Relative expression of hormone receptors by endothelial and smooth muscle cells in proliferative and non-proliferative areas of congenital arteriovenous malformations.

BACKGROUND: Episodic growth due to microvascular proliferations (MVP) has been reported in congenital arteriovenous malformations (AVM), which are normally quiescent lesions composed of mature malformed vessels. Since AVM also may worsen under conditions of hormonal dysregulation, we hypothesized that hormonal influences may stimulate this process of vasoproliferative growth through potential interactions with hormone receptors (HR). METHODS: 13 Cases of AVM tissue with histologically documented vasoproliferative growth were analyzed quantitatively for the presence and tissue localization of estrogen receptor (ER), progesterone receptor (PGR), growth hormone receptor (GHR) and follicle-stimulating hormone receptor (FSHR) in relation to resident cells of interest (endothelial cells (EC), smooth muscle cells (SMC) and mast cells (MC)) by applying multiplex immunohistochemistry (IHC) staining. Expression patterns in lesions with MVP and mature vessels were quantified and compared. Available fresh frozen tissues of 3 AVM samples were used to confirm the presence of HR using Reverse-Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR). RESULTS: All four HR studied were expressed in all cases within EC and SMC in areas of MVP and mature vessels, but not in normal skin tissue. ER, GHR, and FSHR showed more expression in EC of MVP and in SMC of mature vessels. RT-qPCR confirmed presence of all 4 HR in both areas. CONCLUSION: Expression of ER, PGR, GHR, and FSHR in vasoproliferative areas of congenital AVM could explain onset of sudden symptomatic growth, as has observed in a subpopulation of patients. These findings may have implications for eventual anti-hormonal targeted therapy in the lesions involved.

Strategies for managing multi-patient 3D mass spectrometry imaging data.

Mass spectrometry imaging (MSI) has emerged as a powerful tool in biomedical research to reveal the localization of a broad scale of compounds ranging from metabolites to proteins in diseased tissues, such as malignant tumors. MSI is most commonly used for the two-dimensional imaging of tissues from multiple patients or for the three-dimensional (3D) imaging of tissue from a single patient. These applications are potentially introducing a sampling bias on a sample or patient level, respectively. The aim of this study is therefore to investigate the consequences of sampling bias on sample representativeness and on the precision of biomarker discovery for histological grading of human bladder cancers by MSI. We therefore submitted formalin-fixed paraffin-embedded tissues from 14 bladder cancer patients with varying histological grades to 3D analysis by matrix-assisted laser desorption/ionization (MALDI) MSI. We found that, after removing 20% of the data based on novel outlier detection routines for 3D-MSI data based on the evaluation of digestion efficacy and z-directed regression, on average 33% of a sample has to be measured in order to obtain sufficient coverage of the existing biological variance within a tissue sample. SIGNIFICANCE: In this study, 3D MALDI-MSI is applied for the first time on a cohort of bladder cancer patients using formalin-fixed paraffin-embedded (FFPE) tissue of bladder cancer resections. This work portrays the reproducibility that can be achieved when employing an optimized sample preparation and subsequent data evaluation approach. Our data shows the influence of sampling bias on the variability of the results, especially for a small patient cohort. Furthermore, the presented data analysis workflow can be used by others as a 3D FFPE data-analysis pipeline working on multi-patient 3D-MSI studies.

S100A8/A9 promotes parenchymal damage and renal fibrosis in obstructive nephropathy.

Despite advances in our understanding of the mechanisms underlying the progression of chronic kidney disease and the development of fibrosis, only limited efficacious therapies exist. The calcium binding protein S100A8/A9 is a damage-associated molecular pattern which can activate Toll-like receptor (TLR)-4 or receptor for advanced glycation end-products (RAGE). Activation of these receptors is involved in the progression of renal fibrosis; however, the role of S100A8/A9 herein remains unknown. Therefore, we analysed S100A8/A9 expression in patients and mice with obstructive nephropathy and subjected wild-type and S100A9 knock-out mice lacking the heterodimer S100A8/A9 to unilateral ureteral obstruction (UUO). We found profound S100A8/A9 expression in granulocytes that infiltrated human and murine kidney, together with enhanced renal expression over time, following UUO. S100A9 KO mice were protected from UUO-induced renal fibrosis, independently of leucocyte infiltration and inflammation. Loss of S100A8/A9 protected tubular epithelial cells from UUO-induced apoptosis and critical epithelial-mesenchymal transition steps. In-vitro studies revealed S100A8/A9 as a novel mediator of epithelial cell injury through loss of cell polarity, cell cycle arrest and subsequent cell death. In conclusion, we demonstrate that S100A8/A9 mediates renal damage and fibrosis, presumably through loss of tubular epithelial cell contacts and irreversible damage. Suppression of S100A8/A9 could be a therapeutic strategy to halt renal fibrosis in patients with chronic kidney disease.

