Increased mass levels of certain serine proteases in the stratum corneum in acute eczematous atopic skin.

Acute eczematous atopic dermatitis (AD) is associated with increases in stratum corneum (SC) serine protease activity. The purpose of this study was to examine whether the increased SC protease activities in acute eczematous atopic dermatitis were associated with increased mass levels of SC proteases. Six subjects with healthy skin and six patients with AD each with non-lesional skin or lesional acute eczematous skin had the mass levels of their extractable SC kallikreins (KLK), plasmin and urokinase quantified using Luminex multiplex bead-based assays from SC tape strippings. The mass levels of KLK5 and KLK14 together with urokinase were not elevated in the SC in atopic skin. However, the mass levels of KLK7 and KLK11 together with plasmin were greatly elevated compared with the extracts from the non-lesional and the healthy skin and correlated with the corresponding enzymatic activities.

Pathways of tumour cell killing by activated macrophages: both tumour cell membrane and nucleus can be the primary target.

Plasma membrane and nucleus can be primary targets of tumour cell killing by activated macrophages (AMø). Necrotic-type cytotoxicity with loss of membrane integrity and cytoplasmic swelling was expressed by AMø from normal and from perforin-deficient mice, indicating that perforin was not involved. Incubation with AMø consistently triggered the release of thymidine from prelabelled targets, whereas chromatin condensation and small DNA fragments were only occasionally detected. It is shown by means of Pulsed-Field Gel Electrophoresis that DNA degradation in target cells is a slowly progressing process that may stop at any time, indicating that nuclear-type killing doesnot necessarily lead to the formation of low molecular weight fragments. Neither Fas nor the p55 tumour necrosis factor receptor appear to be involved in signalling nuclear-type killing. Accordingly, AMø do mediate membrane- and nuclear-type killing but the mechanisms differ from those identified in T cell cytotoxicity.

Mitogenic signal transduction in T lymphocytes in microgravity.

The activation by concanavalin A Con A of human peripheral blood lymphocytes (PBLs) in the presence of monocytes as accessory cells was investigated in cultures exposed to microgravity conditions in Spacelab. Activation of T cells was measured as incorporation of [3H]thymidine into DNA, secretion of interleukin-2 (IL-2), and interferon-gamma, and expression of IL-2 receptors. Whereas, as discovered in earlier experiments, the activation of resuspended T cells is strongly inhibited, activation of cells attached to microcarrier beads is more than doubled in microgravity. The results suggest that the depression of the activation in resuspended cells may be attributed to a malfunction of monocytes acting as accessory cells. In fact, although the ultrastructure of resuspended monocytes is not altered in microgravity, the secretion of IL-1 is strongly inhibited. Our data suggest that (1) IL-2 is produced independently of IL-1, (2) IL-1 production is triggered only when monocytes (and lymphocytes?) adhere to microcarriers, (3) the expression of IL-2 receptors depends on IL-1, and (4) provided sufficient IL-1 is available, activation is enhanced in microgravity. Finally, cultures of resuspended PBLs and monocytes in microgravity constitute a complete and natural system in which monocytes are not operational. This may be useful for studies of the role of accessory cells and cell-cell interactions in T lymphocyte activation.

Anti-HIV IgM antibody analysis during early manifestations of HIV infections.

In a prospective study we tested the appearance of IgG and IgM positive viral protein bands in Western blots from six people who seroconverted for anti-HIV antibody. Quantification of the immunoblotted bands was performed by reading the Western blot stripes in Camag scanner and analysed on a 350 computer (digital equipment). In the first serum, all people were negative for anti-HIV antibodies. In the second serum, after 16 to 122 days, all people showed IgM HIV-antibodies to p24. IgG HIV-antibodies were detectable in all people after 18 to 114 days after the second collection. Our data clearly demonstrated that for early analysis of HIV infection only the detection of IgM antibodies to viral protein bands of the Western blot technique provides reliable results and that scanning and advanced integration analysis of the Western blot peaks offer the advantage of direct quantitative comparison of the results, not just qualitative description. Further, this direct quantitative comparison of antibodies to HIV virus protein bands can be used as a prognostic marker for disease states.

