Incidence of and risk factors for sentinel lymph node metastasis in patients with a postoperative diagnosis of ductal carcinoma in situ.

BACKGROUND: Positive sentinel lymph nodes (SLNs) are found in up to 13 per cent of women with a preoperative diagnosis of ductal carcinoma in situ (DCIS) of the breast and in up to 4 per cent of those with a postoperative diagnosis. This retrospective national register study investigated the incidence of positive SLNs in women with a postoperative diagnosis of DCIS, and the value of additional tumour sectioning to identify occult tumour invasion. METHODS: All surgical patients with a final histopathological diagnosis of pure DCIS registered in the Swedish national breast cancer register in 2008 and 2009 were eligible. Additional sectioning was performed on archived primary tumour tissue from women with SLN metastasis (including cases of isolated tumour cells) and matched SLN-negative control patients with the aim of detecting occult invasion. RESULTS: SLN tumour deposits were reported in 11 of 753 women who had SLN biopsy (macrometastases, 2; micrometastases, 3; isolated tumour cells, 6), resulting in a SLN positivity rate of 0·7 per cent (5 of 753). Occult invasion was found in one (9 per cent) of these 11 patients and in two (10 per cent) of 21 control patients. No risk factors for SLN metastasis were identified. CONCLUSION: SLN positivity is rare in women with a histopathological diagnosis of pure DCIS. Additional primary tumour assessment may reveal occult invasion in both SLN metastasis-positive and -negative patients. The value of performing SLN biopsy in the setting of a preoperative diagnosis of DCIS was limited, and current Swedish practice should therefore be questioned.

Hyperplastic polyposis coli syndrome and colorectal carcinoma.

BACKGROUND: Patients with hyperplastic polyposis coli syndrome (HPCS) have a propensity to develop colorectal carcinoma (CRC). PATIENTS AND METHODS: Details were retrieved from the files of patients attending our hospital between 1988 and 2004 who fulfilled the World Health Organization criteria for HPCS. RESULTS: Over a period of 16 years, 10 cases of HPCS were identified at our hospital (0.625 cases/year or one case every 1.6 years). A mean of 40.3 hyperplastic polyps per patient were found (range 6-159). Other colorectal lesions were found as follows: two patients each had one mixed polyp; there were 15 serrated adenomas in eight patients; and there were 30 tubular, tubulovillous, or villous adenomas in eight patients. Among the 10 patients with HPCS, seven developed a CRC. Of the four villous adenomas, three were associated with a CRC, but only one of the 15 serrated adenomas was associated with a CRC. The pathway of cancer evolution in HPCS patients remains unresolved. CONCLUSIONS: Similarly to our results, a review of the literature indicates a high incidence of CRCs in HPCS patients. These patients are at a high risk of developing a CRC and should therefore receive regular colonoscopic surveillance.

Expression of interleukin-15 in mouse and human atherosclerotic lesions.

Atherosclerotic lesions are characterized by prominent macrophage and T-cell infiltration and atherosclerosis is widely recognized as an inflammatory disease. The cytokine interleukin-15 (IL-15) has T-cell chemotactic and pro-inflammatory properties and promotes the recruitment of T cells to sites of inflammation. We have therefore examined IL-15 expression in the atherosclerotic ApoE-deficient mouse model as well as in human atherosclerotic lesions. In gene expression arrays, a transcript corresponding to IL-15 mRNA was elevated in atherosclerotic aortas of ApoE-deficient mice fed a Western diet for 10 and 20 weeks, corresponding to lesions of the fatty streak and fibrofatty plaque stage, respectively. Immunostaining for IL-15 localized to aortic smooth muscle cells in nonatherosclerotic C57BL/6 mice, whereas both macrophages and smooth muscle cells stained positive for IL-15 in atherosclerotic lesions of ApoE-deficient mice. Finally, advanced atherosclerotic lesions of human carotid arteries were immunostained to determine whether IL-15 is involved in human disease. IL-15 protein was present also in the human lesions with a distribution primarily overlapping that of macrophages. In conclusion, IL-15 is up-regulated in both human and animal atherosclerotic lesions and may contribute to the recruitment of T cells and their activation during atherogenesis.

Gene expression in atherosclerotic lesion of ApoE deficient mice.

