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Encouraged by recent findings showing the usefulness of nonreplicating antigen delivery systems in the induction ofmucosal immune responses, we investigated microspheres as a means to deliver LW 50020, an immunomodulator consisting of lysates of seven common respiratory pathogens. BALB/c mice were orally immunized with LW 50020 encapsulated into poly-(D,L-lactide-co-glycolide) (PLG) and poly-(L-lactic acid) (PLA) microspheres prepared by either a solvent-evaporation or a solvent-extraction double-emulsion technique. Particle uptake into intestinal Peyer's patches, induction of antibodies in sera and secretion of immunoglobulins by isolated Peyer's patches, lungs and spleen lymphocytes were investigated. Our results revealed size and surface characteristic-dependent uptake of microspheres into Peyer's patches. Microsphere translocation into Peyer's patches was efficient for 0.8-microm microspheres, but poor for 2.0-microm and surface-modified microspheres. We showed an enhanced immune response in the lungs and sera following oral immunization with 0.8-microm PLG solvent-evaporation microspheres. The immunomodulation was statistically significant as compared to free LW 50020. In contrast, oral immunization with other preparations caused reduced or absent modulation of the immune response compared to 0.8-microm microspheres and free antigen. These findings indicate that microspheres displaying small particle sizes, rapid antigen release and high antigen content provide optimal tools to deliver orally applied antigens.

Summary Untreated control OS chickens develop spontaneous autoimmune thyroiditis (SAT). In contrast, OS chickens treated with a monoclonal anti-CD4 antibody failed to develop SAT. The preventive effect of anti-CD4 antibody on SAT was associated with the marked depletion of CD4+ T-cells by anti-CD4 treatment. These results indicate that CD4+ T-cells play a crucial role in SAT of OS chickens.

The effects of surface preparation and sterilization treatments of cpTi on in vitro osteoblast cell attachment were investigated. Surface characterization by XPS and AES and determination of wetting angles indicated that sterilization by exposure to ultraviolet light or gamma irradiation resulted in highly energetic, relatively contaminant-free cpTi surfaces which supported high levels of osteoblast cell attachment. Steam autoclaving, however, resulted in relatively low energy surfaces with high levels of O-C-and N-containing contaminants. Other inorganic contaminants were also detected on the surface of selected samples. As a result, steam-autoclaved surfaces supported significantly lower levels of cell attachment. At 2 hours after initial attachment, the osteoblast cells failed to spread and integrate onto the surfaces sterilized by steam autoclaving. These results confirm previous studies regarding the contamination resulting from steam autoclaving and indicate that non-contaminating steriliza...

Arteriosclerotic lesions can be induced in normocholesterolaemic rabbits by immunisation with heat-shock protein (hsp) 65, a stress protein expressed in high concentrations in human atherosclerotic lesions. If an immune reaction to hsp65 also plays a part in human atherogenesis, it should be possible to detect anti-hsp65 antibodies in patients with atherosclerotic lesions. To study the possible relation between immune reaction to hsp65 and atherosclerosis, 867 normal inhabitants of South Tyrol, aged 40-79 years, were selected randomly for determination of serum antibodies against hsp65, simultaneous sonographic assessment of carotid atherosclerotic lesions, and evaluation of established risk factors--ie, blood cholesterol, hypertension, smoking, diabetes mellitus, and obesity. Autoantibodies to nuclear antigens, thyroid antigens, and rheumatoid factors were also measured. Serum anti-hsp65 antibodies were significantly (p0.05) increased in subjects aged 60-79 years with carotid atherosclerosis compared with those without lesions, and increased antibody concentration was independent of age, sex, and other established risk factors. On the other hand, the incidence and titres of autoantibodies did not correlate with carotid atherosclerotic lesions. Our data provide the first evidence of a strong correlation between anti-hsp65 antibodies and carotid atherosclerosis, suggesting that hsp65 might be involved in the pathogenesis of atherosclerosis.