Digital microscopy as valid alternative to conventional microscopy for histological evaluation of Barrett's esophagus biopsies.

Management of Barrett's esophagus (BE) relies heavily on histopathological assessment of biopsies, associated with significant intra- and interobserver variability. Guidelines recommend biopsy review by an expert in case of dysplasia. Conventional review of biopsies, however, is impractical and does not allow for teleconferencing or annotations. An expert digital review platform might overcome these limitations. We compared diagnostic agreement of digital and conventional microscopy for diagnosing BE ± dysplasia. Sixty BE biopsy glass slides (non-dysplastic BE (NDBE); n = 25, low-grade dysplasia (LGD); n = 20; high-grade dysplasia (HGD); n = 15) were scanned at ×20 magnification. The slides were assessed four times by five expert BE pathologists, all practicing histopathologists (range: 5-30 years), in 2 alternating rounds of digital and conventional microscopy, each in randomized order and sequence of slides. Intraobserver and pairwise interobserver agreement were calculated, using custom weighted Cohen's kappa, adjusted for the maximum possible kappa scores. Split into three categories (NDBE, IND, LGD+HGD), the mean intraobserver agreement was 0.75 and 0.84 for digital and conventional assessment, respectively (p = 0.35). Mean pairwise interobserver agreement was 0.80 for digital and 0.85 for conventional microscopy (p = 0.17). In 47/60 (78%) of digital microscopy reviews a majority vote of ≥3 pathologists was reached before consensus meeting. After group discussion, a majority vote was achieved in all cases (60/60). Diagnostic agreement of digital microscopy is comparable to that of conventional microscopy. These outcomes justify the use of digital slides in a nationwide, web-based BE revision platform in the Netherlands. This will overcome the practical issues associated with conventional histologic review by multiple pathologists.

Vimentin over-expression and carbonic anhydrase IX under-expression are independent predictors of recurrence, specific and overall survival in non-metastatic clear-cell renal carcinoma: a validation study.

PURPOSE: Clinical outcomes prognostic markers are awaited in clear-cell renal carcinoma (ccRCC) to improve patient-tailored management and to assess six different markers' influence on clinical outcomes from ccRCC specimen and their incremental value combined with TNM staging. MATERIALS AND METHODS: This is a retrospective, multicenter study. One hundred and forty-three patients with pT1b-pT3N0M0 ccRCC were included. Pathology specimens from surgeries were centrally reviewed, mounted on a tissue micro-array and stained with six markers: CAIX, c-MYC, Ki67, p53, vimentin and PTEN. Images were captured through an Ultra Fast Scanner. Tumor expression was measured with Image Pro Plus. Cytoplasmic markers (PTEN, CAIX, vimentin, c-MYC) were expressed as surface percentage of expression. Nuclear markers (Ki67, p53) were expressed as number of cells/mm2. Clinical data and markers expression were compared with clinical outcomes. Each variable was included in the Cox proportional multivariate analyses if p < 0.10 on univariate analyses. Discrimination of the new marker was calculated with Harrell's concordance index. RESULTS: At median follow-up of 63 months (IQR 35.0-91.8), on multivariate analysis, CAIX under-expression and vimentin over-expression were associated with worse survival (recurrence, specific and overall survival). A categorical marker CAIX-/Vimentin+ with cutoff points for CAIX and vimentin of 30 and 50 %, respectively, was designed. The new CAIX-/Vimentin+ marker presented a good concordance and comparable calibration to the reference model. Limitations are the retrospective design, the need for external validation and the large study period. CONCLUSION: Using an automated technique of measurement, CAIX and vimentin are independent predictors of clinical outcomes in ccRCC.

Platelet and endothelial cell P-selectin are required for host defense against Klebsiella pneumoniae-induced pneumosepsis.