BACKGROUND: Atherosclerosis, the major cause of mortality and invalidity in industrialized countries, is a multifactorial disease associated with high plasma cholesterol levels and inflammation in the vessel wall. Many different genes have previously been demonstrated in atherosclerosis, although limited numbers of genes are dealt with in each study. In general, data on dynamic gene expression during disease progress is limited and large-scale evaluation of gene expression patterns during atherogenesis could lead to a better understanding of the key events in the pathogenesis of atherosclerosis. We have therefore applied a mouse gene filter array to analyze gene expression in atherosclerotic ApoE-deficient mice. MATERIALS AND METHODS: ApoE-deficient mice were fed atherogenic western diet for 10 or 20 weeks and aortas isolated. C57BL/6 mice on normal chow were used as controls. The mRNAs of 15 animals were pooled and hybridized onto commercially available Clontech mouse gene array filters. RESULTS: The overall gene expression in the ApoE-deficient and control mice correlated well at both time points. Gene expression profiling showed varying patterns including genes up-regulated at 10 or 20 weeks only. At 20 weeks of diet, an increasing number of up-regulated genes were found in ApoE-deficient mice. CONCLUSIONS: The gene expression in atherogenesis is not a linear process with a maximal expression at advanced lesion stage. Instead, several genes demonstrate a dynamic expression pattern with peaks at the intermediate lesions stage. Thus, detailed evaluation of gene expression at several time points should help understanding the development of atherosclerosis and establishment of preventive intervention.

Quantitative analysis of specific mRNA species in minute cell samples by RT-PCR and flow cytometry.

A method for the quantitative determination of specific mRNAs in small numbers of cells, freshly isolated from tissues or early cell cultures, was developed by combining quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative flow cytometry. Freshly isolated umbilical vein endothelial cells were sorted by flow cytometry and then lysed. The number of cells in the lysate was determined by counting of nuclei after propidium iodide staining using flow cytometry. The number of plasminogen activator inhibitor-1 (PAI-1) mRNA copies per cell was determined by quantitative RT-PCR using point-mutated PAI-1 cRNA as an internal standard. The cells were shown to contain 400-900 copies of PAI-1 mRNA molecules per cell which confirms that endothelial cells in vivo express PAI-1. PAI-1 mRNA expression was also analyzed in small numbers of endothelial cells in primary culture in basal conditions and after incubation with different interleukins. The method allowed reliable and reproducible estimation of the number of mRNA copies per cell from original cell samples containing less than 1000 cells. This method can be used for the quantitative determination of various mRNA species in specified cell populations from small tissue samples or cultured cells.

[Pathogen and resistance spectrum in intraoral infections of the jaw-facial area with special reference to anaerobic bacteria]. / Erreger- und Resistenzspektrum bei intraoralen Infektionen des Kiefer-Gesichts-Bereichs unter besonderer Berücksichtigung der anaeroben Keimflora.

The aim of the study was to obtain more knowledge about the aerobic and anaerobic species causing maxillofacial infections and their resistance patterns today. Samples of pus or infectious tissue obtained from 110 patients of maxillofacial surgery were investigated microbiologically by means of aerobic and anaerobic cultivation. After incubation, the cultivated species were isolated and identified. The resistance patterns of all bacteria to penicillin, doxycyclin, and clindamycin were determined. Additionally, the resistance of aerobic species to cefuroxim was documented, and the MICs of cefoxitin and metronidazole to the anaerobic species were assessed. The most frequent disease was periodontitis apicalis (70 patients). Aerobic species alone were found in 23% of the samples, 14% of the infections harbored only anaerobes, but 63% were mixed infections caused by aerobic and anaerobic bacteria. In case of detection of aerobic species, streptococci were always identified. Five patients were infected by Staphylococcus aureus and gram-negative aerobic rods were found in eight patients. Most of the anaerobic species were black pigmented prevotella species (62), nonpigmented prevotellae (56), and fusobacteria (37). Metronidazole and clindamycin were highly efficient to gram-negative anaerobic rods. Most of the oral species were resistant to penicillin and doxycyclin. The indication for applying antibiotics should always be noticed and these drugs should only be used after determination of the pathogenic microorganisms and their susceptibility to the antimicrobials.

T-cell recognition of lipid peroxidation products breaks tolerance to self proteins.

