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The prognostication of patient outcome is one of the greatest challenges in the management of early stage oral tongue squamous cell carcinoma (OTSCC). This study introduces a simple histopathological model for the prognostication of survival in patients with early OTSCC. A total of 311 cases (from Finland and Brazil) with clinically evaluated early stage OTSCC (cT1-T2cN0cM0) were included in this multicentre retrospective study. Tumour budding (B) and depth of invasion (D) were scored on haematoxylin-eosin-stained cancer slides. The cut-off point for tumour budding was set at 5 buds (low

The aim of this study was to investigate the expression of Nrf2, sulfiredoxin and DJ1 in pancreatic cancer. The expression of Nrf2, sulfiredoxin and DJ1 was studied immunohistochemically in a large set of pancreatic adenocarcinomas consisting of 103 cases. Eighty six percent of the cases showed cytoplasmic Nrf2 and 24% nuclear Nrf2 positivity. Sulfiredoxin positivity was observed in 54% and DJ1 positivity in all cases. Nuclear Nrf2 positivity had an association with sulfiredoxin (p=0.019) and was associated with a poor survival (p=0.010). Stage IV tumors tended to have a more nuclear Nrf2 expression (p=0.080). DJ1 expression was more often found in well-differentiated tumors (p=0.012), and DJ1 expression was associated with better survival (p=0.020). According to the results, nuclear Nrf2 expression predicts a worse survival in pancreatic adenocarcinoma, which is in keeping with its protection of cells against oxidative or xenobiotic stress. In accordance with Nrf2's regulation of the synthesis of sulfiredoxin, there was an association between them (p=0.019). DJ1 had no association with Nrf2, and its expression predicted a better survival of patients.

It has been proposed that an epithelial injury may be one of the multiple primary events in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The aim of this study was to characterize the tight junction and adherens junction proteins in normal human lung, IPF, cryptogenic organizing pneumonia, and asbestosis. We determined the immunohistochemical cell-specific expression of tight junction proteins claudin-1, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-7, as well as 3 adherens junction proteins, E-cadherin, N-cadherin, and β-catenin. We further analyzed the expression of claudin-1, claudin-3, and claudin-4 and E-cadherin, N-cadherin, and β-catenin at the transcriptional level by quantitative real-time reverse transcriptase polymerase chain reaction. The expression levels of both tight junction and adherens junction proteins were elevated in regenerative alveolar epithelium in pulmonary fibrosis as compared with the expression of these proteins in normal alveolar epithelium. In particular, the expression levels of claudins-1 and claudin-3 were clearly elevated in all diseases. Furthermore, the amounts of adherens junction proteins messenger RNAs (mRNAs) were also all increased in pulmonary fibroses in comparison with healthy controls, with N-cadherin showing the greatest increase in mRNA levels in all diseases. However, the amounts of claudin-1, claudin-3, and claudin-4 mRNAs in fibrotic lung were similar to or even lower than those measured in the healthy controls. It is possible that the diminished capacity to produce claudin mRNAs may be one explanation for poor repair capacity of alveolar epithelial cells in IPF.

Background Hypoxia inducible factor-1alpha (HIF-1α) is a transcription factor that regulates the transcription of genes associated with cell proliferation and angiogenesis. The purpose of this study is to clarify the correlation of HIF-1α protein expression with vascular endothelial growth factor (VEGF) and inducible (iNOS), endothelial (eNOS), and neuronal nitric oxide synthase (nNOS) expression in esophageal tumors. Additionally, vascular density in tumor tissue was assessed. Materials and Methods Eighty-eight esophageal carcinomas were analyzed by immunohistochemistry in paraffin embedded sections. Results HIF-1α immunoreactivity was seen in 71.2 % of the tumors. Squamous cell carcinomas expressed more often HIF-1α than adenocarcinomas (P = 0.009). HIF-1α immunoreactivity was associated with iNOS (P = 0.049), and iNOS positivity was also more commonly seen in squamous cell carcinomas than adenocarcinomas (P = 0.016). VEGF immunoreaction tended to associate with HIF-1α (P = 0.073) and iNOS (P = 0.08). ENOS did not associate with HIF-1α, but tended to associate with VEGF (P = 0.072). T1-T2 tumors were more often VEGF negative than T3-T4 tumors (P = 0.063). In the subgroup of 78 operatively treated ECs patients with HIF-1α positivity (>+) had more often distant metastases (P = 0.036). There was no association between iNOS, eNOS, nNOS, or VEGF, and microvessel density in tumor tissue, tumor marginal zone, or in peripheral tissue. Conclusions These results show that there is a link in expression between HIF-1α, iNOS, (eNOS), and VEGF in esophageal cancer. This is in line with the fact of HIF-1α's function as a transcriptional factor for these angiogenic factors. Results also show that squamous cell and adenocarcinomas differ in their expression of HIF-1α and iNOS. VEGF appear to have association with depth of invasion in esophageal carcinomas. In our material HIF-1α positivity was associated with distant metastases, but not with patient survival.

