Christina Mayerl

Cross-Reactive B-Cell Epitopes of Microbial and Human Heat Shock Protein 60/65 in Atherosclerosis

Objective—Growing evidence suggests that immune reactions to heat shock protein 60 (HSP60) are involved in atherogenesis. Because of the high phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for breaking the tolerance to self-HSP60, which is expressed on the surface of stressed arterial endothelial cells. Methods and Results—We purified serum antibodies to Escherichia coli HSP60 (GroEL), the 60-kD chlamydial HSP, and HSP65 of Mycobacterium tuberculosis by affinity chromatography from clinically healthy subjects with sonographically proven carotid atherosclerosis. Reactivity of the purified antibodies with overlapping human HSP60 peptides was measured, and 8 shared common epitopes, recognized by all anti-bacterial HSP60/65 antibodies, were identified. Antisera specific for these cross-reactive epitopes were produced by immunizing rabbits with peptides derived from human HSP60. By immunohistochemistry, the epitopes were found to be present in the arterial wall of young subjects during the earliest stages of the disease. Conclusions—Antibodies to microbial HSP60/65 recognize specific epitopes on human HSP60. These cross-reactive epitopes were shown to serve as autoimmune targets in incipient atherosclerosis and might provide further insights into the mechanisms of early atherogenesis.

The vascular-associated lymphoid tissue: a new site of local immunity.

Recent data suggest that atherosclerosis might be a systemic (auto)immune reaction against heat shock protein 60, first occurring at notorious local predilection sites, i.e. the intima at arterial branching points. The local infiltration of mononuclear cells, mainly macrophage-derived foam cells, T cells and smooth-muscle cells in atheromatous plaques, have long been described. During the past few years, research has been concentrated on the early stages in the development of atherosclerosis, and on healthy arteries from young individuals unaffected by arterial disease. In this review, we summarize data characterizing pre-existing mononuclear cell infiltrations in healthy arteries from children and teenagers. These arterial accumulations at regions known to be predilection sites for the later development of atherosclerosis consist mostly of activated T cells, macrophages and dendritic cells, with only a few mast cells and virtually no B or natural killer cells. In analogy to the mucosa-associated lymphoid tissue, we termed these accumulations ‘vascular-associated lymphoid tissue’, and assumed a similar function as a local immunosurveillance system, monitoring the bloodstream for potentially harmful endogenous or exogenous antigens. In addition to the remarkable accumulation of mononuclear cells, the vascular-associated lymphoid tissue regions are characterized by a typical distribution of extracellular matrix proteins: collagen type I, collagen type III, fibronectin and tenascin are expressed preferentially in the vascular-associated lymphoid tissue region, whereas collagen type IV, collagen type V, collagen type VI and laminin show a homogenous distribution throughout all regions of the intima. Vascular adhesion molecules type 1, intercellular adhesion molecules type 1 and P-selectin are already present on the healthy endothelial cells of young children. Interactions between adhesion molecules, extracellular matrix components and cellular elements of the vascular-associated lymphoid tissue may provide the basis for the cellular accumulations in the vascular-associated lymphoid tissue regions and the possible development of atherosclerotic lesions later in life.

Prognostic significance of tenascin-C expression in superficial and invasive bladder cancer

Aims: Tenascin-C (Tn-C) is an extracellular matrix glycoprotein that is upregulated in malignant tumours. Tn-C promotes cell growth, cell migration, and angiogenesis. It has been suggested to be a prognostic factor in various types of malignant tumours, but there is little information on its significance in bladder cancer with regard to overall survival (OS) and recurrence free survival (RFS). Methods: Tn-C expression was studied in 106 patients with bladder cancer diagnosed between 1994 and 1997. Immunohistochemistry was performed using a monoclonal antibody against Tn-C. RFS and OS were estimated by the Kaplan–Meier method and compared by the log rank test in univariate analysis and by the Cox multistep regression method in multivariate analysis. Results: Within the mean follow up period of 126 months, patients with diffuse Tn-C staining in the tumour stroma had a significantly worse OS than those with negative staining or only moderate Tn-C expression (p  =  0.025). Patients with cytoplasmic expression of Tn-C had a significantly better OS than those without (p  =  0.001). Multivariate analysis, taking into consideration age, grade, stage, tumour associated carcinoma in situ, progression, and Tn-C staining in tumour stroma, showed that only expression of Tn-C in invasive tumour cells was an independent positive prognostic factor for OS (p  =  0.049). Conclusions: Tn-C may provide important prognostic information in bladder cancer depending on the expression pattern in the tumour stroma or cytoplasm of the tumour cells.

