Georg Wick

Association of serum antibodies to heat-shock protein 65 with carotid atherosclerosis

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 (p < 0.05) 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.

Infections, Immunity, and Atherosclerosis Associations of Antibodies to Chlamydia pneumoniae, Helicobacter pylori, and Cytomegalovirus With Immune Reactions to Heat-Shock Protein 60 and Carotid or Femoral Atherosclerosis

BACKGROUND Atherogenesis involves inflammatory processes in which infections are incriminated as possible contributors. METHODS AND RESULTS 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 (P<0.0001). Concomitantly, serum antibodies to mycobacterial heat-shock protein 65 (mHSP65) correlated with seropositivity to C pneumoniae and H pylori but not to cytomegalovirus. CONCLUSIONS 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.

Association of Serum Antibodies to Heat-Shock Protein 65 With Carotid Atherosclerosis Clinical Significance Determined in a Follow-Up Study

BACKGROUND Previous work has proved that increased titers of antibodies against heat-shock protein (hsp) 65 are associated with atherosclerotic lesions independently of other established risk factors. The present follow-up study was designed to further scrutinize the association of hsp antibodies and atherosclerosis and evaluate the possible predictive value of these antibodies for the development and/or progression of lesions in the same population. METHODS AND RESULTS A total of 750 subjects 45 to 74 years old were recruited, and the rate of participation was 93.6%; 58 subjects died between 1990 and 1995. All participants were subjected to determination of serum antibodies against hsp65 and sonography to assess carotid atherosclerotic lesions and evaluate other risk factors, ie, age, sex, body mass index, blood cholesterol, apolipoprotein B, apolipoprotein A, triglycerides, lipoprotein(a), fibrinogen, leukocyte number, antithrombin III, ESR, ferritin, hypertension, smoking, and diabetes mellitus. Our data show that hsp65 antibody titers in the population emerged as highly consistent over a 5-year observation period (r=0.78, P<0.0001). Titers were significantly elevated in subjects with progressive carotid atherosclerosis and correlated with intima/media thickness. Multiple linear regression analysis documented these associations to be independent of age, sex, and other risk factors. Subanalyses revealed a preferential association of hsp65 antibody titers with advanced lesions (odds ratio, 1.42; 95% CI, 1.02 to 1.98; P=0.039). Other risk factors neither confounded nor modified this association. Finally, hsp65 antibody titers significantly predicted the 5-year mortality (hazard ratio, 1.52; 95% CI, 1.14 to 2.03; P<0.001). CONCLUSIONS These findings indicate a sustained existence of anti-hsp65 antibodies in subjects with severe atherosclerosis, which is predictive for mortality.

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.

Network of Vascular-Associated Dendritic Cells in Intima of Healthy Young Individuals

In earlier studies, our group has established a new “immunological” hypothesis for atherogenesis supported by experimental and clinical studies showing that inflammatory immunological reactions against heat shock protein 60 initiate the development of atherosclerosis. In the present study, we describe the discovery of a so-far-unknown network of dendritic cells in the innermost layer of arteries, the intima, but not veins of healthy humans and rabbits. The number of these dendritic cells is comparable to that of Langerhans cells in the skin, and dendritic cells show a similar phenotype (CD1a+ S-100+ lag+ CD31− CD83− CD86− and no staining for von Willebrand factor or smooth muscle cell myosin). These vascular-associated dendritic cells accumulate most densely in those arterial regions that are subjected to major hemodynamic stress by turbulent flow conditions and are known to be predisposed for the later development of atherosclerosis. These results open new perspectives for the activation of the immune system within the arterial wall.

Inhibition of Neointima Hyperplasia of Mouse Vein Grafts by Locally Applied Suramin

BACKGROUND Saphenous vein grafts are widely used for aortocoronary bypass surgery as treatment for severe atherosclerosis and often are complicated by subsequent occlusion of the graft vessel. METHODS AND RESULTS We described a mouse model of venous bypass graft arteriosclerosis that can be effectively retarded by locally applied suramin, a growth factor receptor antagonist. Mouse isogeneic vessels of the vena cava veins pretreated with suramin were grafted end to end into the carotid arteries and enveloped with a mixture of suramin (1 mmol/L) and pluronic-127 gel. In the untreated group, vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-fold and 10-fold the original thickness in grafted veins at 4 and 8 weeks, respectively. Pluronic-127 gel alone did not influence neointima formation. Suramin treatment reduced the neointima hyperplasia 50% to 70% compared with untreated controls. Immunohistochemical studies demonstrated that a significant proliferation of vascular smooth muscle cells (SMCs) constituted neointimal lesions between 4 and 8 weeks. The majority of SMCs expressed platelet-derived growth factor (PDGF) receptors-alpha and -beta, which were significantly reduced by suramin treatment. In vitro studies indicated that suramin completely blocked PDGF receptor activation or phosphorylation stimulated by PDGF-AB, inhibited activation of mitogen-activated protein kinase (ERK) kinases (MEK1/2) and ERK1/2, and abrogated transcription factor AP-1 DNA-binding activity. CONCLUSIONS Suramin inhibited SMC migration and proliferation in vivo and in vitro by blocking PDGF-initiated PDGF receptor and MAPK-AP-1 signaling. These findings indicate that locally applied suramin is effective in a mouse model of venous bypass graft arteriosclerosis.