Especially since the (re-)discovery of T cell subpopulations with specialized regulatory activities, mechanisms of anti-inflammatory T cell regulation are studied very actively and are expected to lead to the development of novel immunotherapeutic approaches, especially in chronic inflammatory diseases. Heat shock proteins (Hsp) are possible targets for regulatory T cells due to their enhanced expression in inflamed (stressed) tissues and the evidence that Hsp induce anti-inflammatory immunoregulatory T cell responses. Initial evidence for an immunoregulatory role of Hsp in chronic inflammation was obtained through analysis of T cell responses in the rat model of adjuvant arthritis and the findings that Hsp immunizations protected against the induction of various forms of autoimmune arthritis in rat and mouse models. Since then, immune reactivity to Hsp was found to result from inflammation in various disease models and human inflammatory conditions, such as rheumatoid arthritis (RA), type 1 diabetes, and atherosclerosis. Now, also in the light of a growing interest in T cell regulation, it is of interest to further explore the mechanisms through which Hsp can be utilized to trigger immunoregulatory pathways, capable of suppressing such a wide and diversified spectrum of inflammatory diseases.

Background Anti-heat-shock protein 60 (HSP60) antibody-levels have been linked to carotid atherosclerosis and cardiovascular risk in a variety of studies. The potential role of cellular immune reactions against HSP60 has so far attracted little attention in epidemiological research. Methods and results In vitro T-cell reactivity to various HSP60s and tuberculin was assessed in blood samples from a elderly subpopulation of the Bruneck study (100 men, 50–69 years) and the young participants of the ARMY study (141 men, 17–18 years), and analyzed for a potential association with common carotoid artery intima-media thickness (IMT). In vivo skin reaction against tuberculin was recorded in subjects of the Bruneck study and correlated with the in vitro proliferative response to tuberculin ( P =0.004). T-cells isolated from peripheral blood of all individuals proliferated upon stimulation with HSP60s. In multivariate linear regression analysis adjusted for standard risk factors, T-cell stimulation was significantly related to IMT in the ARMY ( P =0.005 for human HSP60 and P =0.064 for mycobacterial HSP60) but not in the Bruneck study. Conclusions T-cell reactivity against HSP60s correlated with IMT in male youngsters but not in men aged 50 and over, indicating a more prominent role of specific cellular immunity to HSP60s in the young and very early stages of atherosclerosis.

Publisher Summary This chapter focuses on atherosclerosis and autoimmune hypothesis of atherogenesis. Heat shock proteins (HSPs) are expressed by prokaryotic and eukaryotic cells as a response to various stress factors. HSP60 is a special case since it is not only an important and highly antigenic component of bacteria and parasites, but also plays a major role in several autoimmune diseases. The autoimmune hypothesis of atherogenesis postulates that pre-existing adaptive and innate immunity to HSP60 exposed on the surface of arterial endothelial cells (ECs) leads to the development of an initial inflammatory stage of this disease. Immunity against HSP60 seems to be an important facet of the defense system, e.g., as reflected by the fact that a considerable proportion of lymphocytes within the vascular-associated lymphoid tissue (VALT) show reactivity against HSP60. Also, every healthy animal and human possesses humoral antibodies against HSP60. It is noted that cells of the innate immune system recognize HSP60 as a danger signal, be it of microbial or autologous origin. The development of atherosclerosis therefore depends on the expression and accessibility of atherogenic HSP60 epitopes on the surface of ECs.