Peroxidation of polyunsaturated fatty acids in lipoproteins and cell membrane phospholipids occurs in many situations in the body, both under normal and pathological conditions. Low-density lipoprotein is particularly prone to oxidation and is believed to be a pathogenetic component in atherogenesis. Both antibody responses and T-cell responses to oxidatively modified lipoproteins have been demonstrated in humans as well as in animal models. However, little is known about how these responses arise or how T cells recognize these antigens. In the present study, mice were immunized with homologous albumin covalently modified with a series of defined aldehydes which are known to be generated during lipid peroxidation. T-cell hybridomas from immunized animals demonstrated major histocompatibility complex-restricted and protein sequence-dependent responses to modified albumin, but not to native albumin. In addition to the response to modified epitopes, some aldehyde modifications resulted in strong antibody responses also to the non-modified protein. This T-cell-dependent break of tolerance constitutes a novel pathway for induction of autoimmunity by lipid peroxidation. The findings have implications in many situations where lipid peroxidation products are generated, including atherosclerosis and inflammatory and infectious diseases.

The macrophage scavenger receptor type A directs modified proteins to antigen presentation.

Scavenger receptors constitute a family of cell surface receptors that internalize endotoxins, oxidized low-density lipoproteins (oxLDL) and other proteins with clustered negative charges for degradation in macrophages. They were recently proposed to play a role in antigen presentation but the type of scavenger receptor involved in this process has not been known. In this report, we have examined the cellular immune responses to modified proteins in mice lacking the SR-A scavenger receptor (SRAKO) and their wild-type (ICR) controls. While spleen cells of ICR mice immunized with maleylated murine serum albumin (Mal-MSA) exhibit strong proliferative responses to the antigen, no such responses were found in SRAKO mice. However, addition of SR-A+ antigen-presenting cells from ICR mice unmasked proliferative responses to Mal-MSA in spleen cultures of immunized SRAKO mice. Similarly, addition of SR-A+ antigen-presenting cells was necessary to detect T cell responses in spleen cultures of oxLDL-immunized SRAKO mice. This indicates that SR-A can mediate uptake of modified antigens for presentation to antigen-specific T cells. The fact that cellular immunity developed in SRAKO mice implies that other scavenger receptor(s) also internalize modified antigens for presentation in vivo. These observations show that scavenger receptors participate in immune recognition of oxidized protein antigens; this system may be important for recognition of damaged macromolecules but could also play a role in autoimmunity.

Hypercholesterolemia is associated with a T helper (Th) 1/Th2 switch of the autoimmune response in atherosclerotic apo E-knockout mice.

Atherosclerosis is an inflammatory-fibrotic response to accumulation of cholesterol in the artery wall. In hypercholesterolemia, low density lipoproteins (LDL) accumulate and are oxidized to proinflammatory compounds in the arterial intima, leading to activation of endothelial cells, macrophages, and T lymphocytes. We have studied immune cell activation and the autoimmune response to oxidized LDL in atherosclerotic apo E-knockout mice. Autoantibodies to oxidized LDL exhibited subclass specificities indicative of T cell help, and the increase in antibody titers in peripheral blood was associated with increased numbers of cytokine-expressing T cells in the spleen. In addition to T cell-dependent antibodies, IgM antibodies to oxidized LDL were also increased in apo E-knockout mice. This suggests that both T cell-dependent and T cell-independent epitopes may be present on oxidized LDL. In moderate hypercholesterolemia, IgG antibodies were largely of the IgG2a isotype, suggesting that T cell help was provided by proinflammatory T helper (Th) 1 cells, which are prominent components of atherosclerotic lesions. In severe hypercholesterolemia induced by cholesterol feeding of apo E-knockout mice, a switch to Th2-dependent help was evident. It was associated with a loss of IFN-gamma-producing Th1 cells in the spleen, whereas IL-4-producing Th2 cells were more resistant to hypercholesterolemia. IFN-gamma but not IL-4 mRNA was detected in atherosclerotic lesions of moderately hypercholesterolemic apo E-knockout mice, but IL-4 mRNA appeared in the lesions when mice were made severely hypercholesterolemic by cholesterol feeding. These data show that IFN-gamma-producing Th1 cells infiltrate atherosclerotic lesions and provide T cell help for autoimmune responses to oxidized LDL in apo E-knockout mice. However, severe hypercholesterolemia is associated with a switch from Th1 to Th2, which results not only in the formation of IgG1 autoantibodies to oxidized LDL, but also in the appearance of Th2-type cytokines in the atherosclerotic lesions. Since the two subsets of T cells counteract each other, this switch may have important consequences for the inflammatory/immune process in atherosclerosis.

Identification and analysis of macrophage-derived foam cells from human atherosclerotic lesions by using a "mock" FL3 channel in flow cytometry.