Tight junctions regulate the paracellular permeability and orientation of cells and claudins are key components of tight junctions.To study the influence of cigarette smoke on claudin expression in vitro and in lung cancer patients.We studied the effect of smoking on claudin expression by exposing a bronchial cell line (BEAS-2B) and two carcinoma cell lines (SK-LU1 and SK-MES1) to tobacco smoke for 48 h and analysed their claudin mRNA expression. The relation between smoked pack years and protein expression of claudins 1-5 and 7 in 344 lung cancer patients was determined by immunohistochemistry.In BEAS-2B cells and SK-LU1 cells, an initial increase was followed by a decline in the mRNA expression of several claudins. In SK-MES1 cells, no evident elevation in claudin expression was observed. Intense claudin 1 and 4 positivity was found more often in cancer samples of smokers and ex-smokers compared to non-smokers (p0.001 and p=0.003, respectively). Heavy smokers with longer than 40 pack-years consumption had more often intense claudin 1 (p=0.011), 4 (p=0.050) or 7 (p=0.058) expression in squamous cell carcinoma compared to non-smokers or smokers with fewer pack-years. Claudin 1 positivity predicted a better survival in adenocarcinoma (p=0.044) and in squamous cell carcinoma (p=0.027) and claudin 4 positivity in adenocarcinoma only (p=0.048). In squamous cell carcinoma, claudin 7 positivity was associated with a better survival (p=0.011).Bronchial BEAS-2B cells and SK-LU1 cells respond to tobacco smoke by changing their claudin mRNA synthesis and resulting tight junction permeability changes may thus contribute to tobacco induced carcinogenesis both during initiation and progression. This concept is strengthened by findings in the clinical tumour material, where tobacco consumption was associated with claudin expression.

Oral (mobile) tongue squamous cell carcinoma (OTSCC) is the most common cancer diagnosed within the oral cavity. Due to the inherent disadvantages of the mobile tongue OTSCC behaves aggressively and is generally associated with higher rates of occult metastasis and neck nodal metastasis than any other cancer of the oral cavity. The prognosis remains relatively poor and is still heavily reliant on TNM (tumor, node, metastasis) staging of the tumor despite a vast array of literature on possible prognostic indicators. This is a two-part article which examines the methods by which the behavior and prognosis of OTSCC has been studied, the prognostics markers, and the relevance and future direction of prognostic studies. In this first part, we discuss the relative merits of the methods used in prognostic studies and the clinicopathologic prognostic factors.

Squamous cell carcinoma of the oral (mobile) tongue (OTSCC) is increasingly regarded as a biologically different entity compared to cancer affecting other oral sites. It is more aggressive and generally associated with a higher rate of metastasis. This is the concluding part of our two-part article that examines the methods by which the behavior and prognosis of OTSCC has been studied, the prognostics markers, and the relevance and future direction of prognostic studies. In this part, we continue our discussion of the histopathologic and molecular prognostic factors, and serum and salivary biomarkers in of OTSCC, and emphasize the need to regard OTSCC as a high risk variant of oral cancer. We conclude with future direction of prognostic studies of OTSCC.

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.

