A new mathematical procedure to evaluate peaks in complex chromatograms.

Automatic peak evaluation in chromatograms and subsequent quantification of compound concentrations is still a challenge in the analysis of complex samples containing hundreds or thousands of compounds. Although a number of software packages for peak evaluation exist, baseline definition and overlapping peaks of different shapes are the main reasons which prevent reliable automatic analysis of complex chromatograms. A new mathematical procedure is presented which uses peak shapes extracted from the chromatogram itself and modified by nonlinear (in fact, hyperbolic) stretching of the peak head and tail. With this approach, the peak parameters are position, height, scale of front, scale of tail, and smoothness of transition from front to tail scaling. This approach is found to give a substantially better fit than traditional analytically defined peak shapes. Together with a good peak finding heuristic and nonlinear optimization of parameters this allows a reliable automatic analysis of chromatograms with a large number of peaks, even with large groups of overlapping peaks. The analysis matches the quality of standard interactive methods, but still permits interactive refinement. This approach has been implemented and tested on a large set of data from chromatography of hydrocarbons in ambient air samples.

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo.

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4+CD8lo intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class I.

Clonal deletion of major histocompatibility complex class I-restricted CD4+CD8+ thymocytes in vitro is independent of the CD95 (APO-1/Fas) ligand.

The CD95 (APO-1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca(2+)-independent cytotoxicity of cells from perforin knock-out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double-positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen-specific deletion of double-positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2b-specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV-peptide 33-41 in a major histocompatibility complex-class I-restricted fashion. Deletion was not blocked by soluble mouse and human CD95-Fc receptor decoys. CD95-Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L-transfected L929 cells in sensitive CD95+ target cells and in thymocytes. These results suggest that TCR-induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC-class I-restricted autoreactive thymocytes.

Intrathymic T cell receptor (TcR) targeting in mice lacking CD4 or major histocompatibility complex (MHC) class II: rescue of CD4 T cell lineage without co-engagement of TcR/CD4 by MHC class II.

A critical step during intrathymic T cell development, termed positive selection, is associated with rescue of short-lived, immature thymocytes from programmed cell death, T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TcR)-major histocompatibility complex (MHC) interactions during positive selection can be closely mimicked by targeting TcR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. Here, we show that antibody-mediated TcR signaling in mice deficient for CD4 or MHC class II expression induces polyclonal differentiation of the CD4 T cell lineage. Following a single TcR signal pulse in situ, a temporal sequence of phenotype changes can be discerned: CD69 up-regulation (< 1 day), CD8 down-regulation, TcR up-regulation (1-1.5 days) and down-regulation of the heat-stable antigen (1.5-2 days). Differentiation of phenotypically and functionally mature CD4 T cells in situ is attained within 3 days. Rescue of CD4 lineage T cells in the absence of TcR/CD4 co-engagement by MHC class II in this experimental system supports the stochastic/selective model of T cell lineage commitment.

Half-life of antigen/major histocompatibility complex class II complexes in vivo: intra- and interorgan variations.

We have determined the half-life in vivo of antigen/MHC class II complexes in different organ microenvironments. Mice were "pulsed" with myoglobin intravenously and MHC class II-positive antigen-presenting cell (APC) populations from different organs were isolated after various time intervals. Specific antigen/MHC complexes were quantitated by co-cultivation of the APC subsets with myoglobin-specific T-T hybridoma cells in vitro. Half-lives of antigen/MHC complexes differed both between organs and between compartments of the same organ. Half-lives in peripheral organs (spleen and bone marrow) ranged between 3 and 8 h, whereas in the thymus half-lives between 13 h (cortical epithelial cells) and 22 h (medullary dendritic cells) were observed. Half lives in vivo were independent of antigen processing, since intact protein or antigenic peptides yielded similar values. The considerably longer half-life of peptide/MHC complexes in the thymus as compared to peripheral organs may reflect the distinct role which antigen presentation plays in both organs, i.e. induction of tolerance versus induction of immunity.

The 39-kilodalton outer membrane protein of Proteus mirabilis is an OmpA protein and mitogen for murine B lymphocytes.

Partial amino acid sequence analysis of a major outer membrane protein of Proteus mirabilis (39-kDa protein) indicates that it is an OmpA protein. The mitogenic activities of the 39-kDa protein for murine lymphocytes were also investigated with T lymphocytes isolated by passing spleen cells over columns of nylon wool fiber and B lymphocytes obtained by treating spleen cells with monoclonal antibodies to Thy1 plus complement. The 39-kDa protein showed little activity in stimulating T cells to proliferate but was strongly mitogenic for B cells.

Distinct requirements of positive and negative selection for selecting cell type and CD8 interaction.

