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Biomedical subjects

G Wick

Publications and source records attributed to G Wick.

At least 127 records · Page 7Linked to original sources
PubMed1995

[Fragments of human chorionic gonadotropin (hCG): diagnosis of pregnancy and tumors].

The pregnancy and tumor marker human chorionic gonadotropin (hCG) belongs to the family of the glycoprotein hormones. Information on epitope forming sequences of hCG and its subunits hCG alpha and hcg beta has significant impact on the examination of intra- and extracellular metabolism and the standardization of diagnostic assay systems. Variants of hCG appear in biological fluids with variable modifications on different parts of the molecule. These changes may influence the binding patterns of monoclonal antibodies (MCA), thereby causing erroneous results in hCG immunoassays. The aim of the present work was to investigate the influence of peptide bond cleavages and the loss of certain segments of the molecule, which were induced by proteases on the expression of the seven hCG alpha-(alpha 1-alpha 7), nine hCG beta- (beta 1-beta 9) and four hCG beta-core-fragment-epitopes (beta 10-beta 13), previously identified by us [1-10]. To this end, we digested hCG alpha and hCG beta with chymotrypsin. Hormone fragments were separated by high performance liquid chromatography (HPLC) and subsequently immunochemically examined by direct binding radioimmunoassay (DB-RIA), competitive RIA and immunoenzymometric assays (IEMA). Fractions containing hCG-like immunoreactivity were sequenced by Edman and carboxypeptidase-Y degradation. It appeared that: (I) Amino acids (AA) alpha 41-47 and the peptide bonds between AA alpha 40/41, alpha 47/48 and alpha 29/30 do not influence the expression of the 7 alpha-epitopes, (II) The absence of the hCG beta N-terminus plays a crucial role for the formation of epitopes beta 10 and beta 13. (III) Neither the presence nor the absence of the C-terminal peptide of hCG beta (hCG beta CTP, AA beta 114-145) has any importance for the expression of epitopes beta 1-beta 7 and beta 10-beta 13 (IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers, Tumor↗
PubMed1994

A novel and sensitive method for the detection of secreted cell products using time-resolved fluorescence.

A new test has been developed for the quantitative detection of products secreted from isolated cells, based on the use of lanthanide- rather than enzyme-linked streptavidin. Used as a label, europium (Eu3+) can be measured with high sensitivity by time-resolved fluorescence. The main advantages of this assay are both an increased sensitivity and measuring range of cell released substances, when compared to the standard "wet" ELISA. Thus, the immunoglobulin secretion rate of 10(5) splenocytes could be easily measured by time-resolved fluoroimmunoassay (TR-FIA), while it remained below the detection limit of the 'wet' ELISA. In contrast to the classical ELISPOT test, this assay does not detect single antibody secreting cells (ASC), but would be useful for precise quantification of secreted cell products, such as immunoglobulins, cytokines, growth factors.

Animals↗
PubMed1994

Ultrastructural analysis of thymic nurse cell epithelium.

Thymic nurse cells (TNC), a paradigmatic cell type of cortical epithelium, are large lymphoid-epithelial cell complexes of thymocytes enclosed within vacuoles lined by the epithelial cell membrane. TNC express major histocompatibility complex (MHC) class I and class II molecules on their surface and vacuole-lining membranes at high density and it was suggested that TNC provide an optimal microenvironment for positive selection of T cells. In this report we present electron microscopical data demonstrating that chicken TNC display morphological structures of exocytosis previously shown for hormone-secreting cells. In TNC, however, exocytosis is restricted to the capillary cleft between the epithelial cell and engulfed thymocytes. Thus, besides physical contact between the epithelial cell and enclosed thymocytes, TNC may additionally influence the development of thymocytes through release of soluble factors in a restricted microenvironment. By employing the 3-(2,4-dinitroanilino)-3'-amino-N-methyl-propylamine technique which at the ultrastructural level detects acidic organelles involved in processing of antigens presented by MHC class II molecules, we also show that TNC contain acidic compartments similar to classical antigen-presenting cells, i.e. early and late endosomes and lysosomes, albeit in a lower amount than in thymic dendritic cells. This fact provides evidence that TNC not only are capable of antigen presentation but also possess the intracellular machinery for antigen processing.

Animals↗
PubMed1994

Thymocyte apoptosis induced by elevated endogenous corticosterone levels.

