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.

Modulation of lipopolysaccharide-induced production of cytokines by methionine-enkephalin.

In the present study, we have examined the effect of opioid peptide methionine enkephalin (MENK) on production of factors with interleukin-1 (IL-1) and tumor necrosis factor (TNF) activity by mouse peritoneal macrophages and assessed whether modification in the production of those cytokines could be related to alteration of phagocytosis by MENK. None of the MENK concentrations examined altered IL-1 or TNF activity alone. However, peritoneal macrophages co-stimulated with 1 microgram of lipopolysaccharide (LPS) and 10(-10) M MENK potentiated IL-1 activity, compared to LPS alone, but abrogated TNF activity induced by LPS. While MENK alone slightly decreased phagocytosis of sheep red blood cells (SRBC) by mouse peritoneal macrophages, cells simultaneously incubated with 1 microgram of LPS and 10(-10) M MENK had increased phagocytosis compared to LPS alone. Moreover, phagocytosis of SRBC by cells incubated overnight with the supernatant of the respective cell culture was significantly augmented. These results provide additional evidence for the immunoregulatory role of neuropeptides and suggest that the modulatory action of MENK could be mediated, at least in part, through the up-regulation of cytokines, most probably IL-1 and TNF.

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.

Met-enkephalin modulates stress-induced alterations of the immune response in mice.

Overnight restraint stress of mice decreased ConA-driven lymphocyte proliferation, plaque-forming cell response to sheep red blood cells (SRBC), and NK activity in the spleen, but the phagocytic activity was enhanced. Injection of methionine-enkephalin (MENK), 10 mg/kg, i.p., 30 min before restraint, abolished these changes (except for the NK activity) and attenuated the stress-induced elevation of glucocorticoids. However, MENK itself affected the immune responses like stress: It decreased NK activity and the PFC response and enhanced phagocytic activity. Contrary to results with stress, MENK had no effect on cell proliferation. The opioid-receptor antagonist naloxone given before restraint reversed the stress-induced enhancement of phagocytosis and the decrease of T-cell proliferation. Alterations of the immune responses induced by restraint stress seem to be mediated by at least two mechanisms: activation of the hypothalamus-pituitary-adrenal (HPA) axis and the secretion of opioid peptides. MENK injected before stress may interfere with either or both mechanisms. T or B lymphocytes seem to be affected by the activation of the HPA axis, and phagocytes by a direct opioid action, whereas NK cells seem to be under the influence of another control mechanism.

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.

In vitro suppression of macrophage spreading caused by supernatants of tumour, thymus, and lymph node cells.

Peritoneal macrophages washed out from C3Hf/Bu mice were cultivated in medium 199 supplemented with 35% foetal calf serum. If the pH of the medium was adjusted to 7.2, there were 70% spread macrophages after six hours of incubation at 37 degrees C. We investigated the effect of tumour cells on macrophage spreading. Supernatants from cell cultures of a fibro-sarcoma of C57BL mice and lymphoma of C3Hf/Bu mice were used to determine the number of spread macrophages after 6 hours of culture in these supernatants. Supernatants from cell cultures of the kidney, thymus and lymph nodes of normal C57BL or C3Hf/Bu mice and peritoneal cells of normal C3Hf/Bu mice in medium 199 alone served as control. We observed that supernatants from both allogeneic and syngeneic tumour cell cultures reduced significantly the percentage of spread macrophages in comparison with the percentage of spread macrophages found in the kidney culture supernatants. However, a similar spreading inhibition was caused also by supernatants from the thymus and lymph node cell cultures. In these experiments we confirmed the earlier observations that tumour cells can suppress the activation of macrophages, and we also pointed to the possibility that this effect is not specific only for malignant tissues.

Phagocytic function in patients with recurrent urinary tract infections.

Phagocytes play an essential role in the host's defence against uropathogenic bacteria which are mostly extracellular pathogens. The functional capacity of peripheral blood phagocytes (predominantly polymorphonuclears, PMN) was investigated in 47 patients (urethrocystitis, acute or chronic pyelonephritis) and in healthy persons. The random mobility of phagocytes was determined by measuring their spontaneous migration from a capillary tube. Using radioactively labelled sheep erythrocytes as targets and the phagocytes as effector cells, ingestion, digestion and extracellular cytotoxicity were determined. All the four phagocytic functions in patients were significantly lower than in healthy controls, especially in patients with chronic pyelonephritis. These results link reduced phagocytosis by blood phagocytes with recurrent urinary tract infections (UTI). Whether the defect is primary or secondary to infection (and only transient) should be the object of further studies.

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.

Allergen-induced CD23 on CD4+ T lymphocytes and CD21 on B lymphocytes in patients with allergic asthma: evidence and regulation.

