, it was possible, using the standard score zeta, to calculate whether each individual result differed significantly from controls. By this procedure the intensity of cell-mediated immunity in individual patients could be estimated.

Dose-response relationship in the migration inhibition test using peritoneal exudate cells and blood leucocytes of tuberculin sensitive guinea pigs.

A comparison of the direct migration inhibition using peritoneal exudate cells and peripheral blood leucocytes from the same tuberculin sensitive guinea pigs was performed. Three antigen concentrations: 3, 15, and 75 microgram of PPD per ml were used. Both type of cells provided similar results except at early incubation intervals when leucocytes, in the presence of lower doses of the antigen, displayed stronger inhibition than peritoneal cells. Thus, in our study, peripheral blood leucocytes are at least equivalent to peritoneal exudate cells in the migration inhibition test.

Particularity of local immunity in the nasopharynx. Parallel study of surface receptors and cell-mediated immune responses in cells derived from palatine or pharyngeal tonsils and blood.

Immunological functions of the pharyngeal tonsil, palatine tonsils and blood leucocytes of children undergoing tonsillectomy were evaluated by determining T or B lymphocytes, the response to mitogens, and the cell-mediated immunological responses to tuberculin. In all the test systems used similar results were obtained with cells derived from either the palatine or pharyngeal tonsils. The mean percentage of T lymphocytes was significantly higher in the peripheral blood than in tonsils, but the reverse was true of B lymphocytes. The reaction to PHA was lower in tonsillar cell culture than in blood cell culture, but tonsillar cells reacted better to Con A than blood cells. In lymphocyte transformation tests tonsillar cells reacted to specific antigen (tuberculin) and this reaction was significantly higher than that of the parallelly tested blood lymphocytes. Further, in about 50% of the children tested, tuberculin caused migration inhibition of the mixture containing tonsillar cells and guinea pig peritoneal cells. Surprisingly, nearly identical results were obtained if migration inhibition test was performed with tonsillar cells alone. Consequently, poorly migrating tonsillar cells are nevertheless usable for direct migration inhibition testing.

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.

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.

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.

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.

Decreased phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) in type-1 diabetes.

Leucocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) were tested in 48 children with type-1 diabetes and in 22 healthy children. Both phagocytosis and ADCC for opsonized 51Cr-erythrocytes significantly decreased in the diabetics. Phagocytosis decreased in well and in poorly balanced diabetics, but the latter, having type-1 diabetes for less than 5 years, exhibited a lower phagocytic capacity than the patients with a longer duration of disease. The decrease of ADCC in poorly balanced patients was greater than in the well balanced ones as compared to the controls. The duration of diabetes was without influence on their leucocytes' ADCC.

Capillary migration of the cells of three murine tumours.

Cells of a murine myeloid leukemia, a lymphoid leukaemia, and a reticulosarcoma, were tested for their ability to migrate from capillary tubes. As compared with normal cells from the speeen, bone marrow, lymph nodes or peritoneal exudate, tumour cells migrated rather poorly. When present, the migration could be attributed to the existence of normal cells in the suspension of tumour cells. No correlation has been found between the migratory behaviour and the mode of growth of the tumour "in vivo".