In this review, we first briefly introduce the reader to our autoimmune hypothesis for the development of atherosclerosis based on experimental and clinical data. This hypothesis postulates that humoral and cellular immunity against heat-shock protein 60 (HSP60), a phylogenetically highly conserved stress protein, is the mechanism that initiates atherogenesis. We then turn to our investigations of arterial specimens from children and young adults. These clearly show that mononuclear cell infiltrations of the intimal layer already occur before the emergence of clear-cut atherosclerotic lesions, a phenomenon we have termed vascular-associated lymphoid tissue (VALT). In early lesions analyzed within the framework of the Pathological Determinants of Atherosclerosis in Youth (PDAY) study, T lymphocytes proved to be a major cellular constituent. In the Bruneck Study, a large, prospective atherosclerosis-prevention study in adults aged 40 years and older, we found a highly significant correlation between serum anti-HSP60 antibody titers and the occurrence and extent of sonographically demonstrable atherosclerotic lesions. However, no such correlation emerged with respect to HSP60-reactive T cells in the peripheral blood. In contrast, the similar Atherosclerosis Risk-Factors in Male Youngsters (ARMY) study, performed on 17- to 18-year-old volunteers, showed a highly statistically significant correlation between arterial intima-media thickening and HSP60 reactivity among peripheral T cells and (less pronounced) anti-HSP60 antibodies, even at this young age. We take this as indirect evidence that both T cell and B cell immunity against HSP60 plays a major role in the earliest stages of the disease. Because VALT can already be observed in healthy children and young adults, we hypothesize that T cells initiate the disease and that humoral antibodies play a facilitating, accelerating role. Finally, we provide initial evidence that smoking, as the most important risk factor for atherogenesis, also exerts its disease-inducing and disease-promoting effects by inducing HSP60 expression by vascular endothelial cells.

Atherogenesis involves inflammatory processes in which infections are incriminated as possible contributors.We evaluated cardiovascular risk factors as well as seropositivity to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus in a population-based study. A significant association between prevalence and severity of atherosclerosis in carotid and femoral arteries and IgA antibodies to C pneumoniae was demonstrated that was not substantially altered after adjustment for established risk factors. For anti-H pylori IgG antibodies, significant correlations to vascular disease were restricted to low social status and lesions in carotid arteries. In addition, the study design allowed us to monitor lesion progression over time. In this prospective analysis, C pneumoniae seropositivity emerged as a significant risk predictor. Antibody titers against cytomegalovirus were not a marker for prevalence or incidence of atherosclerosis in this population. Further infection parameters added to the predictive value of chlamydial serology in risk assessment: Mean odds ratios for the prevalence of carotid atherosclerosis were 4.2 and 6.3 for seropositive subjects with elevated C-reactive protein levels and clinical evidence for chronic respiratory infection, respectively. For subjects with all 3 infection parameters, the odds ratio of carotid atherosclerosis reached 10.3 (P0.0001). Concomitantly, serum antibodies to mycobacterial heat-shock protein 65 (mHSP65) correlated with seropositivity to C pneumoniae and H pylori but not to cytomegalovirus.This prospective population-based study provides strong evidence for a potential atherogenic role of persistent bacterial infection, especially C pneumoniae, as indicated by serological and clinical data and demonstrates a correlation between immune reactions to mHSP65 and bacterial infections in atherogenesis.

Acute rejection of the heart allograft is the major cause of heart failure in the first month after transplantation. Most studies on the prevention of acute rejection have concentrated on immune suppression of the recipients, whereas little is known about the effects of genetically manipulated donor organs on heart allograft survival. Herein, we describe a mouse model of heart allografts donated by p53-/- mice that can prolong the survival time of the grafts.Hearts of p53-/- or p53+/+ C57BL/6J mice were grafted to the neck carotid artery and jugular vein of BALB/c mice using a cuff technique. The graft survival was observed daily. The hearts were analyzed using several techniques, including histology, immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and Western blot analysis.p53+/+ allografts ceased beating at 7.6+/-0.5 days, whereas p53-/- hearts were beating at 10.5+/-1.1 days after transplantation (P0.01). Mean histological rejection scores were significantly lower in allografts donated by p53-deficient mice. Furthermore, apoptotic cells, determined by TUNEL and a reagent kit for detection of cardiac apoptosis, were of high numbers in the allograft sections from wild-type hearts but rare in p53-/- allografts (4.2+/-1.3 vs. 0.7+/-0.5/250x field). Immunofluorescence staining and Western blot analysis revealed that high levels of p53 and proapoptotic protein Bax were expressed in wild-type grafts but not p53-/- allografts. Interestingly, Bcl-2, an antiapoptotic protein, was abundant in cardiac allografts from p53-/- mice and almost undetectable in grafts from wild-type mice.Thus, p53 is involved in cardiac apoptosis induced by alloimmune reaction, and prolonged survival of heart allografts can be achieved when p53 is lacking.