A well-known model of apoptosis is induction in thymocytes by injection of pharmacological doses of exogenous steroids. The aim of this study was to investigate whether this process also occurs under physiological conditions, i.e. by stimulation of endogenous glucocorticoid release, using the chicken as an experimental model. Endogenous glucocorticoid levels can be elevated by immunization with exogenous antigens or by injection of conditioned medium, e.g. supernatant of mitogen-stimulated spleen cells. This effect is mediated by so-called glucocorticoid-increasing factors, and is considered to act as an immunoregulatory principle. Thymocyte DNA of so treated birds showed a typical "ladder" pattern after electrophoresis in a 1.8% agarose gel, and degradation could be prevented by RU 38,486. This provides evidence that apoptosis can be induced by elevating endogenous corticosterone levels in vivo. By means of in situ nick translation (ISNT) and simultaneous immunofluorescence tests, it was possible to analyze various thymic subpopulations during apoptosis after treatment with exogenous glucocorticoids. Additionally, using confocal microscopical techniques, apoptosis of the same cells as analyzed by ISNT is shown. The possible role of elevated concentrations of endogenous glucocorticoids in regulating thymocyte cell death and autoimmune diseases is discussed.

Animals↗
PubMed1994

A single injection of Staphylococcus aureus enterotoxin B reduces autoimmunity in MRL/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice carry a mutation in the Fas gene whose product is involved in the regulation of lymphocyte apoptosis. This mutation is associated with the lpr phenomenon, i.e., a massive expansion of phenotypically abnormal CD4-CD8- cells ("double negative," DN) alpha/beta T cells (lpr cells) that becomes manifest at 3-4 months of age. As in normal mice, intravenous SEB injection into 2- or 6-month-old female MRL/lpr mice causes a transient expansion of SEB-reactive V beta 8+ T cells, followed by a deletion of this subset. In contrast, in the same animals, the frequency of abnormal V beta 8+CD4-CD8- cells is not modulated by SEB. Whereas DN T cells are completely resistant to SEB-mediated deletion in vivo, their precursors appear susceptible to SEB-induced deletion. Thus, a single injection of SEB prior to the surge of DN T cells in peripheral lymphoid organs, at 2 months of age, is sufficient to cause a stable long-term (6 months) deletion of DN cells. This is accompanied by a significant amelioration of autoimmune parameters (autoantibody titers, incidence of arthritis and nephritis), thus pointing to the feasibility of employing superantigens for simple manipulations of the immune repertoire that result in the long-term prophylaxis of autoimmune diseases.

Animals↗
PubMed1994

Genetically determined target organ susceptibility in the pathogenesis of spontaneous autoimmune thyroiditis: aberrant expression of MHC-class II antigens and the possible role of virus.

Considerable controversy exists concerning the role of aberrant MHC-class II antigen expression in the pathogenesis of organ-specific auto-immune disease. Since Obese strain (OS) chickens are afflicted with a spontaneously occurring autoimmune thyroiditis (SAT), we have readdressed this pivotal question by investigating the chronical appearance of MHC-class II antigens on thyroid epithelial cells (TEC) of OS and normal healthy CB chickens before onset of overt clinical symptoms in the former. Among the candidates as potent inducers of aberrant MHC-class II antigen expression, interest in our studies focussed on the potential role of viruses in the development of SAT. Since aberrant MHC-class II antigen expression could prove to be an epiphenomenon of virally afflicted TEC, we determined 2,5-oligoadenylate synthetase and 2,5-oligoadenylatepolymer cytosol levels in both chicken lines. Our results indicate that the presence of infiltrating lymphocytes does not necessarily represent a prerequisite for the aberrant expression of MHC-class II antigens but coincides in most cases. However, the phenomenon seems to play a perpetuating rather than a causative role. Moreover, in support of a possible viral involvement, elevated levels of the 2,5-oligoadenylate synthetase and 2,5-oligomers could be demonstrated in TEC cytosol of OS chickens.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Interleukin-1 receptor deficiency in brains from NZB and (NZB/NZW)F1 autoimmune mice.