Summary Sarcoidosis, atypical mycobacteriosis, and tuberculosis are common diseases of human lung with a typical feature of formation of granulomas. The structure of granulomas has not been elucidated completely. We studied the expression of tenascin-C, precursor proteins of collagens I and III, and the presence of myofibroblasts in granulomas of sarcoidosis, atypical mycobacteriosis, and tuberculosis of human lung. Twenty-five histologic samples of lung were analyzed by immunohistochemistry using antibodies to tenascin-C and aminoterminal propeptides of collagens I and III. To identify the myofibroblast-type cells in granulomas, the sections were also stained with antibodies against α –smooth muscle actin, vimentin, and desmin. In every case, tenascin-C and precursor proteins of collagens I and III were expressed around granulomas. Precursor protein of collagen I was expressed also within them. In tuberculosis and atypical mycobacteriosis, expression of tenascin-C and precursor protein of collagen I was stronger than in sarcoidosis. The cells demarcating granulomas and, thus, colocalizing with tenascin-C and both collagen precursors were positive for α –smooth muscle actin and vimentin, which suggests that these cells are myofibroblasts. They were also more abundantly present in tuberculosis and atypical mycobacteriosis, as suggested by α –smooth muscle actin staining. We concluded that tenascin-C and precursor proteins of collagens I and III are expressed around granulomas in sarcoidosis, atypical mycobacteriosis, and tuberculosis of the lung; and furthermore, their expression colocalize with the expression of myofibroblasts. Our results further point to the fact that fibrogenesis and matrix turnover is stronger in tuberculosis and atypical mycobacteriosis than in sarcoidosis.

Objective. Matrix metalloproteinases have long been associated with aggressive behavior of several malignancies, but their role in endometrial cancer has not been conclusively established. This study aimed to evaluate the roles of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) as prognostic factors in endometrial carcinoma and their association with CA 125 and other conventional prognostic markers. Methods. The MMP-2 and MMP-9 immunoreactive proteins were evaluated from primary tumors of endometrial carcinoma in 266 specimens by using a specific monoclonal antibody in immunohistochemical stainings. The median follow-up time was 79 months. Results. Expression of the MMP-2 and MMP-9 proteins was found in 88% and 70% of the primary tumors, respectively. Positive MMP-2 immunostaining was associated with a shortened recurrence-free (P=0.04) and cancer-specific survival (P=0.05). MMP-2 negativity was linked with a favorable prognosis; only one patient developed recurrent disease and died during the follow-up. Preoperative serum levels of CA 125 were higher in the patients presenting with tumors positive for MMP-2 than in those with negative immunostaining (P=0.03). Conclusions. We suggest that MMP-2 is linked with biologically aggressive nature of this cancer type. It seems that MMP-2, but not MMP-9, has some prognostic value in endometrial carcinoma. However, the conventional prognostic markers are superior to MMP-2 in assessing aggressive behavior and cancer-specific survival in endometrial cancer.

Changes in the collagenous matrix may contribute to the pathogenesis and progression of human aortic valve stenosis (AS). To evaluate the significance of collagen I and III in the pathogenesis of AS, we studied their synthesis in diseased valves. Type I and type III collagen mRNA expression and the immunohistochemical localization of the collagen antigens were studied from 36 AS and 2 normal aortic valves. The concentrations of propeptides and telopeptide structure of type I (PINP, PICP, and ICTP) and those of III collagens (PIIINP and IIINTP) were measured by radioimmunoassays in soluble tissue extracts and trypsin-solubilized calcified and non-calcified matrices of 11 AS and 24 healthy aortic valves of different ages. The synthesis of type I collagen, localized in the myofibroblasts adjacent to calcified nodules, was two- to three-fold in the AS samples compared to the controls. The proportion of collagen in the total protein fraction was 90% in the healthy valves, 50% in the non-calcified matrix, and 10% in the calcified matrix of AS valves. In the calcified valves, the ICTP content was six-fold compared to the age-matched controls and two-fold compared to the young control group. In the controls, the amount of ICTP in type I collagen decreased with age (r=-0.908, p