This paper illustrates a new and cost effective approach to the design and manufacture of intelligent sensors and control electronics that reliably operate up to 300 Degrees Centigrade. A description of this Silicon-On-Insulator (SOI) technology for creating both the sensors and integrated circuits is provided. Measured results that demonstrate the inherent reliability of this technology at high temperatures are presented. Representative distributed control architectures and applications such as engine control units; valve position sensors and exhaust gas re-circulation sensor electronics are illustrated.

Glucocorticoid hormones are known to act as important modulatory factors in the development of autoimmune diseases, and to play an important role in thymic T-cell selection. There seems to be a finely balanced equilibrium between the apoptosis-inducing effects of glucocorticoid and T cell receptor ligand binding. Here we are investigating whether glucocorticoid-induced T cell apoptosis is mainly dependent on circulating glucocorticoid levels or if the thymus itself is able to produce glucocorticoids. To this end, we attempted to demonstrate enzyme activities of the whole set of steroidogenic enzymes for the synthesis of glucocorticoids in murine thymic tissue. We isolated steroidogenic organelles from thymic tissue, incubated these with radioactive (precursor) steroids in vitro, and visualized the resulting products by thin-layer chromatography. Our results show that the thymus possesses all enzymes and cofactors required for glucocorticoid production. However, an intact thymic architecture is necessary for glucocorticoid production, since 11beta-hydroxylase was not detected in irradiated thymi or in a thymic epithelial cell line. The results of these experiments show that the whole glucocorticoid metabolism takes place within the thymus. This finding provides the biochemical basis for the in situ effects of glucocorticoid hormones on thymocyte development and selection.

BACKGROUND: Studies from our laboratory have shown that the earliest stages of atherosclerosis may be mediated by an autoimmune reaction against heat shock protein 60 (Hsp60). The interactions of Hsp60-specific T cells with arterial endothelial cells (EC) require expression of both Hsp60 and certain adhesion molecules shown to be induced simultaneously in EC by mechanical and other types of stress. Recently, it was shown that suppression of T cell-mediated immune responses by cyclosporin A (CyA) enhanced atherosclerotic lesion formation in mice. In contrast, aspirin was found to lower the risk of myocardial infarction in men. These conflicting observations may be due to different effects of anti-inflammatory agents on adhesion molecule and Hsp expression in EC, respectively. MATERIAL AND METHODS: In the present study, we analyzed the effects of CyA, aspirin, and indomethacin on T cell proliferation using a proliferation assay. To explore the expression of adhesion molecules, monocyte chemoattractant protein-1 (MCP-1), and Hsp60 in human umbilical vein endothelial cells (HUVECs), Northern blot analyses were used. To examine the activation status of the transcription factors nuclear factor kappaB (NF-kappaB) and heat shock factor-1 (HSF-1), electrophoretic mobility shift assays were performed. RESULTS: With the exception of indomethacin, the used immunosuppressive and anti-inflammatory agents significantly inhibited T cell proliferation in response to influenza virus antigen in a dose-dependent manner. Interestingly, CyA and indomethacin did not suppress tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression on HUVECs, whereas aspirin had an inhibitory effect. These observations correlated with the modulation of NF-kappaB activity in EC. All agents tested induced expression of Hsp60 6 hr after application. In addition, aspirin and indomethacin, but not CyA, induced Hsp70 expression in HUVECs that correlated with induction of HSF-1 activity. CONCLUSION: Our results show that the tested agents (except indomethacin) are inhibitors of the T cell-mediated immune response, as expected, that aspirin is an effective suppressor of adhesion molecule expression, and that all three agents can induce Hsp60 in HUVECs. These data provide the molecular basis for the notion that (1) part of the anti-atherogenic effect of aspirin may be due to the prevention of the adhesion of sensitized T cells to stressed EC; (2) that part of the atherosclerosis-promoting effect of CyA may be due to its potential as an inducer of Hsp60 expression and its inability to down-regulate adhesion molecule expression on EC; and (3) that down-regulation of MCP-1 expression by aspirin may result in decreased recruitment of monocytes into the arterial intima beneath stressed EC.