Macrophages are one of the major cell types in atherosclerotic lesions. They are believed to play an important role in the pathogenesis and development of the lesion, but their functional state and phenotypic characteristics are not well understood. Using flow cytometry, we analyzed surface markers of macrophages extracted from tissue digests. However, conventional techniques were hampered by the abundance of cell debris and extracellular lipids, which co-localized with double-positive cells in all fluorescent plots. We therefore developed a method to overcome this problem by using a novel gating technique in multiparameter flow cytometry. This method utilized the third fluorescence channel (FL3) as a "mock" channel, since no antibody conjugated to an FL3-specific fluorochrome was added to the samples. Cells single-positive for macrophage-specific monoclonal antibodies (mAb) conjugated to phycoerythrin (PE) (FL2) were separated from non-specific fluorescent particles in the FL2 versus FL3 fluorescent plot and a region excluding debris could be set. This was then used as a gate to exclude debris also in the first fluorescence channel (FL1) vs. FL2 plot in which expression of a panel of activation markers identified by fluorescein isothiocyanate (FITC)-conjugated mAb was analyzed. Using this strategy, we were able to identify and analyze the phenotype of macrophages from human atherosclerotic lesions. We were also able to sort these cells and in this way obtained a preparation of macrophage-derived foam cells from the tissue with little contamination of debris.

Increased expression of B7-1 costimulatory molecule on cerebrospinal fluid cells of patients with multiple sclerosis and infectious central nervous system disease.

The expression of the costimulatory molecule B7-1 (BB-1; CD80) and its ligand CD28 was investigated on peripheral blood (PB) and cerebrospinal fluid (CSF) T and B lymphocytes and monocytes in 11 patients with relapsing-remitting multiple sclerosis (MS) 21 age-matched healthy controls and 10 patients with central nervous system (CNS) infectious disease (CID). Three channel flow cytometry was used with a novel gating technique in order to unambiguously identify the low numbers of B lymphocytes present in normal CSF. There was a significantly higher fraction of B7-1+ B lymphocytes in the CSF of patients with MS (72%) and CID (69%) when compared with healthy individuals (53%; p < 0.0001 and p < 0.002, respectively). Furthermore, two patients with a clinical picture of encephalitis showed a profoundly increased B7-1 expression on CSF monocytes. Comparison of absolute numbers of B7-1+ B lymphocytes/mL CSF between MS patients and healthy controls revealed a highly increased frequency of these cells among MS patients (235 cells/mL in MS patients versus 3.9 cells/mL in controls; p < 0.0001) with no overlap between the groups, which was otherwise seen for all other analyzed cell populations. We therefore hypothesize that activated B lymphocytes expressing high levels of B7-1 may be of pathogenetic importance in the development and maintenance of the MS disease.

Surface Force Studies of Langmuir-Blodgett Cellulose Films

The interactions between cellulose surfaces, between chitosan-coated surfaces, and between one cellulose surface and one chitosan-coated surface have been studied using the interferometric surface force apparatus (SFA). The cellulose surfaces were prepared by depositing trimethylsilyl cellulose on hydrophobized mica using the Langmuir-Blodgett technique. The surfaces were desilylated in a humid HCl atmosphere to obtain regenerated cellulose. ESCA measurements and wetting studies demonstrated that the desilylation process was effective. Scanning force microscopy studies showed the cellulose surfaces to be smooth with a root-mean-square roughness of 0.16 nm. Surface force and ellipsometry measurements illustrate that the cellulose film swells considerably in humid air and in water, suggesting that it is mostly amorphous. The interaction between two cellulose surfaces is dominated by a steric repulsion caused by a few dangling tails. On separation an attractive force was present both between two cellulose surfaces and between one cellulose surface and one chitosan surface. The long-range interaction between cellulose and chitosan was shown to be attractive.

Evidence for a local immune response in atherosclerosis. CD4+ T cells infiltrate lesions of apolipoprotein-E-deficient mice.