BACKGROUND: Sepsis is associated with activation of platelets and endothelial cells accompanied by enhanced P-selectin surface expression. Both platelet- and endothelial P-selectin have been associated with leukocyte recruitment and induction of inflammatory alterations. Klebsiella (K.) pneumoniae is a common human sepsis pathogen, particularly in the context of pneumonia. METHODS: Wild-type (WT) and P-selectin-deficient (Selp(-/-) ) mice or bone marrow chimeric mice were infected with K. pneumoniae via the airways to induce pneumosepsis. Mice were sacrificed during early (12 h after infection) or late-stage (44 h) sepsis for analyses, or followed in a survival study. RESULTS: Selp(-/-) mice displayed 10-1000-fold higher bacterial burdens in the lungs, blood and distant organs during late-stage sepsis. P-selectin deficiency did not influence leukocyte recruitment to the lungs, but was associated with decreased platelet-monocyte complexes and increased cytokine release. Bone marrow transfer studies revealed a role for both platelet and endothelial cell P-selectin as mice deficient in platelet or endothelial cell P-selectin displayed an intermediate phenotype in bacterial loads and survival compared with full wild-type or full knockout control mice. CONCLUSION: Both platelet and endothelial cell P-selectin contribute to host defense during Klebsiella pneumosepsis.

A change in inflammatory footprint precedes plaque instability: a systematic evaluation of cellular aspects of the adaptive immune response in human atherosclerosis.

BACKGROUND: Experimental studies characterize adaptive immune response as a critical factor in the progression and complications of atherosclerosis. Yet, it is unclear whether these observations translate to the human situation. This study systematically evaluates cellular components of the adaptive immune response in a biobank of human aortas covering the full spectrum of atherosclerotic disease. METHODS AND RESULTS: A systematic analysis was performed on 114 well-characterized perirenal aortic specimens with immunostaining for T-cell subsets (CD3/4/8/45RA/45RO/FoxP3) and the Th1/non-Th1/Th17 ratio (CD4(+)T-bet(+)/CD4(+)T-bet(-)/CD4(+)/interleukin-17(+) double staining). CD20 and CD138 were used to identify B cells and plasma cells, while B-cell maturation was evaluated by AID/CD21 staining and expression of lymphoid homeostatic CXCL13. Scattered CD4 and CD8 cells with a T memory subtype were found in normal aorta and early, nonprogressive lesions. The total number of T cells increases in progressive atherosclerotic lesions (≈1:5 CD4/CD8 T-cell ratio). A further increase in medial and adventitial T cells is found upon progression to vulnerable lesions.This critical stage is further hallmarked by de novo formation of adventitial lymphoidlike structures containing B cells and plasma cells, a process accompanied by transient expression of CXCL13. A dramatic reduction of T-cell subsets, disappearance of lymphoid structures, and loss of CXCL13 expression characterize postruptured lesions. FoxP3 and Th17 T cells were minimally present throughout the atherosclerotic process. CONCLUSIONS: Transient CXCL13 expression, restricted presence of B cells in human atherosclerosis, along with formation of nonfunctional extranodal lymphoid structures in the phase preceding plaque rupture, indicates a "critical" change in the inflammatory footprint before and during plaque destabilization.

Effect of recombinant ADAMTS-13 on microthrombosis and brain injury after experimental subarachnoid hemorrhage.

BACKGROUND: A common complication after aneurysmal subarachnoid hemorrhage (SAH) is delayed cerebral ischemia (DCI), which is associated with vasospasm and other mechanisms such as microthrombosis. ADAMTS-13 activity plays a role in the prevention of thrombus formation in the cerebral microvasculature. Previously, we observed that patients with DCI have lower levels of ADAMTS-13. OBJECTIVES: To examine whether recombinant human ADAMTS-13 (rADAMTS-13) reduces cerebral microthrombus formation and brain injury in an experimental mouse model of SAH including wild-type and ADAMTS-13(-/-) mice. METHODS: Experimental SAH was induced with the prechiasmatic blood injection model. The following experimental groups were investigated: (i) C57BL/6J mice (n = 10); (ii) C57BL/6J mice (n = 10) treated with rADAMTS-13 20 min after SAH; (iii) ADAMTS-13(-/-) mice (n = 10); and (iv) ADAMTS-13(-/-) mice (n = 10) treated with rADAMTS-13 20 min after SAH. Mice were killed at 48 h. Results are presented as means with standard errors of the mean. RESULTS: Infusion with rADAMTS-13 reduced the extent of microthrombosis by ~ 50% in both wild-type mice (mean fibrinogen area: 0.28% ± 0.09% vs. 0.15% ± 0.04%; P = 0.20) and ADAMTS-13(-/-) mice (mean fibrinogen area: 0.32% ± 0.05% vs. 0.16% ± 0.03%; P = 0.016). In addition, rADAMTS-13 reduced brain injury by > 60% in both wild-type mice (mean microglia area: 0.65% ± 0.18% vs. 0.18% ± 0.04%; P = 0.013) and ADAMTS-13(-/-) mice (mean microglia area: 1.24% ± 0.36% vs. 0.42% ± 0.13%; P = 0.077). CONCLUSIONS: Our results support the further study of rADAMTS-13 as a treatment option for the prevention of microthrombosis and brain injury after SAH.