Thymocytes form unique multicellular complexes with epithelial cells (thymic nurse cells, TNC) and rosettes (ROS) with macrophages, epithelial cells and dendritic cells. To investigate the role of differentiation checkpoints in the formation of the thymic heterotypic complexes in vivo, we used mutant mice which have genetically defined blocks at early and late stages of T cell development. We show that RAG-1-/-, TCRbeta-/- , and p56lck-/- mice lack thymocyte ROS formation with epithelial cells, macrophages, or dendritic cells. TNC formation was not affected by TCRbeta and p56lck gene mutations but partially decreased in RAG-1-/- mice, indicating that TNC are the earliest thymocyte-stromal cell complexes formed in development, whereas ROS only appear after thymocytes have rearranged and expressed a functional TCRbeta chain. Genetic blocks in CD8 lineage commitment (CD8-/- and IFN regulatory factor-1-/- mice) and positive and negative T cell selection (CD45-/-, TCRalpha-/-, and CD30-/- mice) did not affect thymocyte-stromal cell complexes. Surprisingly, CD4-/- mice, but not MHC class II-/- mice, had significantly reduced numbers of TNC and ROS, in particular, a severe defect in ROS formation with thymic dendritic cells. The CD4-/- block in ROS and TNC formation was rescued by the introduction of a human CD4 transgene. Moreover, we show that the adhesion receptors CD44 and LFA-1 cooperate in the formation of the thymic microenvironment. These results provide genetic evidence on the role of defined stages in T cell development and adhesion molecules on thymocyte/stromal cell interactions in vitro.

Recent data from different laboratories have provided evidence that the first stages of atherosclerosis are inflammatory in nature. Research in the last decades on this multifactorial disease has primarily focussed on the role of lipids, with only a few anecdotal findings suggesting the involvement of the immune system in atherogenesis. Within the group of antigens that may be responsible for this immunoactivation during atherogenesis, heat shock protein (hsp) 65/60 became a serious candidate based on the fact that immunization] of normocholesterolemic rabbits with hsp65 leads to the development of arteriosclerotic lesions in the aortic intima and these primary inflammatory lesions are aggravated by a cholesterol-rich diet, thus completely resembling human fatty streaks and atherosclerotic plaques. Furthermore, T cells in atherosclerotic lesions of rabbits have been shown to react specially with mycobacterial hsp65, suggesting that cell-mediated immune responses to hsp60 are also involved in the pathogenesis of this disease In a large epidemiological study we demonstrated that serum antibodies to mycobacterial hsp65 were significantly increased in clinically healthy subjects with sonographically demonstrable carotid atherosclerosis. These antibodies crossreact with human hsp60. Thus further elucidation of the role of the role of the immune system in atherogenesis could enhance our understanding of the mechanism of this vascular disorder, and may lead to new therapeutic strategies for atherosclerosis.