Increase of GTP cyclohydrolase I activity in mononuclear blood cells by stimulation: detection of heterozygotes of GTP cyclohydrolase I deficiency.

An assay is described for GTP cyclohydrolase I activity in human mononuclear cells isolated from 20 ml of heparinized blood. The activity of this enzyme was low in unstimulated cells and increased 5-10 times after stimulation by phytohemagglutinin (formation of 0.8-1.3 pmol dihydroneopterin triphosphate/min per mg protein at 37 degrees C, n = 15) or mixed lymphocyte culture. No activity was detected in phytohemagglutinin-stimulated mononuclear cells of a patient with proven GTP cyclohydrolase I deficiency in liver; the samples from the father and mother of the patient showed 30 and 46%, respectively, of the mean of 15 healthy controls. In unstimulated cells, neopterin was the main component of the total intracellular pterins (after oxidation). After stimulation, dihydroneopterin triphosphate, measured as neopterin triphosphate by high performance liquid chromatography, was increased 10-30 times; neopterin and pterin were increased only 2- to 6-fold. Since the immunoreactive cells from this patient were unable to produce pterins and all immunological tests on the patient were normal, it is concluded that neither dihydroneopterin triphosphate, nor one of its metabolites are of primary importance for an immune reaction. The assay described can be used for the detection of heterozygotes of GTP cyclohydrolase I deficiency.

Effect of long-term physical exercise on lymphocyte reactivity: similarity to spaceflight reactions.

The response of critical immunological parameters in seven athletes to the sustained physical stress of marathon running was assessed. Variables analysed were the responsiveness of lymphocytes (measured as mitogenic response to concanavalin A), the numbers of lymphocytes, their subsets, and leukocyte numbers. In addition, blood levels of cortisol, epinephrine, and norepinephrine were determined. After the run, lymphocyte responsiveness was severely depressed to 1-70% of the resting values, even though the lymphocyte counts did not change. Leukocyte counts were elevated 2.8-fold. No dramatic changes were found within the lymphocyte subsets, although an increase in pan T-cells and the helper/inducer subset 2 d after the run was significant. In addition, the numbers of B-cells decreased significantly. No change was observed within the suppressor/cytotoxic subset. Cortisol increased 2.1-fold, epinephrine 3.2-fold and norepinephrine 2.7-fold. All these parameters returned to baseline values within 2 d. These data were compared with data obtained during and after spaceflight. We conclude that prolonged physical stress of marathon running induces changes in immunological responsiveness that are strikingly similar to those arising from the stress of spaceflight.

Stress-compensation by a food supplement based on yeast plasmolysate in mitogen-activated T lymphocytes under simulated low-gravity.

T lymphocyte function is strongly depressed in vitro and in vivo under low-g conditions in space as well as simulated in clinostat. Here we describe the effect of a food supplement based on yeast plasmolysate on T cells activated in vitro with Concanavalin A and cultured in a random positioning machine. The mitotic index was measured by 3H-thymidine incorporation into DNA, the expression of activation markers CD25, CD69 and HLA-DR on the cell surface by cytofluorimetry and the secretion of the IL-2R by an enzyme immunoassay. Our data indicate that the food supplement used is capable to modulate T lymphocyte function. The addition of the food supplement increased the expression of activation markers in activated and non-activated cells. Cultivation under low-gravity conditions reduced the expression of the activation markers, but this expression was partly restored or even increased upon addition of yeast plasmolysate. On the other hand, cell proliferation and secretion of soluble IL-2 receptor was reduced after addition of the food supplement in all samples.

Interim evaluation of two cooperative studies assessing the effects of intravenous immunoglobulin (i.v. IgG) on childhood idiopathic thrombocytopenic purpura (ITP): I. Randomized study comparing i.v. IgG with oral corticosteroids in previously untreated acute ITP. II. Prospective study of i.v. IgG in acute and chronic ITP unresponsive to previous treatment.

Since in a pilot study i.v. IgG was shown to induce a rapid rise of thrombocytes in children with ITP two prospective multicenter ITP studies were started: one comparing i.v. IgG with oral corticosteroids in previously untreated acute ITP, the other investigating the response to i.v. IgG in pretreated acute or chronic ITP in childhood. In this report preliminary results of both studies are summarized. i.v. IgG treatment of acute and chronic ITP is at least as effective as corticosteroid therapy but is not associated with significant side effects. At least some patients with chronic ITP may benefit from i.v. IgG. Longer observation periods are required for further analysis.

