Albert Amberger

Is atherosclerosis an immunologically mediated disease

In contrast to general beliefs, recent data from different laboratories have provided evidence that the first stages of atherosclerosis are of an inflammatory nature. Here, Georg Wick and colleagues suggest that an autoimmune reaction against heat shock protein 60 (Hsp60), expressed by endothelial cells in areas that are subject to increased haemodynamic stress, is the initiating event in atherogenesis. Humoral and T-cell-mediated immune responses against Hsp60 have both been demonstrated early in disease. This inflammatory stage, which is reversible and has even been found in children, may progress into fully developed atherosclerotic lesions, displaying all the classical pathohistological and functional consequences, if additional risk factors such as high blood cholesterol levels, smoking and obesity, are present.

Prognostic value of indoleamine 2,3-dioxygenase expression in colorectal cancer: effect on tumor-infiltrating T cells.

PURPOSE: The pathologic interactions between tumor and host immune cells within the tumor microenvironment create an immunosuppressive network that promotes tumor growth and protects the tumor from immune attack. In this study, we examined the contribution of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) on this phenomenon. EXPERIMENTAL DESIGN: Expression of IDO was analyzed in colorectal cancer cell lines by reverse transcription-PCR and functional enzyme activity was assessed by high-pressure liquid chromatography. Semiquantitative immunohistochemistry was used to evaluate IDO expression in the tissue samples of 143 patients with colorectal carcinoma, and was then correlated with the number of tumor-infiltrating T cells and clinical variables. RESULTS: In vitro IDO expression and functional enzyme activity in colorectal cancer cells was found to be strictly dependent on IFN-gamma stimulation. Immunohistochemical scores revealed IDO-high expression in 56 of 143 (39.2%) tumor specimens, whereas 87 of 143 (60.8%) cases showed low IDO expression levels. IDO-high expression was associated with a significant reduction of CD3+ infiltrating T cells (46.02 +/- 7.25) as compared with tissue samples expressing low IDO (19.42 +/- 2.50; P = 0.0003). Furthermore, IDO-high immunoreactivity significantly correlated with the frequency of liver metastases (P = 0.003). Kaplan-Meier analysis showed the crossing of survival curves at 45 months. By multivariate Coxs analysis, IDO-high expression emerged as an independent prognostic variable (<45 months, P = 0.006; >45 months, P = 0.04). CONCLUSION: IDO-high expression by colorectal tumor cells enables certain cancer subsets to initially avoid immune attack and defeat the invasion of T cells via local tryptophan depletion and the production of proapoptotic tryptophan catabolites. Thus, IDO significantly contributes to disease progression and overall survival in patients with colorectal cancer.

Atherosclerosis, autoimmunity, and vascular-associated lymphoid tissue.