Intraplaque hemorrhage in cardiac allograft vasculopathy.

Plaque hemorrhage, inflammation and microvessel density are key determinants of plaque vulnerability in native coronary atherosclerosis (ATS). This study investigates the role of intraplaque hemorrhage (IPH) and its relation with inflammation and microvessels in cardiac allograft vasculopathy (CAV) in posttransplanted patients. Seventy coronary plaques were obtained from 12 patients who died because of CAV. For each patient we collected both native heart and the allograft, at the time of transplantation and autopsy, respectively. Intralesion inflammation, microvessels and IPH were assessed semi-quantitatively. IPH was observed in 21/35 (60%) CAV lesions and in 8/35 (22.9%) native ATS plaques, with a strong association between fibrocellular lesions and IPH (p = 0.0142). Microvessels were detected in 26/35 (74.3%) of CAV lesions with perivascular leakage as sign of endothelial damage in 18/26 (69.2%). IPH was strongly associated with microvessels (p < 0.0001). Inflammation was present in 31/35 (88.6%) of CAV lesions. CAV IPH+ lesions were characterized by presence of both fresh and old hemorrhage in 12/21 (57.1%). IPH, associated with microvessel damage and inflammation, is an important feature of CAV. Fresh and old intralesion hemorrhage suggests ongoing remodeling processes promoting the lesion progression and vulnerability.

Protease activated receptor 4 limits bacterial growth and lung pathology during late stage Streptococcus pneumoniae induced pneumonia in mice.

Streptococcus pneumoniae is a common causative pathogen of pneumonia and sepsis. Pneumonia and sepsis are associated with enhanced activation of coagulation, resulting in the production of several host-derived proteases at the primary site of infection and in the circulation. Serine proteases cleave protease activated receptors (PARs), which form a molecular link between coagulation and inflammation. PAR4 is one of four subtypes of PARs and is widely expressed by multiple cell types in the respiratory tract implicated in pulmonary inflammation, by immune cells and by platelets. In mice, mouse (m)PAR4 is the only thrombin receptor expressed by platelets. We here sought to determine the contribution of mPAR4 to the host response during pneumococcal pneumonia. Pneumonia was induced by intranasal inoculation with S. pneumoniae in mPAR4-deficient (par4-/-) and wild-type mice. Mice were sacrificed after 6, 24 or 48 hours (h). Blood, lungs, liver and spleen were collected for analyses. Ex vivo stimulation assays were performed with S. pneumoniae and mPAR4 activating peptides. At 48 h after infection, higher bacterial loads were found in the lungs and blood of par4-/- mice (p < 0.05), accompanied by higher histopathology scores and increased cytokine levels (p < 0.05) in the lungs. Ex vivo, co-stimulation with mPAR4 activating peptide enhanced the whole blood cytokine response to S. pneumoniae. Thrombin inhibition resulted in decreased cytokine release after S. pneumoniae stimulation in human whole blood. Our findings suggest that mPAR4 contributes to antibacterial defence during murine pneumococcal pneumonia.

Early onset of endothelial cell proliferation in coronary thrombi of patients with an acute myocardial infarction: implications for plaque healing.