Macrophage response to microbial pathogens: modulation of the expression of adhesion, CD14, and MHC class II molecules by viruses, bacteria, protozoa and fungi.

The ability of inactivated viruses, bacteria, protozoa and fungi to modulate the expression of CD14, CD49d, CD49f, CD11a (LFA-1), and CD54 (ICAM-1) molecules in unprimed bone marrow-derived mononuclear phagocytes (BMM phi) was investigated by means of flow cytometry. Incubation with bacterial agents resulted in the large majority of experimental situations in enhanced expression of these macrophage surface molecules. In contrast, viruses and fungi down-regulated the expression of several adhesion receptors, especially integrins. Amplification of MHC class II expression triggered in macrophages by interferon gamma was clearly inhibited by viruses, bacteria, protozoa and fungi. The findings explain earlier results showing that, under the same experimental conditions, bacterial agents are, for the most part, potent stimulators of secretory and cell-mediated macrophage activities while viruses, protozoa and fungi are poor in this respect.

[Immunologic laboratory tests in acquired immunodeficiency syndrome (AIDS) and suspected AIDS]. / Immunologische Laboruntersuchungen bei Patienten mit erworbenem Immunmangel-syndrom (AIDS) und bei AIDS-Verdacht.

Since AIDS-specific laboratory tests are not yet commercially available, laboratory diagnoses of AIDS or of the AIDS-related complex (ARC) are based on "surrogate markers". While single tests are of limited diagnostic value, test combinations are of greater help. However, these tests should be applied restrictively and stepwise. The following parameters were analyzed in respect of their diagnostic and differential-diagnostic value: absolute number of lymphocytes, delayed type hypersensitivity skin tests to seven recall antigens, beta-2-microglobulin, serum-neopterin, C-reactive protein, complement factor B, circulating immune complexes, immunoglobulins, hepatitis B markers, and the ratio of T helper to T suppressor cells. 14 AIDS patients, 11 ARC patients, 23 healthy homosexuals, 6 iv drug users, 6 hemophiliacs and 35 patients with various other disorders were investigated. To analyse the value of a given test or of test combinations in the diagnosis of AIDS and ARC, a discrimination index was introduced and defined as the difference between the percentage of pathological values in one patient group compared to the percentage of pathological values in the other group. A discrimination index of 100 means that a given test is pathologic in all members of one group and negative in all members of the other group. A discrimination index of 60 may mean 80% of pathological values in one group versus 20% in the other. To distinguish AIDS patients from ARC patients the test combination yielding the highest mean discrimination index included serum neopterin, complement factor B and C-reactive protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Mode of action of plasmolysed yeast on lymphocytes under microgravity stress.

A newly developed device to simulate microgravity for space biological investigations under laboratory conditions allowed us to apply a reproducible environmental stress on immunologically active cells. Cell proliferation, soluble IL-2 receptor in the culture supernatant, lymphocyte surface activation markers like CD25 (IL-2R), CD69 and HLA-Dr were the endpoints measured. Untreated donor lymphocyte reactions under microgravity were compared to the same cells treated with an immunomodulator from herbal plasmolysed yeast (Bio-Strath Food Supplement). The main finding is the enhancement of the proliferation inhibition under microgravitational stress by the herbal plasmolysed yeast.

Macrophage response to bacteria and bacterial products: modulation of Fc gamma receptors and secretory and cellular activities.

The ability of bacteria and bacterial products to modulate the expression of Fc gamma receptors and major histocompatibility complex (MHC) class II molecules in resting rat bone marrow-derived mononuclear phagocytes (BMM phi) was determined by means of flow cytometry (FCM). Binding of IgG via Fc gamma receptors was considerably enhanced by most microbial agents; bacterial lipopolysaccharide, lipoteichoic acid and some intact bacteria proved to be as active as interferon-gamma (IFN-gamma) and augmented binding of IgG via high- and low-affinity Fc gamma receptors. In contrast, expression of MHC class II molecules by BMM phi was only slightly affected by the microbial agents. Additional findings attest that resting unprimed rat BMM phi are able to respond directly to Gram-negative and Gram-positive bacteria and to some of their products with the expression of marked secretory [in particular tumour necrosis factor-alpha (TNF-alpha) and nitrite] and cellular activities (TNF-alpha-independent tumoricidal activity). This extensive, direct type of macrophage activation may substantially amplify the capability of these cells to cope with these infectious agents in first-line, non-specific host defence.