The obese strain of chickens: an animal model with spontaneous autoimmune thyroiditis

Publisher Summary Thyroid glands of Obese Strain (OS) chickens become severely infiltrated by mononuclear cells and the occurrence of numerous germinal centers is a characteristic histological hallmark of the disease. Autoimmune thyroid destruction leads to thyroid hormone deficiency that entails the development of hypothyroid symptoms, such as small body size; massive subcutaneous and abdominal fat deposits (hence the name); lipid serum; long, silky feathers; small combs; cold sensitivity; low fertility; and poor hatching ability. The serum of 0s chickens contains AAbs to Tg, microsomal antigens, and T3 and T4. Tg-AAb formation is not because of polyclonal activation, but requires the presence of the autoantigen. The infiltrated thyroid itself has been identified as the major Tg-AAb-producing organ and can be the site of the formation of antibodies against exogenous antigens. OS chickens also produce AAbs against a variety of other organ-specific and non-organ-specific antigens, albeit with significantly lower frequency and titers, and without concomitant histopathological or functional lesions, such as Addison-like disease or diabetes mellitus. Additionally, factors extrinsic to the immune system that also affect immunoregulation have been found to be altered in the OS. Thus, OS chickens display a significantly decreased glucocorticoid tonus because of an increased serum concentration of corticosteroid-binding globulin and therefore markedly decreased free, metabolically active glucocorticoids.

Increased Risk of Atherosclerosis Is Confined to CagA-Positive Helicobacter pylori Strains Prospective Results From the Bruneck Study

Background and Purpose— Accumulating evidence indicates that a variety of infections contribute to the pathogenesis of atherosclerosis, but there is controversy concerning the impact of Helicobacter pylori infections in atherosclerosis. Methods— We evaluated seropositivity to H pylori and to its cytotoxin-associated gene A (CagA) product in a large, prospective, population-based study (n=684). Intima-media thickness and atherosclerosis of carotid arteries were thoroughly assessed by high-resolution duplex scanning. Results— In our study population, H pylori infections defined by seropositivity have no relationship with levels of classic cardiovascular risk factors or markers of systemic inflammation, except for elevated levels of immune reactions to mycobacterial heat shock protein 65. The latter showed a trend toward highest levels in those harboring virulent H pylori strains (P =0.08). Common carotid artery intima-media thickness—both absolute values and changes between 1995 and 2000—were significantly enhanced in subjects seropositive to CagA but not in those infected with CagA-negative H pylori strains. There was a clear dose-response relation between anti-CagA antibodies and both intima-media thickness and atherosclerosis risk. Notably, the risk of atherosclerosis associated with CagA seropositivity was amplified by elevated C-reactive protein levels. Conclusions— Infections with virulent CagA-bearing H pylori strains may contribute to the pathogenesis of early atherosclerosis by aggravating immune-inflammatory reactions.

Primary and secondary alterations of immune reactivity in the elderly: impact of dietary factors and disease

Summary: The function of the immune system declines with age. It is the aim of the present review to demonstrate that it makes sense to distinguish between primary and secondary alterations of immune reactivity in the elderly. Primary changes occur as the result of an age‐dependent intrinsic decline of immune responsiveness. They also occur in healthy persons, i.e. persons, selected according to the criteria of the SENIEUR protocol of the European Communitys Concerted Action Program on Aging (EURAGE). T lymphocytes are hereby more severely affected than B cells or antigen presenting cells, possibly due to the involution of the thymus, which is almost complete at the age of 60. Secondary immunological changes occur as the result of environmental factors including diet, drug intake, physical activity etc. or arc alternatively due to underlying diseases. In this article, the effects of high lipid intake as well as the impact of diseases, such as for instance Alzheimers disease and atherosclerosis, will be addressed. The results underline the complexity of immunological alterations to be expected in old age. Changes in the aging immune system represent an opportunity for increased frequency and severity of disease and endanger the protective effect of vaccination.

Staining of endothelial cells and macrophages in atherosclerotic lesions with human heat-shock protein-reactive antisera.

Our previous epidemiological studies have shown that levels of serum antibodies against mycobacterial heat-shock protein (hsp) 65 correlate positively with carotid atherosclerosis in subjects aged 40 to 79 years. To determine whether these high-titer sera also react with homologous human hsp60 and/or cell components of atherosclerotic lesions, we selected 15 human sera samples, each with high or low titers to recombinant mycobacterial hsp65, and investigated their reactivity with human arterial lesion components by immunoblotting and immunofluorescence techniques. All five higher-titer sera against hsp65 reacted with a 60-kDa band of atherosclerotic lesion proteins and human recombinant hsp60 on Western blots. Pooled sera with low antibody titers to hsp65 diluted similarly as high-titer sera did not show reactivity with atherosclerotic lesion and media proteins. By immunohistochemistry and immunofluorescence with human immunoglobulin G isolated from different sera, labeled with biotin, and visualized with a streptavidin conjugate, positive staining was observed in sections of fatty streaks and atherosclerotic plaques of carotid arteries, and weak staining was observed in the normal intima. Double immunofluorescence identified the majority of positively stained cells as macrophages, endothelial cells, and a few smooth muscle cells. In summary, serum antibodies against hsp65 cross-react with the human 60-kDa homologue present in high levels in atherosclerotic lesions and are mainly reacting with macrophages and endothelial cells, supporting our concept of a possible involvement of humoral-mediated immune reaction against hsp60 in atherogenesis.