AIMS: Coronary thrombotic occlusion in ST-segment elevation myocardial infarction (STEMI) patients is often preceded by episodes of progressive growth of the thrombus mass. Similar to wound healing, the organization of thrombus could depend on ingrowth of microvessels in order to stabilize its structure. We investigated the patterns of neovascularization in different stages of coronary thrombus evolution. MATERIAL AND METHODS: Thrombectomy materials obtained from STEMI patients were histologically classified according to thrombus age in three groups: fresh (< 1 day), lytic (1-5 days) or organized (> 5 days) thrombi. Forty thrombi of each group were randomly collected. Neovascularization in the thrombi was evaluated histomorphologically and with immunodouble stains to visualize various differentiation antigens of endothelial cells (ECs) and primitive cells. RESULTS: Morphologically, ECs in the coronary thrombi manifested as: single cells, cell clusters or microvessels. CD31+/CD34+ ECs were present in 98% of all the thrombi. In addition, endothelial clusters were found in 63% of the fresh thrombi (< 1 day). CD105+, Ki67+, or C-kit+ ECs (active, proliferating cells) were observed in all the stages, but significantly more in organized thrombi (> 5 days) compared with fresh and lytic ones (< 5 days), and mainly as cell clusters (P ≤ 0.05 for all). CD133+ primitive cells were found only sporadically in 11% of all the samples. CONCLUSION: EC proliferation is initiated very early, and gradually progresses during the organization process of thrombus after coronary plaque disruption, with only a limited contribution of primitive cells in this process.

Multiple bacteria contribute to intraplaque T-cell activation in atherosclerosis.

BACKGROUND: Infection with microorganisms is considered a pathogenic factor in atherogenesis. Several studies have shown the presence of a broad spectrum of bacterial species in atherosclerotic plaques, which could trigger local inflammation. Because T cells contribute to atherosclerotic plaque inflammation, we studied the responsiveness of human plaque derived T-cell cultures to bacteria of different species. MATERIALS AND METHODS: Primary polyclonal T-cell cultures were generated from both carotid endarterectomy tissue and peripheral blood of nine patients, and the peripheral blood of eight matched controls. The in vitro proliferative responses of the T-cell cultures against H. pylori, N. meningitidis, N. lactamica, S. aureus, S. pneumoniae, S. epidermidis and E. coli were analysed. T-cell proliferation was measured by (3)H-thymidine incorporation and expressed as a stimulation index. Selective outgrowth of intraplaque microbial specific T cells was studied by calculating the ratio of plaque T-cell SI and peripheral blood T-cell SI in each patient. RESULTS: All patients showed T-cell responsiveness to multiple bacteria in their plaque tissue. Stimulation indices were in the range of 0.3-30, and this degree of reactivity with the different species was heterogeneous among patients. Selective outgrowth (plaque/peripheral blood ratio) of T cells against multiple bacteria was observed in six out of nine patients. CONCLUSIONS: T cells in atherosclerotic plaques have the capacity to selectively respond to antigens of a wide variety of microbial antigens. This supports the view that such mechanisms could contribute to the atherosclerotic inflammatory response.

Interleukin-15 expression in atherosclerotic plaques: an alternative pathway for T-cell activation in atherosclerosis?

T-cell activation in atherosclerotic plaques is thought to be initiated by plaque-derived antigens, such as oxidized LDL (oxLDL). An alternative pathway of T-cell activation independent of antigen stimulation, mediated by the cytokine interleukin (IL)-15, was recently described. We investigated IL-15 expression in atherosclerotic plaques in relation to plaque morphology, inflammatory cells, T-cell activation, and oxidation-specific epitopes by use of immunohistochemistry. In situ hybridization was used to evaluate IL-15 mRNA expression. We also studied the proliferative response of plaque-derived T-cell lines to IL-15 in vitro using [(3)H]thymidine incorporation. Fresh-frozen specimens were classified as fibrous (n=9), fibrolipid (n=8), and lipid-rich (n=14) plaques; normal vessels (n=4) served as reference. Expression of IL-15 mRNA and protein was found almost solely in fibrolipid and lipid-rich plaques, associated with oxLDL-positive macrophages. Sequential immunostains revealed colocalization between IL-15- and CD40L-positive T cells. Moreover, plaque-derived T-cell lines were highly responsive to IL-15. Hence, IL-15 could provide a pathway for antigen-independent T-cell activation.

Immunohistochemical detection of interferon-gamma: fake or fact?