Immunopharmacological in vitro effects of Eleutherococcus senticosus extracts.

The objective of these investigations was to further elucidate the immunopharmacological profile of fluid extracts of Eleutherococcus senticosus and to identify the specific role of its characteristic eleutherosides B and E. An ethanolic dry extract of Eleutherococcus senticosus was used as starting material for the isolation of the eleutherosides B and E. Immunopharmacological studies included expression of major histocompatibility complex class I and II molecules by rat bone marrow-derived mononuclear phagocytes, human lymphocyte marker flow cytometry, and in vitro testing of human lymphocyte functions. In contrast to the isolated eleutherosides B and eleutherosides E and the re-mixed eleutherosides B and E, the whole ethanolic fluid extract of Eleutherococcus senticosus was able to induce and enhance interleukin-1 and interleukin-6 but not interleukin-2 production in vitro. The effective concentration of the whole ethanolic extract ranged from 1.0-0.1 mg/ml for the enhancement of interleuking-1 alpha production and 1.0-0.03 mg/ml for the enhancement of interleukin-6 production. It is concluded that the observed enhancing immunopharmacological activities on acute phase response mediators are best exhibited by the induction with whole ethanolic extracts whereas the species-specific and characteristic eleutherosides B and E are not associated with these activities.

Surface phenotype of rat bone marrow-derived mononuclear phagocytes.

Utilizing a panel of currently available monoclonal antibodies, the surface phenotype of a pure population of resting rat bone marrow-derived mononuclear phagocytes (BMM phi) was analyzed by means of flow cytometry. The present work provides an extensive list of surface markers expressed by BMM phi and also outlines advantages and limitations of flow cytometric analysis of this cell type. The results show that the majority of surface markers considered to be expressed selectively by T lymphocytes, such as Thy-1, CD2 and CD5 antigens, leukosialin (W3/13), or an alloantigen of peripheral T cells, are not expressed by BMM phi. On the other hand, the CD8 antigen and the leukocyte common antigen recognized by MRC OX-33, considered to represent specific markers of cytotoxic T cells and/or peripheral B cells, are expressed on a variable, often considerable proportion of BMM phi. Monoclonal antibodies W3/25, MRC OX-35, and MRC OX-38, directed against epitopes on the CD4 molecule, labeled a variable proportion of BMM phi. Among the 39 monoclonal antibodies examined, none appeared to recognize an epitope which is expressed selectively by mononuclear phagocytes.

Coordinate up- and down-modulation of inducible nitric oxide synthase, nitric oxide production, and tumoricidal activity in rat bone-marrow-derived mononuclear phagocytes by lipopolysaccharide and gram-negative bacteria.

Simultaneous incubation of primary rat bone-marrow-derived mononuclear phagocytes (BMMo) and tumor cells with gram-negative agents triggers within 24 h interferon gamma (IFN gamma)- and tumor necrosis factor (TNF alpha)-independent tumoricidal activity. On the other hand, BMMo that had been incubated for 24 h with gram-negative agents prior to re-exposure to the same agent had largely lost their ability to generate tumoricidal activity, although their ability to bind lipopolysaccharide (LPS) was not diminished. Parallel measurements of the kinetics of inducible nitric oxide synthase (iNOS), nitrite secretion, and tumoricidal activity triggered in primary BMMo by LPS revealed that these parameters take a coordinate course, reaching a peak within 24 h and then rapidly decaying. Down-regulation of expression of NOS protein and iNOS activity could be attributed neither to down-regulation of LPS receptors nor to L-arginine depletion.

Mononuclear phagocytes from human bone marrow progenitor cells; morphology, surface phenotype, and functional properties of resting and activated cells.