Objective Tetracycline derivatives affect many cellular functions relevant to chronic cardiovascular pathologies, including cell proliferation, migration and matrix remodelling. Accordingly, we sought to determine whether they may modulate the pathologic characteristics known to be significantly involved in human aortic valve stenosis, such as gelatinase production, apoptosis, expression of vascular endothelial growth factor (VEGF) and tumour necrosis factor-α (TNF-α). Methods The effects of tetracycline derivatives (tetracycline and CMTs-3, -5, -8) on MMP-2 and -9 and their endogenous tissue inhibitor (TIMP-1 and -2) production profiles in explanted human aortic valve pieces were examined by means of gelatine zymography and reverse zymography. Chemiluminescent ELISA was performed to assess VEGF and TNF-α concentrations in the medium, and in order to evaluate programmed cell death, in situ labelling of the 3′-ends of the DNA fragments generated by apoptosis-associated endonucleases was performed. Results CMT-3 and -8 lowered the MMP-9 and VEGF levels significantly in a drug-, dose-, and time-dependent manner. MMP-2 and TIMPs remained unchanged, emphasizing the specificity of CMTs to MMP-9 production on the one hand and restoring the beneficial equilibrium of MMP-9 and TIMPs on the other. Tetracycline was the only drug with a significant impact on net gelatinolytic activity, suggesting that the effect of tetracycline is more extensive concerning total MMP activity. Conclusions Tetracycline derivatives may have therapeutic effects on the pathologic remodellation of advanced human aortic stenosis through the inhibition of MMP-9 and VEGF production.

Matrix metalloproteinases (MMPs) are involved in tumor growth and spreading. Here, we investigated the tumor immunoreactive protein of MMP-2, MMP-9 and TIMP-1 as well as the levels of circulating total TIMP-1 and MMP-2/TIMP-2-complex as prognostic factors in lung cancer patients. The material included 59 patients, 30 with a squamous cell carcinoma, 21 with an adenocarcinoma and eight with other histology. Circulating antigens were measured by ELISA assay and the protein expression in primary tumors was analyzed by streptavidin–biotin immunohistochemical staining using specific monoclonal antibodies. The strong positivity for MMP-2 or MMP-9 in tumor predicted poor prognosis. The 5-year survival rates were 83 or 85% in patients negative for MMP-2 or MMP-9, respectively. Only 17% of the patients with a tumor highly positive for MMP-2 and 43% of those with a high positivity for MMP-9 survived at that time (Cox regression P=0.042 for MMP-2 and log rank P=0.046 for MMP-9). On the contrary, strong tissue positivity for TIMP-1 demonstrated a tendency for a favorable survival, although the difference did not reach statistical significance. In patients with a squamous cell carcinoma Stage I, low serum TIMP-1 (≤300 ng/ml) also predicted unfavorable survival (log rank P=0.033). Further in subgroup of patients treated by lobectomy (n=29), the high levels of circulating MMP-2/TIMP-2-complex (≥300 ng/ml) associated with an increased survival rate, the 5-year survival being 81 versus 34% (log rank P=0.069) in patients with high or low serum levels for MMP-2/TIMP-2-complex, respectively. Tissue MMP-2 correlated to high expression of MMP-9 immunoreactive protein (P=0.003), but the serum levels of MMP-2/TIMP-2-complex or TIMP-1 did not correlate to the immunostaining of the corresponding tumors. We conclude that in lung carcinoma the best prognostic value is achieved by using immunohistochemistry for MMP-2 and MMP-9. In early disease, however, serum TIMP-1 or MMP-2/TIMP-2-complex could offer some further prognostic value.

The prognostic and predictive relevance of p53 immunoreactivity is used here as a tentative approach for defining more accurately the benefit of adjuvant hormonal therapy in postmenopausal node-positive breast cancer patients. Ninety-seven postmenopausal patients with axillary lymph node metastasis were treated with an antiestrogen for a period of 3 years after primary surgery and radiotherapy. The p53 status of the primary tumor was assessed by immunohistochemistry and 24% of the samples showed positive expression of p53. Within the average follow-up time of 59 months, disease recurrence was diagnosed in 34 patients (35%). Multivariate analysis showed high clinical stage, negative estrogen receptor status and p53 positivity to be independent prognostic factors predicting both shortened disease-free survival and worse overall survival. p53 immunoreactivity was associated with worse clinical outcome irrespective of hormone receptor status. The data suggest that adjuvant therapy with antiestrogens is insufficient in this patient population with p53-positive tumors.