Immunohistochemistry is a widely accepted tool to investigate the presence and immunolocalization of cytokines in tissue sections at the protein level. We have tested the specificity and reproducibility of IFNgamma immunohistochemistry on tissue sections with a large panel of anti-IFNgamma antibodies. Thirteen different commercially available anti-IFNgamma antibodies, including seven advertised and/or regularly applied for immunohistochemistry/-cytochemistry, were tested using a three-step streptavidin-biotin-peroxidase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzyme double staining was used to identify the IFNgamma-positive cells. Serial cryostat sections were used of human reactive hyperplastic tonsils, rheumatoid synovium, and inflammatory abdominal aortic aneurysms, known to possess a prominent Th1-type immune response. In vitro phorbol myristate acetate/ionomycin-stimulated T-cells served as positive control; unstimulated cells served as negative control. Cultured T-cells were used adhered to glass slides (immunocytochemistry), in suspension (FACS), or snap-frozen and sectioned (immunohistochemistry). Immunocytochemistry and FACS analysis on stimulated cultured T-cells showed positive staining results with 12 of 13 anti-IFNgamma antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFNgamma immunohistochemical single- and double staining analysis of the tissue sections showed huge variations in staining patterns, including positivity for smooth muscle cells (n = 8), endothelial cells (n = 4), extracellular matrix (n = 4), and CD138+ plasma cells (n = 12). Specific staining of T-cells, as the sole positive staining, was not achieved with any of the 13 antibodies. IFNgamma-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFNgamma antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFNgamma-immunohistochemistry must be interpreted with great caution.

The role of inflammation and infection in coronary artery disease.

New insights into atherosclerosis, the most common disease affecting coronary arteries, may change therapeutic strategies from largely symptomatic to causal. Atherosclerotic plaques contain a lipid-related, immune-mediated inflammation, with release of secretory products capable of changing plaque morphology. Plaques prone to complications contain large numbers of inflammatory cells; stable plaques contain little inflammation. Similarly, atherectomy specimens from patients with coronary syndromes revealed more inflammatory cells in unstable than in stable patients. These observations, and the fact that acute coronary syndromes are associated with increased blood levels of inflammatory markers, have renewed interest in the possible relationship between infection and atherogenesis. Of all potential candidate antigens, Chlamydia pneumoniae presently is considered the most likely because a substantial number of patients with unstable syndromes contain C. pneumoniae-reactive T cells, both in blood and within the atherosclerotic plaque, suggesting enhancement of intraplaque inflammation.

Adventitial infiltrates associated with advanced atherosclerotic plaques: structural organization suggests generation of local humoral immune responses.

Advanced atherosclerotic lesions often contain adventitial lymphoid infiltrates, which occasionally contain nodular aggregates resembling lymphoid follicles. The structural organization suggests that local maturation of B cells may take place at these sites, as described for the mucosa-associated lymphoid tissue (MALT). This concept was evaluated by studying the micro-anatomy and cellular composition of adventitial infiltrates associated with advanced atherosclerosis of the aorta. Sections of 22 atherosclerotic aortas were stained immunohistochemically for cellular markers characteristic for lymphoid follicles, such as HECA-452-positive endothelial cells, CD20-positive B cells, CD21-positive follicular dendritic cells, and CD68-positive macrophages. Ki-67 was used as a proliferation marker. The TUNEL technique was used to study the presence of apoptotic cells. Specimens containing MALT served as comparison and positive controls. Seven of the 22 atherosclerotic aortas contained adventitial infiltrates resembling lymphoid follicles. The organized nodular centres were composed of CD45RA+ B cells, follicular dendritic cells (CD21+), a few T lymphocytes (CD3+) and 'tingible body' macrophages (CD68+). A large number of cells were Ki-67-positive; apoptotic bodies were numerous and phagocytosed by macrophages. The parafollicular area contained CD45RO-positive T cells and HECA-452-positive vessels. Vessels elsewhere were always HECA-452-negative. Specimens with MALT showed similar features. This study reveals a close resemblance between adventitial lymphoid infiltrates in advanced atherosclerotic aortic disease and MALT, suggesting local generation of a humoral immune response, likely to be initiated by antigens released during a process of long-standing tissue injury and inflammation as part of advanced atherosclerosis.

Unstable atherosclerotic plaques contain T-cells that respond to Chlamydia pneumoniae.