Interaction of CD4+ T cells and B cells is necessary for IgE production. It has been recently demonstrated that cell surface antigen CD21 is a ligand for CD23 (Fc epsilon RII) and that the pairing of these molecules may participate in the control of IgE production. In this study we investigated the effect of the Dermatophagoides pteronyssinus (Dpt) allergen and recombinant interleukin(rIL)-4 on the expression of CD21 and CD23 on T and B cells of asthmatic patients allergic to Dpt and of healthy controls. Peripheral blood mononuclear cells (PBMC) were incubated alone or with Dpt allergen (100 biological units/ml) and/or rIL-4 (100 U/ml) for up to 7 days. The flow-cytometric analysis of double-fluorescence staining revealed that Dpt allergen and/or rIL-4 induced CD23 on CD4+ T lymphocytes only in allergic patients. The allergen-induced CD23 on T cells is de novo synthesized antigen since no induction of CD23 on T cells was observed in cultures with 0.4 microgram/ml actinomycin D. Moreover, 100 U/ml of interferon-gamma inhibited the induction of CD23 on CD4+ T cells. T cells obtained from healthy donors did not express CD23 or CD21 antigen upon incubation with allergen and/or rIL-4. Although rIL-4 also induced CD23 in controls, the expression was only observed on CD20+ cells. The allergen alone induced a significant elevation of the mean fluorescence intensity of both CD21 and CD23 only in allergic individuals. When the cell proliferation was analyzed, a slightly increased stimulation index upon cultivation of PBMC was obtained from non-allergic donors as well, but less than in allergic patients. The co-expression of major histocompatibility complex class II molecules and CD23 on CD4+ T lymphocytes in allergic patients, as assessed by the three-color immunofluorescence analysis, indicates that these cells were activated. We conclude that CD4+ T lymphocytes possess a unique capability to express CD23 upon exposure to allergen. Moreover, the allergen-mediated induction of CD23 on T cells observed only in allergic patients may be the reason for the increase of IgE production. This would not occur in non-allergic individuals as there is no CD23 expression on T cells.

Detection of cellular immunity to tumor-associated antigen(s) in mice by the macrophage spreading inhibition test.

Soluble preparations of antigens from a methylcholanthrene-induced fibrosarcoma of C57BI mice were prepared by homogenization of tumor tissue and high-speed centrifugation of the homogenate. These preparations were able to sensitize syngeneic mice to tumor-associated antigens (TAA) of the fibrosarcoma, and to provoke a delayed hypersensitivity reaction when injected into the footpad of sensitized mice. Furthermore, the same soluble preparations could inhibit in vitro the spreading of peritoneal macrophages from mice sensitized to TAA. A similar inhibition of macrophage spreading was obtained when peritoneal cells from C57BI mice, bearing transplants of the fibrosarcoma, were incubated with the preparations. We conclude that the macrophage spreading inhibition test, like other in vitro assays, can detect cell-mediated immunity to tumor-associated antigens.

A characterization of the in vivo immunomodulation by Met-enkephalin in mice.

Single intraperitoneal injections of Met-enkephalin (MENK) into CBA mice decreased the phagocytic activity of peritoneal macrophages, Con-A-induced proliferation and NK-activity of spleen cells. Conversely, in mice which had been immunized with sheep erythrocytes, treatment with MENK was associated with enhancement of phagocytosis, and no effect on lymphoid proliferation and NK-cytotoxicity of spleen cells. MENK-induced inhibition of cellular functions in nonimmunized mice was associated with a decrease of plasma ACTH level, whereas MENK-induced stimulation of phagocytosis in immunized mice was paralleled with an elevation of ACTH, suggesting a role of corticoids in immunomodulation by MENK. MENK-induced modulation (suppression and stimulation) of phagocytosis, as well as inhibition of spleen cell proliferation, was not observable in adrenalectomized mice, although a reduced NK-cytotoxicity was still present.

CD21-CD23 ligand pair expression in children with allergic asthma.

The CD23 antigen, a low affinity receptor for IgE, was recently shown to interact with another ligand, CD21, and the pairing of these molecules is important in T cell-B cell interaction and control of IgE production. Here, we analysed the expression of CD21 and CD23 on CD4+ and CD20+ lymphocytes in 25 allergic children and 12 age-matched non-allergic controls. Both the percentage (P < 0.01) and the absolute number (P < 0.001) of CD23+ cells were increased in allergic children. There was no difference of CD21+ cells. Double positive CD4+ CD23+ cells (2.5%) were only detected in one patient, in others all CD23 being expressed on B cells. The CD21 antigen was expressed only on B cells. Furthermore, allergic children had an increased mean fluorescence intensity of both the CD21 (P < 0.001) and the CD23 (P < 0.001) receptor. To analyse the possible difference in B cell subsets expressing CD21 and CD23 antigens, three-colour fluorescence analysis was performed. In allergic children the subset of CD20+ CD21- cells expressed more CD23 than in controls (P < 0.001). These results may mean an impaired expression and possibly regulation of CD21-CD23 interaction in allergic conditions.

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.

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.

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.

Defective phagocytosis in chronic trichophytosis.

Chronic trichophytosis as a primary clinical entity (primary chronic trichophytosis, PCT) is increasingly encountered in the literature. Its prevalent cause in Croatia is the anthropophilic form of Trichophyton interdigitale. Chronicity of the disease may result from immunological defects of the patients. Thus, in 62 patients with PCT: skin testing, enumeration of T- and B-lymphocytes in the peripheral blood, quantitation of immunoglobulin classes, and phagocytosis (random mobility, ingestion, digestion and extracellular killing) by peripheral blood leukocytes was tested. The findings were compared to those in healthy persons. Defective phagocytosis (random mobility, ingestion and digestion) was found in patients with PCT (P < 0.001). All the other results were within the control range. Therefore, PCT seems to be associated with defective phagocytosis of peripheral blood leukocytes in affected persons.