Claudins are involved in the formation of tight junctions in epithelial and endothelial cells. The present study evaluated the expression of claudin 2, 3, 4, and 5 in 20 cases of Paget's disease (13 mammary and 7 extramammary cases), and compared the results with those of other neoplastic skin lesions, including actinic keratoses, basal cell carcinomas, and malignant melanomas. To compare claudin expression in Paget's disease and breast neoplasia, it was also studied in a large set of breast carcinomas. Membrane-bound claudin 3 and 4 expression was seen in all cases of Paget's disease, whereas claudin 5 was seen in 50% of cases and claudin 2 was seen in 32% of cases. In contrast, claudins 3, 4, and 5 were not seen in the other skin lesions, and claudin 2 was seen in most of them, suggesting an inverse expression of these claudins between Paget's disease and epidermal and nevocytic lesions. Claudin expression in breast carcinomas was claudin 2 in 52%, claudin 3 in 93%, claudin 4 in 92%, and claudin 5 in 47%. Claudins 2 and 5 were found more often in ductal carcinomas than in lobular carcinomas. Expression of claudins were frequently associated with each other. They were not associated with estrogen or progesterone receptor status or with tumor grade. No significant differences were found between claudin expression in Paget's disease and breast carcinomas. The results demonstrate that claudins could be useful in diagnosing Paget's disease and in differentiating these lesions from other epidermal lesions, such as actinic keratoses, basal cell carcinomas, and nevocytic lesions. The lack of difference in claudin expression between Paget's disease and breast tumors suggests that changes in the phenotype of claudins 2, 3, 4, and 5 are not necessary for epidermal invasion.

Glutaredoxins (Grx) are thiol-disulfide oxidoreductases with antioxidant capacity and catalytic functions closely associated with glutathione, an antioxidant abundantly present in human lung. The present study investigated the expression of both human glutaredoxins in cultured human lung cells and lung homogenates by reverse-transcription polymerase chain reaction and Western blotting. Immunohistochemical studies were conducted with 38 human lung specimens, including healthy lung, parenchymal sarcoidosis, extrinsic allergic alveolitis, and usual interstitial pneumonia (UIP). The ultrastructural localization of Grx1 was assessed by immunoelectron microscopy. In addition, cultured airway epithelial cells were exposed to tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta. Both Grx1 and Grx2 could be detected at the mRNA and protein level in cultured human lung cells, but only Grx1 was prominently expressed in lung homogenates and alveolar macrophages. Immunohistochemically, Grx1 was highly concentrated to alveolar macrophages and weakly positive in the bronchial epithelium. Grx1 was ultrastructurally localized to the plasma membrane, cytoplasmic vacuoles, and nucleus. The expression of Grx1 decreased in alveolar macrophages of sarcoidosis and allergic alveolitis compared with the case for controls (P0.001), and bronchial epithelium of these diseases revealed no Grx1 immunoreactivity. Fibroblast foci and other fibrotic areas in UIP were mainly negative. In A549 cells, Grx1 was down-regulated by TGF-beta, whereas TNF-alpha caused no clear effect. Overall, high expression of Grx1 in alveolar macrophages suggests its importance in the primary defense of human lung. Decreased expression of Grx1 further suggests the impairment of this system both in inflammatory and fibrotic lung diseases, consistent with the down-regulation of Grx1 by TGF-beta in vitro.

The pathogenesis of interstitial lung diseases (ILDs) is known to be associated with reactive oxygen and nitrogen metabolites and increased oxidant stress. One of the major antioxidants in human lung is glutathione (GSH) and enzymes linked to its synthesis. The rate-limiting enzyme of GSH synthesis is gamma-glutamylcysteine synthetase (γ-GCS) containing catalytically active heavy (γ-GCSh) and regulatory light (γ-GCSl) subunits. It can be hypothesized that γ-GCS is the major determinant in explaining reduced GSH levels in fibrotic lung disorders. We investigated the regulation of γ-GCS by transforming growth factor beta 1 (TGF-β 1 ) and tumor necrosis factor alpha (TNF-α) in human lung cells and its expression and distribution in fibrotic (biopsy-proven idiopathic pulmonary fibrosis, for instance, usual interstitial pneumonia, UIP, n=15), inflammatory, and granulomatous diseases of human lung parenchyma (desquamative interstitial pneumonia, n=10; ILD associated with collagen diseases, n=10; sarcoidosis, n=19 and allergic alveolitis, n=8). In human lung alveolar epithelial cells, γ-GCSh was decreased by TGF-β 1 , whereas TNF-α caused a transient enzyme induction. In normal lung, γ-GCS was mainly localized to the bronchiolar epithelium. In UIP, the highest immunoreactivities were observed in the airway epithelium and metaplastic alveolar epithelium, but fibroblastic foci were negative. In sarcoidosis, the highest reactivities were detected in the epithelium, alveolar macrophages and pulmonary granulomas. γ-GCS was ultrastructurally localized to the cytoplasm of regenerating type II pneumocytes and macrophages. In conclusion, γ-GCS is widely expressed in sarcoidosis and regenerating epithelium but is low in the fibrotic areas of usual interstitial pneumonia, probably because of enzyme down-regulation.