We investigated the requirements of positive and negative selection in the thymus for CD8 interaction and the selecting cell type. Thymic epithelial cells are known to mediate positive selection, whereas thymocytes fail to do so. The reason for this failure could be either the low amount of MHC class I molecules on thymocytes or the lack of other properties required for positive selection. To address this question a CD2.Kb transgenic mouse was prepared in which the expression of the Kb gene is under control of the CD2 promoter. In these mice the thymocytes exhibited very high levels of Kb. The mice were crossed with two TCR transgenic mice expressing either the Des.TCR (anti-Kb), which is positively selected on Kk and negatively on Kb, and the 2C.TCR (anti-Ld) with positive selection on Kb and negative on Ld. Despite the high Kb expression on thymocytes in CD2.Kb x 2C.TCR mice no positive selection was observed, whereas efficient negative selection was found in CD2.Kb x Des.TCR F1 mice. Thus, although thymocytes can negatively select, even a strong increase of MHC class I expression cannot convert them into positively selecting cells. This is consistent with the notion that thymic epithelium is specialized for positive selection, but the respective difference between thymocytes and thymic epithelium is not clear. The influence of CD8 was investigated using transgenic mice expressing a Kk/A2 or a Kb/A2 hybrid gene in which the promoter, the alpha 1, alpha 2 domains were of mouse origin and the alpha 3 domain of human origin. Because the murine CD8 molecule does not efficiently bind to human HLA class I, in these mice the CD8 interaction was impaired. In Kb/A2 x 2C.TCR and Kk/A2 x Des.TCR mice no positive selection of the respective TCR was found, whereas in Kb/A2 x Des.TCR mice negative selection was still functional. Altogether, the results indicate that positive selection depends more strictly on CD8 interaction and cell type than negative selection.

T cell receptor targeting to thymic cortical epithelial cells in vivo induces survival, activation and differentiation of immature thymocytes.

We report that targeting of T cell receptors (TcR) to non-major histocompatibility complex (MHC) molecules on thymic cortical epithelial cells by hybrid antibodies in vivo and in fetal thymic organ cultures results in phenotypic and functional differentiation of thymocytes. A single pulse with hybrid antibodies rescues immature, CD4/8 double-positive thymocytes from their programmed death in vivo, induces expression of the early activation antigen CD69 followed by TcR up-regulation, concomitant down-regulation of CD8 or CD4 and their conversion to functional mature T cells by day 3. This temporal sequence of maturation only affects small thymocytes without co-induction of blastogenesis. TcR targeting to MHC class II-positive epithelial cells predominantly induces CD4-positive T cells. This generation of CD4 single-positive T cells occurs also in MHC class II-deficient mice and thus is independent of CD4-MHC class II interactions. Moreover, in the presence of a specific deleting antigen (Mls 1a), TcR targeting results in transient activation of immature thymocytes, however, not in subsequent TcR (V beta 6) up-regulation and development of single-positive T cells. Our findings imply that TcR cross-linking to cortical epithelial cells is sufficient to confer a differentiation signal to immature thymocytes. Furthermore, this approach distinguishes two independent TcR-mediated intrathymic events: activation and subsequent deletion of the same thymocyte subset.

Tolerance induction by clonal deletion of CD4+8+ thymocytes in vitro does not require dedicated antigen-presenting cells.

The cellular requirements of T cell tolerance induction in the thymus by clonal deletion was investigated by using an in vitro assay: thymocytes from mice expressing a transgenic TcR specific for lymphocytic choriomeningitis virus (LCMV) and H-2Db were co-cultured with various H-2b cell types as antigen-presenting cells in the presence of the antigenic LCMV peptide. The results revealed that all cell lines examined including embryonic and transformed fibroblasts, melanoma cells, cortical thymic epithelial cells, lymphomas and neuronal cells induced an antigen dose-dependent deletion of CD4+8+ thymocytes. Similarly, highly enriched accessory cell populations from thymus and spleen (macrophages, dendritic and cortical epithelial cells, i.e. thymic nurse cells) could induce antigen-specific depletion of immature CD4+8+ thymocytes. Depending on the cell type examined micromolar to picomolar concentration of LCMV peptide were required to induce deletion. The effectiveness of deletion by the different cell types did not correlate with their major histocompatibility class I expression level; it was, however, influenced by the presence of ICAM-1 adhesion molecules.

Thymocytes can tolerize thymocytes by clonal deletion in vitro.

Clonal deletion of thymocytes bearing TCR for self antigens is one major mechanism of T cell tolerance induction. Peptide antigen-induced deletion of thymocytes from alpha beta TCR transgenic mice has been studied using single cell suspension cultures. The results show that antigen-presenting immature CD4+CD8+ thymocytes can tolerize antigen-reactive immature thymocytes in vitro by programmed cell death (apoptosis) 6-8 h after antigen exposure. Antigen-induced apoptosis of immature thymocytes was inhibited by antibodies specific for the alpha beta TCR, CD3, CD8, and LFA-1 molecules. This implies that clonal elimination of self-reactive CD4+CD8+ thymocytes does not depend on specialized deleting cell types in the thymus and occurs whenever the TCR of immature thymocytes bind antigen fragments presented by MHC molecules.