Altered systemic serologic parameters in patients with silicone mammary implants

BACKGROUND The most common local complication in patients with silicone mammary implants (SMIs) is excessive peri-SMI connective tissue capsule formation and its subsequent contracture. However, considerable controversy remains as to whether these implants also cause systemic side effects. The present study was undertaken to identify possible alterations of serological markers in SMI patients that may herald systemic side effects. METHODS We investigated several systemic serological parameters in 143 individuals, 93 of whom had received SMIs and 50 were controls. The patients were grouped according to the severity of capsular contracture (Baker scores I-IV) and the duration of SMI implants (less than 1 year, between 1 and 5 years, more than 5 years). We also included control groups (female blood donors, nurses with possible professional silicone exposure). Patients with breast cancer and subsequent SMI-reconstruction were excluded from the study since they are generally considered immunocompromised. The following parameters were determined: anti-neutrophil cytoplasmatic autoantibodies (cANCA), anti-nuclear autoantibodies (ANA), anti-cardiolipin antibodies (CL-Ab), rheumatoid factor (RF), complement components (C3, C4), circulating immune complexes (CIC), procollagen III (a marker of active fibrosis), anti-polymer antibodies (APA) and soluble intercellular adhesion molecule-1 (sICAM-1). RESULTS The following parameters were increased in the sera of SMI patients: CIC, procollagen III, APA, sICAM-1. CONCLUSIONS We found a set of parameters in serum that correlate with fibrosis development and the duration of the implants in otherwise healthy SMI carriers. Future studies will clarify whether these serological abnormalities will be useful in predicting clinical disease, and also further assess the sensitivity and specificity of these parameters. Our present recommendation as a result of this study is that SMI patients with persistent abnormal serological parameters should be monitored closely by a clinical team that includes rheumatologists.

Immunosenescence and juvenile idiopathic arthritis

Aging of the immune system (immunosenescence) is characterized by diminished thymus function, decreased output of recent thymic emigrants, compensatory peripheral proliferation of mature T cells and oligoclonal expansions of specific CD28(-) T cells. Clinical consequences are poor responses to new antigens or vaccinations, increased infection rates with higher morbidity and mortality, and increasing incidence of autoimmune diseases with advancing age. Premature immunosenescence is suggested to play a role in the pathogenesis of adult rheumatoid arthritis and in children with juvenile idiopathic arthritis (JIA). However, so far, there is not enough evidence for supporting one of the two theories: the first, favoring premature immunosenscence in children developing autoimmune conditions as the primary defect causing break-down of self-tolerance; the second, that premature immunosenescence in children with autoimmune disorders is secondary to chronic stimulation and activation of the immune system by inflammatory processes by the autoimmune disease itself. This contradictory view of etiology and pathogenesis of autoimmune diseases in the very young underlines the need for population-based longitudinal studies on immune-risk factors for autoimmune diseases beginning at infancy.

Large-Scale Analysis of Cell Cycle Regulators in Urothelial Bladder Cancer Identifies p16 and p27 as Potentially Useful Prognostic Markers

Aims: We investigated the value of multiple cell cycle markers for their prognostic impact on overall survival and recurrence-free survival in urothelial carcinoma (UC). Methods: A tissue microarray consisting of 99 UCs was evaluated for the expression of p53, p16, p21, p27, cyclin D1, cyclin E , Bcl-2, Ki-67 and PCNA. Statistical analysis was performed applying Kaplan-Meier and Cox regression models using receiver operator characteristic curves for determination of markers’ cutoffs. Results: Expression above the cutoffs of Ki-67, p53 and p27, particularly in high-grade and early-stage UC, was associated with worse overall survival, while expression of p16 indicated a better outcome in low-grade and low-stage tumors. Recurrence-free survival was better in patients with high-grade UC expressing PCNA, p16 and cyclin E, and low-grade UC expressing Bcl-2 above the cutoffs, but worse in all tumors with high Ki-67. Conclusion: Cell cycle deregulation in UC is complex and the prognostic value of the various involved proteins should be differentially regarded with respect to this complexity and other tumor characteristics such as grade and stage. Our results point towards the role of p16- and p27-associated pathways in tumor progression and indicate that, by using standardized approaches for tissue antigen expression, evaluation and cutoff determination, single potentially useful prognostic markers could be identified.

Progression of arteriovenous bypass restenosis in mice exposed to a 50 Hz magnetic field.

Abstract The controversy over whether magnetic fields (MF) produced by electrical wiring and appliances contribute to diseases such as cancer has been debated in the literature for more than 2 decades. These extremely low frequency fields at 50 or 60 Hz are omnipresent in the industrialized world and have been linked to various forms of cancer by epidemiological studies. Little has been published investigating any possible role of MF and cardiovascular disease, and this is the first study looking specifically at the effect of exposure to high-intensity MF on the development and progression of restenosis. A mouse arteriovenous bypass model was used, and mice were exposed to MF for periods of 1, 2, or 3 weeks. Neointima formation, infiltration of mononuclear cells, and heat shock protein 60 expression were all studied at the conclusion of the exposure regimen. Animals exposed to the MF for 1 week showed significantly smaller neointima formation compared with control mice exposed to a null field, although this difference was not observed in mice exposed for 2 or 3 weeks. No difference was found between mice exposed to MF and controls in any of the other parameters investigated.