Encouraged by recent findings showing the usefulness of nonreplicating antigen delivery systems in the induction ofmucosal immune responses, we investigated microspheres as a means to deliver LW 50020, an immunomodulator consisting of lysates of seven common respiratory pathogens. BALB/c mice were orally immunized with LW 50020 encapsulated into poly-(D,L-lactide-co-glycolide) (PLG) and poly-(L-lactic acid) (PLA) microspheres prepared by either a solvent-evaporation or a solvent-extraction double-emulsion technique. Particle uptake into intestinal Peyer's patches, induction of antibodies in sera and secretion of immunoglobulins by isolated Peyer's patches, lungs and spleen lymphocytes were investigated. Our results revealed size and surface characteristic-dependent uptake of microspheres into Peyer's patches. Microsphere translocation into Peyer's patches was efficient for 0.8-microm microspheres, but poor for 2.0-microm and surface-modified microspheres. We showed an enhanced immune response in the lungs and sera following oral immunization with 0.8-microm PLG solvent-evaporation microspheres. The immunomodulation was statistically significant as compared to free LW 50020. In contrast, oral immunization with other preparations caused reduced or absent modulation of the immune response compared to 0.8-microm microspheres and free antigen. These findings indicate that microspheres displaying small particle sizes, rapid antigen release and high antigen content provide optimal tools to deliver orally applied antigens.

Bacterial cell-wall lipopolysaccharide (LPS) is the main endotoxin contributing to local inflammation and systemic toxicity during Gram-negative infections and induces aortic endothelial injury with or without cell death and replication followed by increased leukocyte adhesion. Heat-shock protein (hsp) 60 is under study in our laboratory as a potential antigen inducing immunologic attack to endothelial cells in atherogenesis. To investigate the mechanism of LPS-induced endothelial injury and the phenotypes of adhering cells, Lewis rats were treated in vivo or, in aortic organ cultures, with LPS to determine the expression of intercellular-adhesion molecule-1 (ICAM-1) and hsp60 on aortic endothelium and to characterize phenotypes of adhering leukocytes. Increased ICAM-1 expression by aortic endothelium was observed as early as 3 hr after LPS injection and persisted up to 72 hr, whereas elevated levels of hsp60 were found between 6 and 48 hr. In vitro application of various types of stress, such as LPS, H2O2, and high temperature, not only stimulated endothelial expression of hsp60 but, concomitantly, that of ICAM-1. The number of adhering leukocytes was significantly increased on aortic endothelium 6 hr after LPS administration, and the predominant leukocytes adhering to stressed endothelium were monocytes (80%) and T lymphocytes (8 to 20%). In organ cultures of rat aortic intimal, LPS, and H2O2 evoked increased leukocyte adhesion, which proved to be selective, because adherent leukocytes were mostly Ia+ monocytes and T cells, i.e., activated. Adhering T cells were gamma/delta antigen-receptor positive in 8 to 16% after LPS stress, whereas these cells amount to only 2 to 4% of peripheral blood T cells. Blocking of adhesion molecules ICAM-1, LFA-1 alpha, and/or LFA-1 beta reduced adhesion up to 34%. Increased coordinated LPS-dependent expression of hsp60 and ICAM-1 correlates with monocyte and T-cell adhesion to aortic endothelium. These observations may be significant for elucidating the mechanism of the initiating events in the development of atherosclerosis.

Correct determination of specific IgE is important for the diagnosis of allergic diseases. Different detection systems are supposed to yield similar results of this standardized parameter. In order to investigate this assumption, 40 sera from allergic patients were sent in eight mailings to 23 laboratories over a 2-year period. Three IgE determinations in each serum had to be performed according to laboratory-specific methods. Whereas 'wrong' results were rare with any of the methods and the overall results to a large degree comparable, sensitivity and specificity seemed to vary considerably between antigens, methods, companies and laboratories. According to our observations, better standardization of test reagents and test methods by the suppliers is essential in order to harmonize the test results and to avoid wrong conclusions. At present, the diversity of techniques that are used for IgE determination leads to quite divergent results, and the allergist has to know that. And finally, the legislator is urged to better control these diagnostic tools, because wrong results may lead to life-threatening situations.

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)