Interleukin-1 receptors (IL-1R) are expressed in the brain and the anterior pituitary of normal mice (C3H/He, Swiss), and appear to be involved in the neuroendocrine control of the immune response. Here we have studied the IL-1R density in the brain and the pituitary from several strains of autoimmune mice (NZB, (NZB/NZW)F1, MRL/MP-lpr), using quantitative autoradiography with recombinant human [125I]IL-1 alpha as a ligand. IL-1R was similar in the brain of C3H/He, Swiss and NZW (controls) and MRL/MP-lpr mice. In NZB mice a profound deficit (10% of control mice) in IL-1R was observed exclusively in the dentate gyrus. In (NZB/NZW)F1 the deficit was about 50%. These observations were independent of sex and age. Pituitary receptors were not affected in all the strains except NZW (30% increase). Competition experiments demonstrated that the affinity of IL-1R was not modified in dentate gyrus of (NZB/NZW)F1 and NZW mice. Thus, the number of IL-1R was the only parameter affected. This deficit was not reversed by corticosterone treatment (0.2 mg/20 g body weight, i.p.) and was poorly modified by lipopolysaccharide treatment (0.1 mg/20 g body weight, i.p.) compared to C3H/He mice. In conclusion, this central IL-1R deficit is unlikely to be the consequence of occupancy by abnormal synthesis of brain IL-1. This abnormality is tissue-specific with hereditary autosomal transmission. The role of central IL-1R in neuroimmunoendocrine interactions and in autoimmunity remains to be clarified.

Age Factors↗
PubMed1994

The suitability of human chorionic gonadotropin (hCG)-based birth-control vaccines.

It has been widely hoped that immunological methods of fertility regulation by active immunization against specific antigens of the oocyte, sperm, zygote and early embryo, and the placental pregnancy hormone human chorionic gonadotropin (hCG), will provide a means to control the problem of worldwide population growth. The most advanced candidate vaccines are based on hCG immunogens and have entered clinical trials. However, during the past few years, increasing evidence has emerged that the current approaches using hCG as the target molecule may have some major drawbacks. On the basis of their recent findings, Stephan Dirnhofer and colleagues raise doubts on the suitability, safety and efficacy of gonadotropin-based immunological contraceptive vaccines.

Chorionic Gonadotropin↗
PubMed1994

Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system.

Adjuvants, Immunologic↗
PubMed1994

Methods for the detection of apoptosis.

Apoptosis is central to many basic and clinically oriented investigations, and this article is a brief overview of the most frequently utilized methods for detection of apoptotic cells, including the study of morphology, analysis of DNA degradation, DNA end labeling techniques, flow cytometric analysis, and nuclease assays. Features and advantages of the different methods are discussed.

Animals↗
PubMed1994

Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelial cells.

Heat-shock protein (hsp) expression can be induced by high temperature, exposure to cytokines or oxygen radicals, ischemia, hemodynamic overload, or viral infections. To determine whether surface expression of hsp60 occurs in aortic endothelial cells stressed by high temperature or cytokines, cells from rat aortas were cultivated and stained with several types of monoclonal antibodies against hsp60. Other antibodies, eg, those against intercellular adhesion molecule-1 (ICAM-1), or immune response-associated antigens were also used as controls. Positive staining of endothelial cells on the surface and in the cytoplasm was observed after pretreatment of the cells with cytokine-containing medium, tumor necrosis factor-alpha (TNF-alpha), or interleukin-1 alpha and labeling with a specific monoclonal antibody against hsp60 (II-13). Fluorescence-activated cell sorter analyses showed that over 80% of living endothelial cells stressed by cytokine-containing medium, by TNF-alpha, or at 42 degrees C, but not by interleukin-1 alpha, were positively surface stained with this antibody. Increased intensity of immunostaining with antibodies to ICAM-1 and immune response-associated antigen was also seen on the cytokine-stressed endothelial cells. Furthermore, when TNF-alpha stimulated endothelial cells labeled with 51Cr were incubated with antibody II-13 in the presence of complement, significant lysis occurred. In summary, endothelial cells stressed by high temperature or certain cytokines, eg, TNF-alpha, express hsp60 in the cytoplasm and on their surfaces, and these cells were susceptible to complement-dependent lysis by hsp60-specific antibody. These observations may be significant for elucidating the mechanisms of the involvement of immune reactions to hsp65/60 in initiating atherosclerosis.

Animals↗
PubMed1994

Asbestos fibers in bronchoalveolar lavage and lung tissue of former asbestos workers.