OBJECTIVE: Atherosclerotic lesions are characterized by an immune mediated chronic inflammation. Seroepidemiological studies support a relationship between atherosclerotic disease and infection with C. pneumoniae; an association further endorsed by immunocytochemical and DNA directed studies. However, the question arises whether C. pneumoniae acts as a causal antigen, or is merely a bystander. For this reason we have analyzed the T lymphocyte population of carotid atherosclerotic plaques of symptomatic patients for their response against C. pneumoniae. METHODS: T cell lines were generated from carotid endarterectomy tissues obtained from eight patients with symptomatic disease. The response of these T cell lines against C. pneumoniae elementary bodies was analyzed by 3H-thymidine incorporation. T cell clones were generated by limiting dilution from the cell lines of three patients and tested for antigen specificity in the same manner. Furthermore, cytokine profiles (Th1/Th0/Th2) were established by measuring the production of IFN-gamma and IL-4. RESULTS: Of the eight T-cell lines five responded to C. pneumoniae. Eighteen of 69 CD4-positive clones, generated from three patients with a positive T cell lines response, responded to C. pneumoniae also. The majority (17/18, 96%) of these clones showed a Th1 cytokine profile. CONCLUSION: These results show that in a subpopulation of symptomatic patients C. pneumoniae can activate T cells within atherosclerotic plaques suggesting that a C. pneumoniae enhanced proinflammatory Th1 response contributes to plaque destabilization in these patients.

Atherosclerosis, inflammation, and infection.

In recent years, it has been shown that inflammation plays an important role in the pathogenesis of atherosclerosis. Activated macrophages, T lymphocytes, and mast cells are present in atherosclerotic plaques, which has led to the notion that the inflammatory response is an immune-mediated process. Complicated lesions, moreover, appear to be associated with an increase in the amount of the inflammatory response and in these patients, increased levels of acute phase proteins are present. The appreciation that atherosclerosis is an immune-mediated inflammatory disease has also led to renewed interest in the potential role of infectious agents in initiating or modulating atherosclerosis. Seroepidemiological studies have shown raised antibody titres against several micro-organisms. However, as yet, there are hardly any data available that provide a sound scientific basis for an infectious origin. Of all potential candidate organisms, Chlamydia pneumoniae appears as the one most likely involved in atherogenesis. C. pneumoniae has been retrieved from atherosclerotic tissues; the level of raised plasma titres correlates with the severity of symptomatic atherosclerotic disease; and the incidence of C. pneumoniae-responsive T cells in peripheral blood is increased in patients with coronary heart disease. It also appears that in some patients T cells generated from atherosclerotic plaques respond to C. pneumoniae. At the present state of knowledge, however, it is fair to state that the relationship between infection, intraplaque inflammation, and atherosclerosis still remains hypothetical, despite the increasing evidence that such a relationship could exist.

Cytokine secretion profiles of cloned T cells from human aortic atherosclerotic plaques.

T cells take part in the chronic inflammatory reaction in atherosclerotic plaques, but their specific role in atherosclerosis has not yet been fully elucidated. Nevertheless, one may anticipate that activated T cells may secrete cytokines capable of modulating the morphology and hence the stability of plaques by regulating cell proliferation, lipid metabolism, and extracellular matrix (ECM) synthesis and/or degradation. This study has been designed to investigate the functional properties of T cells in atherosclerotic lesions. For this purpose, T-cell clones were generated from atherosclerotic plaques isolated from human aortas obtained at autopsy from six subjects. Cloned cells were activated with PMA and OKT-3 to initiate cytokine production and cytokine profiles of CD4-positive clones were measured by ELISA. The majority of the T-cell clones (125/155, 81 per cent) produced both interferon (IFN)-gamma and interleukin (IL)-4 (type 0 cytokine profile). Moreover, the production of IFN-gamma was dominant in the majority of these clones. A type 1 cytokine profile (high levels of IFN-gamma and low levels of IL-4) was found in 17 per cent of the clones (27/155). Only three clones (2 per cent) showed a type 2 cytokine secretion pattern (high levels of IL-4 and low levels of IFN-gamma). No cytolytic activity could be established in plaque-derived T cells. Our results show that the T-cell population in atherosclerotic lesions is heterogeneous, but the most dominant T cell by far is the one with a type 0 cytokine profile. The dominant secretion of IFN-gamma by T-cell clones suggest an important role for plaque T cells in modulating the growth and differentiation of other cells, such as macrophages and smooth muscle cells in atherosclerotic plaques.