[Anti-oncogram-oriented polychemotherapy in metastasizing squamous cell carcinoma of the skin: impressive partial remission]. / Antionkogramm-orientierte Polychemotherapie bei metastasierendem Plattenepithelkarzinom der Haut: eindrucksvolle partielle Remission.

Metastatic squamous cell carcinoma of the skin is associated with a poor prognosis even after surgical treatment or irradiation. Polychemotherapy can be used as an alternative regimen. We present a 65-year-old male patient with extensive metastatic squamous cell carcinoma of the skin after multiple surgical treatment and radiation. A combination of bleomycin, methotrexate and cisplatin, planned according to the anti-oncogram, resulted in rapid partial remission of the cutaneous metastases over several months.

Cell morphology, proliferation and collagen synthesis of human fibroblasts cultured on sepiolite-collagen complexes.

The growth and morphology as well as collagen biosynthesis of human fibroblasts obtained and cultured on sepiolite-collagen complexes have been studied. No differences on cell morphology and growth properties nor collagen synthesis were observed when compared with standard culture substrates. The type I/type III ratio of biosynthesized collagen by fibroblasts cultured on sepiolite-collagen complexes was about 5-6 with no difference when compared to control conditions. This normal behavior was also observed for the type I/type III procollagens. According to these studies the sepiolite-collagen complexes do not modify the studied features of the fibroblasts.

Studies on collagen metabolism in the Marfan syndrome.

The pattern of collagen metabolism was studied in nine fibroblast cultures from Marfan patients. The cellular synthesis of collagen and non-collagenous proteins was significantly increased, whereas secretion and degradation remained unchanged. Other steps of post-translational processing such as hydroxylation of prolyl or lysyl residues, affecting triple helix stability, were found to be normal. Furthermore, peptide mapping of isolated a 1(I), a 2(I) and a 1(III) gave no evidence for structural defects. Hence, our study would support the notion that defects other than those affecting collagen type I or III metabolism must represent the molecular basis of the Marfan syndrome.

[Psychogenic bronchial asthma]. / Zur Psychogenie des Asthma bronchiale.

The extensively discussed problem of psychopathological components in bronchial asthma has been investigated in patients of the authors' hospital. The results are compared with the most frequent opinions published. Using a questionnaire, the intensity of bodily complaints and psychic alterations was compared in following groups: asthmatics, neurotics, and patients with acute lung diseases. Considering bodily complaints, the intensity is comparable in asthmatics and neurotics, and higher than in patients with acute lung diseases. Psychic alterations are more pronounced in neurotics, and there is no difference between asthmatics and patients with acute lung diseases. The importance of these results with regard to the general assessment and the immediate medical care for patients with bronchial asthma is discussed.

Tests for the absorption of 75Se-labelled homocholic acid conjugated with taurine (75Se-HCAT).

The absorption of selenomethionine Se 75-labelled homocholic acid conjugated with taurine (75-SE-HCAT) was tested in 46 patients. Retention measurements using (1) an uncollimated gamma camera and (2) a measuring arrangement similar to a human-body counter were compared in order to obtain a quantitative assessment of the absorption capacity of the terminal ileum for bile acids. The retention curve obtained after the oral administration of the 75Se-labelled bile-acid analogue showed a monoexponential decline; in the case of unimpaired absorption, the half-life was greater than 2.5 days. When more than 30 cm of the ileum had been eliminated by inflammatory infection or resection, the measured half-life was below 0.5 days due to malabsorption. We also performed a quantitative determination of the hepatic secretion of 75-Se-HCAT into the gall bladder. If more than 80% of the activity administered is found in the gall bladder, disturbed absorption of bile acids in the terminal ileum can be excluded. Values smaller than 80%, however, do not provide proof of disturbed absorption.

[The sociodynamogram--a contribution to the objectivization of group process]. / Das Soziodynamogramm--Ein Beitrag zur Objektivierung von Gruppenvorgängen.

Two simple sociometric techniques for practical use are described which are derived from the data given in the sociogram. Compared with the conventional sociogram, the group rank score we use everyday therapy has the advantage that it can be published. It permits members of the group to better identify the social situations which cause specific psychic impulses. The sociogram is reserved for the therapist. In our opinion the sociodynamogram has certain advantages over both methods because it provides an insight into spheres which are inaccessible in the sociogram and group rank score.

The application of radioactive labeled leucocytes for the proof of inflammations.

The methods we have tested are reliable for the labeling of granulocytes and monocytes as well as with colloidal substances and with soluble 99mTc-phytate. Supravital colorations and electronmicroscopic pictures do not show any damage of the cells caused by the labeling procedures. In the clinical application after the reinjection of the labeled granulocytes and monocytes, inflammations in the abdomen and in the bone could be proved and localized exactly. A final evaluation of the described methods and its differentiation from other diagnostic possibilities will only be possible after further investigations.