Atherosclerosis is a multifactorial disease induced by the effects of various risk factors on appropriate genetic backgrounds. It is characterized by vascular areas containing mononuclear and proliferating smooth muscle cells,as well as extracellularmatrix (ECM)2 components resulting in hardening and thickening (arteriosclerosis) of the arterial wall. In a strict sense, atherosclerotic lesions are localized in the inlima; they also contain foam cells and deposits of cholesterolcrystalsmanifested as fatty streaks and, finally, as atheroscleroticplaques. Fatty streaks are whitish, cushion-like lesions of the arterial intima harboring abundant lipid-laden macrophages, so-called foam cells, which are considered precursors of the fully developed plaques that may finally exulcerate and even calcif’. The main theories of atherogenesis are the “response to injury” (1) and the “response to altered lipoprotein” (2) hypotheses. The response to injury hypothesis postulates an alteration of the intima by various risk factors (mechanical injury, chemically altered low density lipoproteins [LDL], viruses, toxins) that initiates a primary endothelial dysfunction and subsequent changes in permeability, expression of adhesion molecules, and release of chemotactic and growth factors. Consequently, platelets and monocytes become activated and attach to these endothelial cells. Bloodderived monocytes transmigrate into the subendothelial space and transform into macrophages; smooth muscle cells (SMC) are attracted from the media to the same site. Both monocytes/macrophages and SMC possess the so-called scavenger receptor, which binds chemically altered (oxidized LDL {oxLDL]), but not native, LDL in a nonsaturable fashion. By uptake of oxLDL, macrophages and SMC develop into foam cells, and the deposition of collagenous and noncollagenous ECM components, especially in the peripheral “shoulder” region and the superficial “cap” area, complete the pathohistological appearance of fatty streaks and atherosclerotic plaques, respectively. The response toaltered lipoprotein hypothesis is based on the concept that lipoproteins can be chemically modified and are then able to induce foam cell formation by monocytes/macrophages and SMC. In recent years it has become evident that modification of lipoproteins does not occur primarily in the circulation or during transgression through the endothelium, but they rather accumulate in native form in the subendothelial space, where the lipoproteins are retained and oxidized. Accumulation of oxLDL, therefore, is not only the result of increased influx from the serum into the arterial intima but, conversely, is also due to a diminished efflux with subsequent foam cell formation. This “response to LDL retention” hypothesis (3) is thus a special variant of the response to altered LDL concept. Thus far, these latter theories have not explained why atherosclerotic lesions develop at certain arterial predilection sites or why the disease does not affect the venous vascular bed.

Co-expression of ICAM-1, VCAM-1, ELAM-1 and Hsp60 in human arterial and venous endothelial cells in response to cytokines and oxidized low-density lipoproteins.

T-cells and monocytes are the first cells infiltrating the arterial intima during the early stages of atherogenesis. Recently our laboratory has provided evidence that T-cells isolated from atherosclerotic intima reacts against heat shock protein 60 (Hsp60). Transmigration of activated T-cells into the intima is mediated by adhesion molecules (ICAM-1; VCAM-1; ELAM-1) expressed on activated endothelial cells. Here we studied the potential of cytokines (TNF-alpha, IFN-gamma, IL-1). Escherichia coli lipopolysaccharide (LPS), native and oxidized low-density lipoprotein (LDL; oxLDL) and high temperature to induce adhesion molecules as well as Hsp60 and Hsp70 expression in human endothelial cells (EC). On Northern blots, a strong signal for ICAM-1, VCAM-1 and ELAM-1 was detected after 4 h, which thereafter declined, but did not reach the basal level of untreated control cells. Heat shock induced the expression of Hsp60 and Hsp70 but not of adhesion molecules. EC were cultivated in serum-free medium, which led to the expression of adhesion molecule transcripts. Addition of LDL or oxLDL to these ECs did not alter the expression of these transcripts. The production of adhesion molecule proteins was analysed by flow cytometry. In human venous endothelial cells (HVEC) and human arterial endothelial cells (HAEC) ICAM-1 and VCAM-1 production was permanently highly induced, whereas the high level of ELAM-1 production at 4 h disappeared after 24 h. Furthermore, only HAEC, but not HVEC, produced ICAM-1, VCAM-1 and ELAM-1 after stress by moderately and highly oxLDL. LDL and oxLDL did not induce the production of Hsp60 and Hsp70. The present study demonstrates the co-expression of Hsp60 and adhesion molecules in arterial and venous EC in response to cytokine and LPS exposure, and that oxLDL is an efficient inducer of adhesion molecules in arterial EC and not in venous EC. These features provide the prerequisites for a cellular immune reaction against Hsp60 expressed by stressed EC in the initial stages of atherosclerosis.

Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells.