Background Recently, aortic valve stenosis has been demonstrated to exhibit increased expression of certain matrix metalloproteinases (MMPs), and this has relevantly raised the question about possible interdependency between these and their tissue inhibitors. We sought to assess the expression of elastolytic MMPs and their inhibitors (TIMPs) in nonrheumatic aortic stenosis. Methods The study comprised 30 stenotic and six noncalcified human aortic valves. To measure the expression levels and the amount and molecular forms of gelatinases (MMP-2, MMP-9) and TIMPs (1, 2), in situ hybridization, gelatin zymography, and reverse zymography were carried out. Antielastin staining by a monoclonal BA-4 antibody was performed to investigate the changes of one of the main substrates of these MMPs, and to substantiate the nature of the putative MMP- synthesizing cell. The cases were also immunostained with an antibody to α-smooth muscle actin. Inflammatory cell characterization was managed by monoclonal mouse antibodies (UCHL-1, L26, and PGM-1). Results Compared with the controls, the calcific valves showed increased mRNA expression and activation of MMP-9, and this was associated with typical characteristics of valve disease. MMP-2 mRNA production was rare, but proMMP-2 protein was detected in all valves. In agreement with the interdependency between MMP-9 and its inhibitors, a suggestive imbalance came out in diseased valves. Conclusions The disproportion between MMP-9 and its tissue inhibitors may favor a persistent MMP activation state within the calcific valve and likely contribute to the valvular remodeling process in the setting of developing aortic stenosis.

Angiogenesis is an essential biological process not only in embryogenesis, but also in the progression of several major diseases, including cancer, diabetes, and inflammation. Excessive vascularization can also contribute to some cardiovascular pathologies, such as atherosclerosis, but contradictory reports still prevail regarding its impact on aortic stenosis. Using immunohistochemical techniques, we assessed the vascular density and distribution of angiogenesis (FVIII) and vascular endothelial growth factor (VEGF) expression as well as the expression of 2 VEGF receptors, Flt-1 and Flk-1, in 55 nonrheumatic and 6 control aortic valves. In the light of the fact that the angiogenic effect of VEGF is mediated by sustained formation of nitric oxide, the samples were also immunostained with 3 nitric oxide synthase (eNOS, iNOS, and nNOS) antibodies. The immunohistochemical findings of VEGF and its receptors were verified by immunoblotting techniques. Vascular density was highest in the cases with moderate valve stenosis, and the mean number of FVIII-positive blood vessels was 1.7 +/- 1.9 vessels/mm(2) in the diseased valves, whereas the normal valves contained no blood vessels. Vascular density was significantly higher in the cases showing chronic inflammation (P = 0.007). Interestingly, the patients receiving statin therapy had significantly lower vascular densities than those not receiving such therapy (P = 0.001). Diseased valves showed distinct VEGF, Flt-1, Flk-1, and eNOS positivity of activated endothelial, stromal fusiform myofibroblastic, and histocytic cells. In contrast, immunoreactivity for iNOS and nNOS was seen only in nonendothelial stromal cells, and their expression was weaker. Enhanced vascular density was significantly associated with increased expression of Flk-1 (P = 0.028 for endothelial and P = 0.009 for stromal cells) and with endothelial eNOS expression (P = 0.024). A similar tendency was also observed for VEGF, but not for Flt-1. Our results show a distinct angiogenic response and the presence of angiogenic factors in nonrheumatic aortic valve stenosis, suggesting that angiogenesis may influence on the evolution of this disease.