It has been suggested that immune responses are involved in the development of atherosclerosis. We have evaluated this possibility by analyzing immunocompetent cells in a murine model of the disease. Apolipoprotein E knockout (apoE -/-) mice are genetically hypercholesterolemic due to targeted disruption of the apolipoprotein E gene and develop severe atherosclerosis. Such mice were fed either standard pellets or a diet containing 1.25% cholesterol. Lesions were analyzed from mice at 9 and 16 weeks of age. Immunohistochemical staining of fatty streaks showed that CD4+ T cells were frequent, both in clusters and as single cells. In advanced atherosclerotic plaques, CD4+ T cells were prominent in the fibrous cap and subendotbelially, whereas CD8+ T cells were sparse. The CD25 subunit of the interleukin-2 receptor, which is a marker for activated T cells, was expressed in CD4-rich areas and the major histocompatibility complex class II antigen, I-A(b), which is induced by cytokines released from activated T cells, was also found in the lesions. These data indicate that CD4+ T cells participate in the formation of atherosclerotic lesions in genetically hypercholesterolemic apoE -/- mice. They suggest that immune activation is part of the disease process, and we speculate that a direct link may exist between cholesterol accumulation and T cell activation, possibly by autoimmune responses to modified lipoproteins.

Lymphocyte phenotype and subset distribution in normal cerebrospinal fluid.

The distribution of lymphocyte subpopulations in cerebrospinal fluid (CSF) and their phenotypic characteristics were extensively investigated in a group of 18 healthy individuals using two- and three-color flow cytometry. Generally, CD3+ T lymphocytes constituted the vast majority of CSF lymphocytes while the number of B lymphocytes and NK cells were low. Most T lymphocytes exhibited the phenotype of memory/primed cells in both the CD4+ and CD8+ subpopulations. Two markers for recent activation, HLA-DR and interleukin-2 receptor (CD25) were not upregulated when compared with peripheral blood (PB) in the majority of CSF T lymphocytes. However, a fraction of T lymphocytes co-expressing the NK cells markers CD56 and/or CD16 showed a pronounced upregulation of HLA-DR in CSF as compared with PB. This study documents that the cellular composition of the normal CSF differs profoundly from PB regarding all major lymphocyte subpopulations. This has to be taken into account in studies addressing questions regarding cellular immune reactions in the central nervous system under pathological conditions.

Expression of the macrophage scavenger receptor in atheroma. Relationship to immune activation and the T-cell cytokine interferon-gamma.

Scavenger receptors mediate internalization of modified lipoproteins and foam cell transformation of monocyte-derived cytokines. We investigated macrophage scavenger receptor (MSR) expression in monocyte-macrophages from human peripheral blood and in atherosclerotic lesions and analyzed its relationship to T lymphocytes and immunoregulatory cytokines by immunohistochemistry and polymerase chain reaction (PCR). Antibodies specific for the two MSR isoforms were generated by immunizing rabbits with isoform-specific synthetic peptides conjugated to keyhole limpet hemocyanin. In human atherosclerotic plaques, these antibodies stained macrophages and foam cells in a pattern that corresponded to the distribution of the macrophage marker CD68. CD3-positive T cells and alpha-actin-positive smooth muscle cells exhibited no reactivity to the anti-MSR antibodies. The frequency of cells stained with antibodies to MSR type I was equal to that of cells stained for type II, suggesting that most macrophages coexpress both isoforms. Reverse transcription (RT)-PCR analysis confirmed that both MSR isoforms were expressed in all plaques examined. There was, however, a tendency toward a lower immunohistochemical staining intensity for MSR type I and a decreased number of lipid-rich foam cells in T cell-rich areas. The mRNAs for interleukin-2 and interferon-gamma, two major products of activated T cells, were detected by RT-PCR in all plaques tested. This indicates that activation of T lymphocytes occurs in atherosclerotic plaques. Since interferon-gamma downregulates MSR expression, these observations suggest a potential mechanism for local regulation of MSR expression in the atherosclerotic plaque.

T lymphocytes from human atherosclerotic plaques recognize oxidized low density lipoprotein.

Atherosclerosis, an underlying cause of myocardial infarction, stroke, and other cardiovascular diseases, consists of focal plaques characterized by cholesterol deposition, fibrosis, and inflammation. The presence of activated T lymphocytes and macrophages and high expression of HLA class II molecules are indicative of a local immunologic activation in the atherosclerotic plaque, but the antigen(s) involved has not yet been identified. We established T-cell clones from human atherosclerotic plaques using polyclonal mitogens as stimuli and exposed the clones to potential antigens in the presence of autologous monocytes as antigen-presenting cells. Four of the 27 CD4+ clones responded to oxidized low density lipoprotein (oxLDL) by proliferation and cytokine secretion; this response was dependent on autologous antigen-presenting cells and restricted by HLA-DR. All clones that responded to oxLDL secreted interferon gamma upon activation, but only one produced interleukin 4, suggesting that the response to oxLDL results in immune activation and inflammation but may not be a strong stimulus to antibody production. No significant response to oxLDL could be detected in CD4+ T-cell clones derived from the peripheral blood of the same individuals. Together, the present data suggest that the inflammatory infiltrate in the atherosclerotic plaque is involved in a T-cell-dependent, autoimmune response to oxLDL.