Immunophenotypic characterization of human T cells after in vitro exposure to different silicone breast implant surfaces

The most common complication of silicone breast implants is capsular contracture (massive scar formation around the implant). We postulate that capsular contracture is always a sequel to inflammatory processes, with both innate and adaptive immune mechanisms participating. In general, fibroblasts and macrophages have been used as cell types to evaluate in vitro the biocompatibility of breast implant surfaces. Moreover, also T cells have been found at the implant site at the initial stage of fibrous capsule formation. However, only few studies have addressed the influence of surfaces with different textures on T-cell responses. The aim of the present study was to investigate the immune response of human peripheral blood mononuclear cells (PBMC) to commercially available silicone breast implants in vitro. PBMC from healthy female blood donors were cultured on each silicone surface for 4 days. Proliferation and phenotype of cultured cells were assessed by flow cytometry. Cytokine levels were determined by multiplex and real-time assay. We found that silicone surfaces do not induce T-cell proliferation, nor do they extensively alter the proportion of T cell subsets (CD4, CD8, naïve, effector memory). Interestingly, cytokine profiling identified matrix specific differences, especially for IL-6 and TNF-α on certain surface topographies that could lead to increased fibrosis.

In vitro immunoregulatory effects of thymoglobulin on human immune cell subpopulations

Thymoglobulin (ATG) is a polyclonal rabbit antibody against human thymocytes used as a T cell-depleting agent in organ transplantation. Its polyclonal character suggests that its effect may go far beyond just T cell depletion. The aim of this study was to further elucidate possible mechanisms underlying the suppressive activity of ATG. For in vitro studies, human peripheral blood mononuclear cells (PBMC) were incubated with ATG or control Ig for various time points. Foxp3+ regulatory cells (Tregs) and monocytes were phenotypically analyzed by flow cytometry and functionally tested by in vitro suppression assays. Cytokine levels were determined by quantitative RT- PCR, Multiplex or ELISA techniques. In vitro, the frequencies of Foxp3+ Tregs increased when human PBMC were stimulated with ATG as compared with stimulation by rabbit Ig or without stimulation. ATG-treated cells suppressed proliferation of autologous PBMC stimulated with anti-CD3 and anti-CD28 monoclonal antibodies and this suppression could be reversed by exogenous IL-2. The Foxp3+ expression dropped down on day 10, which suggests that it is transient. Monocytes and natural killer cells stimulated with ATG down-modulated CD16. Monocytes suppressed the proliferation of autologous PBMC. However, there were not statistically significant differences in IL-10, TNF-α and TGF-β1 secretion by monocytes stimulated with ATG or control rabbit Ig. These findings suggest that ATG has immunomodulatory effects that go beyond T cell depletion and induction of Foxp3+ Tregs. The induction of immunosuppressive monocytes might have a protective role in delaying transplant rejection.

An Interstitial Network of Podoplanin-Expressing Cells in the Human Endolymphatic Duct

The human endolymphatic duct (ED) with encompassing interstitial connective tissue (CT) is believed to be important for endolymph resorption and fluid pressure regulation of the inner ear. The periductal CT cells are interconnected via numerous cellular extensions, but do not form vessel structures. Here we report that the periductal CT is populated by two distinct cell phenotypes; one expressing podoplanin, a protein otherwise found on lymph endothelia and on epithelia involved in fluid fluxes, and a second expressing a fibroblast marker. A majority of the interstitial cells expressed podoplanin but not the lymphatic endothelial cell markers hyaluronan receptor (LYVE-1) or vascular endothelial growth factor receptor-3 (VEGFR-3). The fibroblast marker positive cells were found close to the ED epithelium. In the mid- and distal parts of the ED, these cells were enriched under folded epithelia. Furthermore, subepithelial CT cells were found to express activated platelet derived growth factor (PDGF)-β receptors. Cultured CT cells from human inner ear periductal and perisaccular explant tissues were identified as fibroblasts. These cells compacted a three-dimensional collagen lattice by a process that could be promoted by PDGF-BB, a factor involved in interstitial fluid pressure regulation. Our results are compatible with the notion that the periductal CT cells are involved in the regulation of inner ear fluid pressure. By active compaction of the periductal CT and by the formation of villous structures, the CT cells could modulate fluid fluxes over the ED epithelium as well as the longitudinal flow of endolymph in the ED.