This study evaluates the prognostic and predictive relevance of a mutated p53 in a series of 254 samples from primary breast cancer patients. C-erbB-2 analysis was defined in a limited subpopulation of 79 patients. p53 and c-erbB-2 status was analysed by immunohistochemical staining of the tumour samples. Positive p53 immunostaining was present in 86 cases (34%) and correlated with a high malignant grade, negative progesterone receptor status and ductal histology of tumour. C-erbB-2 positivity was seen in 38 samples (48%). Within an average follow-up time of 74 months, 121 patients developed recurrent or metastatic disease. Patients with mutated p53 showed a statistically significant shorter overall survival and disease-free survival in both univariate and multivariate analyses. The worst clinical outcome was seen in patients who were both p53- and c-erbB-2-positive. The response rate to anthracycline-based chemotherapy in metastatic disease was low in the p53-positive cases. Our results help to clarify the independent prognostic role of a mutated p53 status in breast cancer patients, indicating that this gene might be predictive of anthracycline resistance. Patients with a mutant p53 status and overexpressing c-erbB-2 should be regarded as high-risk cases.

We investigated immunohistochemical expression of manganese superoxide dismutase (MnSOD) and three hydrogen peroxide (H(2)O(2)) scavenging pathways, i.e. catalase (CAT), gamma-glutamyl cysteine synthetase (gammaGCS) and thioredoxin (Trx) system in normal bronchial epithelium, bronchial metaplasia and dysplasia and correlated their expression with NF-kappaB activation (p50) and proliferation (Ki67). Normal bronchial epithelium was positive for MnSOD, heavy and light subunits of gammaGCS, CAT and Trx and TrxR. Metaplastic epithelium showed strongest expression of gammaGCSh and Trx, whereas dysplastic epithelium expressed most prominently MnSOD and CAT. There was a significant correlation between expression of gammaGCSh and gammaGCSl (P=0.034) and Trx and TrxR (P=0.037). Trx expression also correlated with gammaGCSh (P0.001) and gammaGCSl (P=0.012) and TrxR with gammaGCSh (P0.001) but not with gammaGCSl immunoreactivity (P=0.744). Expression of p50 was highest in metaplastic epithelium while Ki67 was highest in dysplastic lesions. Expression of Trx and gammaGCSh correlated inversely with age of the patients (R=-0.6038, P0.001 for Trx and R=-0.6162, P0.001 for gammaGCSh). Changes in the expression of these enzymes in bronchial lesions might be due to alterations of antioxidative mechanisms due to irritation via exogenous toxins and activation of reactive oxygen species (ROS) known to be associated with induction of metaplasia and dysplasia in the bronchial tree.

Mesothelioma is a fatal tumor resistant to all treatment modalities for reasons that are still unresolved. Glutathione (GSH)-associated pathways are induced by oxidants and cytotoxic drugs, and they are also involved in the progression and resistance of some tumor cells in vitro. The rate-limiting enzyme in GSH biosynthesis is gamma-glutamylcysteine synthetase (gamma GCS). However, the expression of this enzyme has not been systematically investigated in malignant tumors, and there are no studies of gamma GCS in biopsy specimens of malignant mesothelioma. We investigated the immunohistochemical distribution and expression of both subunits of gamma GCS in healthy pleural mesothelium, pleural mesothelioma tumor biopsy samples (34 cases), and mesothelioma cells in culture (7 cell lines). Nonmalignant mesothelium showed no immunoreactivity for either subunit in any of the cases. The heavy (catalytic) subunit of gamma GCS was highly immunostained in 29 and weakly positive in 5 cases. High-moderate and weak immunoreactivity of the light (regulatory) subunit of gamma GCS was found in 15 and 7 tumors, respectively, whereas 12 cases showed no reactivity. There was no correlation with either catalytic or regulatory subunit expression and patient survival. There was, however, a significant correlation between the heavy chain and multidrug resistance protein (MRP) 2 (P =.048), whereas no correlation was observed between the light chain and MRP1 or MRP2. Treatment of cultured mesothelioma cells with buthionine sulfoximine (BSO), to inhibit gamma GCS, significantly potentiated cisplatin-induced cytotoxicity mainly by nonapoptotic mechanism when assessed by counting the living cells, TUNEL (terminal deoxytransferase-mediated dUTP nick-end labeling) assay, and caspase-3 cleavage. In conclusion, gamma GCS is highly positive in most cases of malignant mesothelioma and may play an important role in the primary drug resistance of this tumor in vivo.