Bronchoalveolar lavage (BAL) provides a simple method of sampling inhaled particles deposited in the lower respiratory tract. We hypothesized that BAL could be used to measure the quantity and quality of lung asbestos burden. This would be true if BAL fluid asbestos fiber content reflected the total content as well as the size distribution of both uncoated and coated asbestos fibers in lung parenchyma. Therefore, we analyzed the asbestos fiber counts of 23 individual sample pairs in both BAL fluid and lung tissue samples obtained from 20 patients with occupational asbestos exposure using transmission electron microscopy (TEM). In addition, fiber type, fiber size, and aspect ratio were compared. Coated asbestos fibers were found in 10 of 23 BAL samples and 16 of 23 biopsies. The mean concentrations of coated asbestos fibers (i.e., asbestos bodies) in BAL and lung parenchyma showed a positive correlation (r = 0.75, p < 0.001). Likewise, the mean amphibole fiber concentrations correlated positively (r = 0.55, p < 0.01). However, there was no relationship between the mean chrysotile fiber counts in BAL and lung parenchyma (r = 0.18, p = 0.40). Asbestos fibers in lung tissue were significantly longer (8.2 +/- 0.5 versus 4.8 +/- 0.6 microns; p < 0.001) but had the same width (0.12 +/- 0.27 versus 0.11 +/- 0.15 microns; p = 0.24) when compared with those retrieved by BAL from the airspace compartment. The aspect ratio (dividing fiber length by width) was much higher in lung tissue than in BAL fluid (66.4 +/- 0.4 versus 42.9 +/- 0.5; p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Asbestos, Amphibole↗
PubMed1994

Retrobulbar T cells from patients with Graves' ophthalmopathy are CD8+ and specifically recognize autologous fibroblasts.

Graves' ophthalmopathy is an autoimmune condition characterized by T cell infiltration of the retrobulbar tissue. Phenotypic and functional analysis of these infiltrating cells may provide insight into the pathogenesis of the disease. IL-2-responsive cells were therefore grown out of the retrobulbar tissue from two patients with severe Graves' ophthalmopathy undergoing orbital decompression surgery, and six T cell lines were established and characterized. They consisted predominantly of CD8 + CD45RO+ cells and secreted IL-4, IFN-gamma, and IL-10 upon activation. When screened for their antigen reactivity, all lines proliferated in response to stimulation with autologous retrobulbar fibroblasts in an HLA class I-restricted manner, but did not recognize autologous peripheral blood mononuclear cells, crude eye muscle extract, allogeneic cells, or purified protein derivate of Mycobacterium tuberculosis. In contrast, PBMC from the same patients responded readily to purified protein derivate of Mycobacterium tuberculosis and allogeneic PBMC, but did not recognize autologous fibroblasts. Interestingly, only one of the six retrobulbar T cell lines displayed cytotoxicity towards its specific target cell population. These results suggest that the retrobulbar fibroblasts are a major T cell target in Graves' ophthalmopathy. Pronounced cytokine production in the absence of target cell cytotoxicity may explain fibroblast proliferation, glycosaminoglycan secretion, and secondary eye muscle enlargement in this condition.

Adult↗
PubMed1994

Free alpha subunit of human chorionic gonadotrophin: molecular basis of immunologically and biologically active domains.

Immunochemical studies were undertaken to identify surface-orientated epitopes of the free alpha subunit of human chorionic gonadotrophin (hCG-alpha) at the amino acid sequence level. We investigated the molecular organization of these epitopes, resolved the immunological topography in terms of spatial arrangement of antigenic domains and related structures to functions such as subunit association or receptor binding. Overlapping synthetic peptides covering the entire amino acid sequence of hCG-alpha, an enzymatically digested hCG-alpha subunit, and a reduced and alkylated hCG-alpha preparation were assayed in a solid-phase one-site enzyme-linked immunoassay, and in a solution-phase competitive radioimmunoassay (RIA). The antigenic topography was mapped by monoclonal antibodies (MCAs) in two-site binding assays (sandwich RIA). On the surface of hCG-alpha, seven different epitopes (alpha 1-alpha 7), arranged in four spatially distinct domains, could be distinguished: A, alpha 1,2,4; B, alpha 3,5; C, alpha 6; D, alpha 7. The peptides spanning hCG-alpha(13-18), hCG-alpha(17-22) and hCG-alpha(33-42) appeared to contribute to the formation of epitopes alpha 2, alpha 4 and alpha 6 respectively. Since epitope alpha 6 is present only on the free non-assembled subunit of different species, we concluded that the region hCG-alpha(33-42), which is evolutionarily highly conserved, represents a subunit assembly site. All but one epitope (alpha 7) are destroyed by reducing and alkylating hCG-alpha. In contrast, chymotryptic digestion of hCG-alpha, leading to release of the heptapeptide hCG-alpha(41-47), did not affect epitope expression, indicating that this sequence is not involved in the formation of antigenic determinants. Addressing the biological properties of hCG-alpha epitopes by radioreceptor assay revealed that the three hCG-alpha peptides corresponding to epitopes alpha 2, alpha 4 and alpha 6 did not displace radiolabelled hCG from its receptor, whereas any of the MCAs directed against determinants (alpha 1-alpha 5), shared by hCG and hCG-alpha, totally inhibited binding. Consistent with this, the antibodies neutralized the biological activity of hCG in terms of testosterone production in a mouse Leydig cell in vitro bioassay. We therefore concluded that hormone antibody-binding sites differ from those of hormone receptor binding, revealing no essential congruence of immunologically and biologically active domains.