Mitochondrial damage with release of cytochrome c is implicated in cell death signalling pathways. To examine mitochondrial function in apoptotic cells, we applied high-resolution respirometry to human leukemia cells arrested in the G1- and S-phase by exposure to the glucocorticoid dexamethasone and nucleotide analogue gemcitabine. At 30% apoptosis, opposite effects were observed on respiratory capacity (71% and 131% of controls, respectively). These changes correlated with alterations in cell size, cytosolic, and mitochondrial marker enzymes. Mitochondrial ATP production and membrane potential were maintained in all treatments, as deduced from high respiratory uncoupling control ratios (UCR). Bcl-2 over-expression did not prevent apoptosis after gemcitabine-treatment, but protected dexamethasone-treated cells from apoptosis, without fully preventing the decline of respiration and cell size. These results, therefore, provide conclusive evidence that alterations in respiratory capacity and enzyme activities per cell are mainly caused by opposite changes in cell size, occurring upon cell cycle arrest triggered by dexamethasone and gemcitabine in the early phase of apoptosis.

Fluid Shear Stress Induces Heat Shock Protein 60 Expression in Endothelial Cells In Vitro and In Vivo

Recent investigations indicate that the initial event in the pathogenesis of atherosclerosis involves an (auto)immunologic injury to the vessel wall. Heat shock proteins (hsps), which are expressed on the endothelial cell surface, constitute possible autoantigens. After being exposed to shear stress of 30 dyne/cm(2) in vitro by means of a rotational viscometer, human umbilical vein endothelial cells were immunohistochemically stained for hsp 60 by the monoclonal antibody ML-30; static control cells were negative. Maximal hsp 60 induction was observed after 12 hours of hemodynamic stress. In Northern blots, the level of hsp 60 mRNA was markedly increased after only 1 hour of shear stress in human umbilical vein endothelial cells compared with static control cells. In vivo investigations in Lewis rats confirmed these in vitro findings: the intima and media of frozen sections of the right common carotid artery exposed to increased wall shear stress (after ligation of the left common carotid artery) were stained for hsp 60. The vessel wall of the left low-shear-stress-exposed side was negative. These findings demonstrate that shear stress results in hsp 60 induction in endothelial cells in vivo and in vitro, providing the prerequisite for humoral and cellular reactions to endothelial hsp in the earliest stages of atherosclerosis.

Changes in mitochondrial redox state, membrane potential and calcium precede mitochondrial dysfunction in doxorubicin-induced cell death

Mitochondria play central roles in cell life as a source of energy and in cell death by inducing apoptosis. Many important functions of mitochondria change in cancer, and these organelles can be a target of chemotherapy. The widely used anticancer drug doxorubicin (DOX) causes cell death, inhibition of cell cycle/proliferation and mitochondrial impairment. However, the mechanism of such impairment is not completely understood. In our study we used confocal and two-photon fluorescence imaging together with enzymatic and respirometric analysis to study short- and long-term effects of doxorubicin on mitochondria in various human carcinoma cells. We show that short-term (<30 min) effects include i) rapid changes in mitochondrial redox potentials towards a more oxidized state (flavoproteins and NADH), ii) mitochondrial depolarization, iii) elevated matrix calcium levels, and iv) mitochondrial ROS production, demonstrating a complex pattern of mitochondrial alterations. Significant inhibition of mitochondrial endogenous and uncoupled respiration, ATP depletion and changes in the activities of marker enzymes were observed after 48 h of DOX treatment (long-term effects) associated with cell cycle arrest and death.

Mutations in FKBP14 cause a variant of Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss.