Interferon-gamma modulates the fibrinolytic response in cultured human endothelial cells.

The fibrinolytic potential of the endothelial cells gives important antithrombotic properties to the vascular wall. Thrombosis is a frequent complication to atherosclerosis and other conditions where inflammatory mediators are present in the vascular wall. Inflammatory agents like lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF alpha) have been demonstrated to modulate the expression of fibrinolytic factors in cultured endothelial cells. In the present study the expression of tissue-type plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) antigen in conditioned medium from cultured human umbilical vein (HUVEC) and human saphenous vein (HSVEC) endothelial cells was investigated under basal conditions and after stimulation with LPS, TNF alpha, interferon-gamma (IFN-gamma) or interleukin-6 (IL-6) alone or in combinations. Stimulation with LPS or TNF alpha increased the expression of PAI-1, u-PA and PAI-2 in HUVEC and HSVEC, while the t-PA response differed between the two cell types. The effects of TNF alpha were modulated by IFN-gamma but not by IL-6. The increased expression of u-PA after stimulation with TNF alpha was reduced by IFN-gamma. In contrast, TNF alpha-induced expression of PAI-2 was synergistically increased by addition of IFN-gamma. These effects of IFN-gamma represent additional mechanisms by which inflammatory mediators may turn the fibrinolytic potential of the endothelium in a prothrombotic direction.

Reduced frequency of memory CD8+ T lymphocytes in cerebrospinal fluid and blood of patients with multiple sclerosis.

Three color flow cytometry was used to analyze immunoregulatory lymphocyte subsets in peripheral blood (PB) and cerebrospinal fluid (CSF) of 21 patients with multiple sclerosis (MS) and 15 age-matched healthy control subjects. Two cell surface antigens associated with T lymphocyte memory and activation, CD45R0 and CD29, were analyzed on the CD4+ and CD8+ subpopulations, respectively. A selective decrease in the expression of the CD45R0 isoform among CD8+ cells was noted in both PB (p < 0.005) and CSF (p > 0.0001) of patients with MS as compared with the control group while the expression of CD29 did not differ between the groups. These changes could indicate a defective differentiation into mature memory CD8+ T lymphocytes in patients with MS. Furthermore, the CD3+CD16/56+ T lymphocyte subset capable of mediating NK cell-like activities was investigated. Although this cell population is quantitatively small, a significant reduction of the proportion of this cell type was detected in both BP and CSF of the MS group compared with the controls (p < 0.01 and p > 0.001, respectively). Further studies are needed to establish the role of these observations in the pathogenesis of MS.

Expression of plasminogen activator inhibitor-1 mRNA in healthy, atherosclerotic and thrombotic human arteries and veins.

Plasminogen activator inhibitor-1 (PAI-1), specific inhibitor of plasminogen activators (PA), plays an important role in the regulation of fibrinolysis. Increased levels of PAI-1 have been associated with vascular disease such as thrombosis and atherosclerosis. In the present study the expression of PAI-1 mRNA in human healthy, atherosclerotic and thrombotic blood vessel walls was quantified by RNA-RNA hybridization in solution and localized by in situ hybridization. The mean expression of PAI-1 mRNA was significantly higher in healthy arteries (0.86 pg/microgram total RNA) than in healthy veins (0.29 pg/microgram total RNA), p < 0.01. The mean PAI-1 mRNA expression in thrombotic arteries (1.72 pg/micrograms total RNA) was significantly higher than that in healthy arteries, p < 0.05, and the mean PAI-1 mRNA expression in thrombotic veins (1.29 pg/micrograms total RNA) was significantly higher than that in healthy veins, p < 0.01. By in situ hybridization PAI-1 mRNA was detected in the intima, media and adventitia of healthy arteries and healthy veins. In atherosclerotic arteries PAI-1 mRNA was detected in the atherosclerotic plaque and in the medial and adventitial layers below the plaque. An increased expression of PAI-1 mRNA was found in the intimal layer of a thrombotic vein. The increased expression of PAI-1 mRNA in thrombotic arteries and veins indicates a role for PAI-1 in thrombogenesis.