Amino Acid Sequence↗
PubMed1994

The molecular basis for epitopes on the free beta-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCG beta-core fragment.

The molecular basis for antigenic determinants on the free beta-subunit of human chorionic gonadotrophin (hCG beta), its carboxyl-terminal peptide (hCG beta CTP) and the hCG beta-core fragment (hCG beta cf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCG beta cf preparation, a synthetic peptide corresponding to the hCG beta CTP (beta 109-145), overlapping synthetic peptides spanning the entire amino acid sequence of hCG beta, and a reduced and alkylated hCG beta preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a soluble-phase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCG beta, nine different epitopes (beta 1-beta 9), arranged in three spatially distinct domains, could be distinguished. Epitopes beta 1-beta 7 were located in a single large domain on both hCG beta and the hCG beta cf whereas hCG beta CTP contained two topographically distant determinants, designated beta 8 and beta 9 respectively. All but the two epitopes located on hCG beta CTP (beta 8 and beta 9) were destroyed by reducing and alkylating hCG beta, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCG beta CTP is linear. At a molecular level, amino acid residues spanning hCG beta 45-52, hCG beta 137-144 and hCG beta 113-116 contributed to the formation of epitopes beta 5, beta 8 and beta 9 respectively. We have also shown that the hCG beta cf represents the immunodominant part of the free beta-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence beta 45-52. The hCG beta CTP does not play a critical role in the immunologically important tertiary structure of hCG beta and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues beta 113-116 and beta 137-144 respectively.

Antibodies, Monoclonal↗
PubMed1993

Serum glycoprotein hormones and their free alpha-subunit in a healthy elderly population selected according to the SENIEUR protocol. Analyses with ultrasensitive time resolved fluoroimmunoassays.

The SENIEUR protocol was elaborated by a working party of European Community's Concerted Action Programme on Aging (EURAGE) to define strict admission criteria for 'healthy' elderly subjects and young controls for immunogerontological studies. This protocol, which is based on case history, laboratory values and drug consumption, intends to limit the influence of underlying disease and/or medication in order to allow analyses of the aging process per se. In a group of 38 male and 37 female individuals we determined the impact of age and classification according to the SENIEUR protocol on luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) and free glycoprotein hormone alpha-subunit serum values. Analyses were performed by a set of ultrasensitive time-resolved immunofluorometric assays (IFMA) using our own panel of monoclonal antibodies (MCA). HLH and hFSH, but also hCG and free alpha serum levels increased highly significantly with age in the female population (P < 0.001). In males hFSH, hLH hCG and the free alpha-subunit increased with age. However, only the rise of hFSH and of Free alpha was statistically significant (P < 0.01). The influence of the SENIEUR status on the respective hormone serum levels was determined using two factor analysis of variance, which revealed no statistically significant difference (P > 0.01) between SENIEUR and NON-SENIEUR individuals for all four analytes in both sexes. We conclude that the age related increase of hLH, hFSH, hCG and free alpha is an intrinsic age-dependent phenomen and is not modified by or due to underlying disease or medication as demonstrated by analyses of SENIEUR individuals. Since SENIEUR and NON-SENIEUR individuals had comparable hormone values, a randomly chosen, 'apparently healthy' population seems to be sufficient for physiological studies on serum GPH levels. Lastly, these age related hormonal changes in an extremely well defined healthy population underline the need for age adjusted 'normal' hormone values as elaborated in this communication.

Aged↗