We report on an autosomal-recessive variant of Ehlers-Danlos syndrome (EDS) characterized by severe muscle hypotonia at birth, progressive scoliosis, joint hypermobility, hyperelastic skin, myopathy, sensorineural hearing impairment, and normal pyridinoline excretion in urine. Clinically, the disorder shares many features with the kyphoscoliotic type of EDS (EDS VIA) and Ullrich congenital muscular dystrophy. Linkage analysis in a large Tyrolean kindred identified a homozygous frameshift mutation in FKBP14 in two affected individuals. Based on the cardinal clinical characteristics of the disorder, four additional individuals originating from different European countries were identified who carried either homozygous or compound heterozygous mutations in FKBP14. FKBP14 belongs to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases). ER-resident FKBPs have been suggested to act as folding catalysts by accelerating cis-trans isomerization of peptidyl-prolyl bonds and to act occasionally also as chaperones. We demonstrate that FKBP14 is localized in the endoplasmic reticulum (ER) and that deficiency of FKBP14 leads to enlarged ER cisterns in dermal fibroblasts in vivo. Furthermore, indirect immunofluorescence of FKBP14-deficient fibroblasts indicated an altered assembly of the extracellular matrix in vitro. These findings suggest that a disturbance of protein folding in the ER affecting one or more components of the extracellular matrix might cause the generalized connective tissue involvement in this disorder. FKBP14 mutation analysis should be considered in all individuals with apparent kyphoscoliotic type of EDS and normal urinary pyridinoline excretion, in particular in conjunction with sensorineural hearing impairment.

Lipocalin-2 Regulates the Inflammatory Response During Ischemia and Reperfusion of the Transplanted Heart

Ischemia and reperfusion (IR) are known to negatively affect early allograft function following solid organ transplantation. Lipocalin‐2 (Lcn‐2) has been described as a marker and potential positive modulator of acute inflammation during these processes. Using a heterotopic murine heart transplant model we previously found that IR resulted in a pronounced upregulation of Lcn‐2 mRNA in the heart at 12 (22.7‐fold increase) and 24 h (9.8‐fold increase) of reperfusion. We now confirm this increase at the protein level and provide evidence for infiltrating polymorphonuclear cells as the primary source of Lcn‐2 protein. Lcn‐2 levels are increased 6.6‐fold at 12 h, 11.4‐fold at 24 h and 6.4 fold at 48 h after reperfusion. In Lcn‐2−/− grafts the number of infiltrating granulocytes is reduced by 54% (p < 0.05) at 2 h, 79% (p < 0.01) at 12 h, 72% (p < 0.01) at 24 h and 52% (p < 0.01) at 48 h after reperfusion compared to Lcn‐2+/+ grafts, without any differences in cardiomyocyte apoptosis. These data suggest a function of Lcn‐2 in the initiation of the inflammatory response. Moreover, an increase in Lcn‐2 is not only restricted to the transplanted heart, but is also observed in the kidney, hinting at a possible involvement of Lcn‐2 in the systemic response to IR.

Ep-CAM expression in pancreatic and ampullary carcinomas: frequency and prognostic relevance

Aims: Pancreatic adenocarcinoma is an aggressive gastrointestinal malignancy with only a few long-term survivors even after radical surgery. Patients with ampullary cancer have a better prognosis but adjuvant therapy needs further improvement. Epithelial cell adhesion molecule (Ep-CAM) is strongly expressed in a variety of epithelial cancers and represents a promising target for immunological tumour therapy. Thus, the aim of this study was to investigate Ep-CAM expression and its potential prognostic impact in pancreatic and ampullary carcinomas. Methods: Ep-CAM expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumour tissue samples from a series of consecutive patients with pancreatic (n = 153) and ampullary cancer (n = 34). Results: Ep-CAM overexpression was observed in 85 of 153 pancreatic cancer specimens (56%) and in 29 of 34 ampullary cancer samples (85%). Overall, Ep-CAM failed to be an independent prognostic marker. However, subgroup analyses showed that Ep-CAM overexpression correlated with shorter overall survival among patients with ampullary cancer and advanced stage pancreatic cancer. In the latter subgroup, survival gradually worsened with increasing Ep-CAM scores. Furthermore, in ampullary cancer, Ep-CAM overexpression was found to correlate with tumour stage. Conclusions: Ep-CAM overexpression was detectable in the majority of cases with pancreatic and ampullary cancer. Therefore, Ep-CAM represents an attractive target for immune-based therapeutic interventions in these tumour entities. However, the prognostic value of Ep-CAM overexpression remains undetermined.