After 3-4 weeks culture of human bone marrow cells in medium supplemented with IL-3, macrophage- (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), the firmly adherent cells exhibited the morphologic features of mononuclear phagocytes and were strongly esterase-positive. Flow cytometric analysis revealed a rather homogeneous cell population with marked autofluorescence; the large majority of the cells expressed CD14, CD11a, b, and c, Fc receptors for IgG, Fc gamma RI, II, and III, and HLA class II molecules. Interferon-gamma (IFN-gamma), bacteria, and bacterial products modulated expression of some of the surface markers, induced and/or enhanced respiratory burst, phagocytic activity, secretion of tumour necrosis factor, and tumouricidal activity; in contrast, these cells were not able to generate reactive nitrogen intermediates.

[Counting of thrombocytes in a medium-sized clinical routine laboratory. Comparative study of a new electronic counting procedure]. / Thrombozytenzählung im mittelgrossen klinischen Routinelabor. Vergleichende Untersuchung eines neuen elektronischen Zählverfahrens.

Three platelet counting methods, i.e. the phase contrast microscopic method, the Thrombocounter (Coulter) and Thrombocell 1000 (Contraves), have been analyzed and compared. Critical evaluation of the results showed marked superiority of the electronic systems over microscopic counting regarding reproducibility and time requirement. The simplicity of sample preparation, which requires no hematocrit correction, and the availability of a capillary blood method renders the Thrombocell 1000 superior to the Coulter Thrombocounter.

Transitory and persistent IgA deficiency. Reevaluation of 19 pediatric patients once found to be deficient in serum IGA.

In 21 out of 5181 serum samples tested between 1968 and 1977 IgA was not detectable. In this subsequent study, 19 individuals could be reinvestigated by clinical and laboratory methods. At reevaluation 26% showed high, 16% normal and 58% subnormal serum IgA. Late maturation of the IgA system therefore occurs frequently. Lack of secretory IgA was proven in 16% and the material used were tears: even in the group with subnormal IgA clinical manifestations are minimal. Development, compensatory mechanisms and criteria for a new definition of selective IgA deficiency are discussed.

[New possibilities for immunoglobulin substitution in antibody deficiency syndrome]. / Neue Möglichkeiten der Immunglobulin-Ersatztherapie bei Antikörpermangel-Syndrom.

The effect of an unmodified immunoglobulin for intravenous substitution in patients with antibody deficiency syndrome (ADS) has been studied. Over 100 doses of Sandoglobulin were administered to ADS patients without any sign of anaphylactoid reaction. There is no upper limit on the quantity infused. Peak values and basal values can be calculated and therapy can be adjusted accordingly. The halflife of the preparation equals that of native IgG in a healthy person. Comparing days with temperature above 38 degrees C (100.4 degrees F), days under antibiotics, and absence from work during equal periods with and without Sandoglobulin, the beneficial effect of this preparation is obvious. Sandoglobulin can be recommended for longterm therapy in ADS.

[Neuraminidase-producing pneumococci in the pathogenesis of hemolytic-uremic syndrome]. / Neuraminidase-produzierende Pneumokokken in der Pathogenese des hämolytisch-urämischen Syndroms.

Hemolytic-uremic syndrome (HUS) accompanied by pneumococcal infections forms a characteristic subgroup of HUS. Pneumococcal neuraminidase splits off neuraminic acid from the glycoproteins present on the surface of red cells, thrombocytes and endothelial cells, and thus exposes the hidden Thomsen cryptantigen (T-Ag). The T-Ag can then react with a complement-fixing antibody of the IgM class which is present in all human plasmas after the age of 6 months. Early diagnosis of T-transformation should be attempted. Highly suggestive hints are: pneumonia, hemolytic anemia, reticulocytopenia, difficulties in ABO typing, a positive direct Coombs test and a positive minor cross-match. The definite diagnosis of T-transformation is established with the aid of anti-T agglutinins from Arachis hypogaea, the common peanut. Two children aged 19 and 22 months with pneumonia, Coombs-positive hemolytic anemia, HUS and exposure of the T-Ag on the red cell membrane are described. In one of them, circulating neuraminidase and circulating pneumococcal antigen of serotype 3 were found. In both children exchange transfusions resulted in elimination of circulating neuraminidase and of T-transformed red cells prone to hemolysis.