Exhaled carbon monoxide (CO), which has been found to be elevated in asthma, is generated primarily by heme oxygenase 1 (HO-1), an enzyme induced by oxidant stress and cytokines. The aim of this study was to assess the distribution and expression of HO-1 in various human lung cells in acute and stable asthma.Normal lung tissue biopsies (from 6 non-smoking subjects operated on for a lung tumour) and macrophages from induced sputum (from 5 healthy controls, 5 untreated asthmatics, 7 stable treated asthmatics and 5 asthmatics recovering from exacerbation and being on systemic steroids) were investigated for HO-1 by immunohistochemistry. The time response of HO-1 induction was examined in cultured monocytes, which are known to maturate into monocyte-derived macrophages in culture. Lung biopsies showed prominent HO-1 immunoreactivity only in alveolar macrophages. Macrophages in the induced sputum of healthy controls showed no HO-1 immunoreactivity, with the exception of one case. Moderate or intense HO-1 immunoreactivity could be observed in alveolar macrophages in 4/5 cases with recent asthma, and 2/7 with stable asthma, but in none of the patients treated with systemic corticosteroids for acute exacerbation. Experiments with cultured cells revealed that HO-1 was induced by oxidants within the first 24 h, but the induction was reversed during the next 48 h. HO-1 is mainly expressed in alveolar macrophages of human lung. Macrophages of induced sputum show prominent but transient HO-1 immunoreactivity, in untreated asthmatics, but not in asthmatics treated with corticosteroids.

We investigated the role of glutathione and nitric oxide synthase (NOS) in fiber-induced cell and DNA toxicity using alkaline (pH 13) single-cell gel electrophoresis (the Comet assay). Transformed cultured human pleural mesothelial (MeT-5A) cells and alveolar epithelial cells (A549) were exposed to crocidolite asbestos fibers (1-10 microg/cm(2)) in the presence of buthionine sulfoximine (BSO) or L-arginine-methyl ester (L-NAME). BSO inhibits gamma-glutamylcysteine synthetase (gamma-GCS) and causes glutathione depletion, and L-NAME inhibits nitric oxide generation. Studies were also conducted to assess the expression of the heavy and light subunits of gamma-GCS in human pleural mesothelium and bronchial epithelium in vivo and the induction of inducible NOS (iNOS) by asbestos fibers. Asbestos fibers caused DNA single-strand breaks, and the process was significantly enhanced by BSO (69% compared to the non-treated cells). A549 cells had a 3.5-fold glutathione content compared to MeT-5A cells, which was consistent with the higher resistance of these cells against oxidants and fibers. Flow cytometry of iNOS showed no change of iNOS by the fibers in either cell type in vitro. L-NAME had no effects on the DNA single-strand breaks in the Comet assay, either. Studies on lung biopsies showed that the immunoreactivities of both gamma-GCS subunits were very low in healthy human mesothelium in vivo. We conclude that glutathione may play an essential role in protecting intact cells against fiber-induced oxidative DNA alterations, and low gamma-GCS reactivity in pleural mesothelium may be associated with the high sensitivity of mesothelial cells to fiber-induced toxicity.

The aim of this work was to evaluate the prognostic and predictive values of c-erbB-2 in breast cancer. 650 patients were enrolled. The amplification/overexpression of c-erbB-2 from fresh frozen or paraffin-embedded breast tumour tissue samples was analysed by polymerase chain reaction (PCR) technique (75%), immunohistochemically (17%) or by Southern blot analysis (8%). 126 patients (19%) were positive for c-erbB-2. 148 patients developed metastatic disease, but only 35 were positive for c-erbB-2. Positivity for c-erbB-2 was significantly associated with node positivity, large tumour size, high grade of malignancy, low receptor status, postmenopausal status, and with a shorter overall survival. In multivariate regression analysis, only tumour size and nodal involvement were risk factors for poor survival when analysed separately together with c-erbB-2 and receptor status. Metastatic patients with c-erbB-2 positivity had a significantly shorter survival and disease-free survival (DFS) than the c-erbB-2-negative patients. 29 advanced patients with c-erbB-2 positivity showed a poor response rate to hormonal, non-anthracycline-based and anthracycline-based therapies. Positivity for the c-erbB-2 is a poor prognostic factor in breast cancer, but it also emerges as predictive of the response to hormonal or chemotherapy treatment once the disease has recurred.

Purpose: This study aimed to characterize the distribution of structural domains of type I and III collagens in the wall of abdominal aortic aneurysms (AAAs), by the use of undilated atherosclerotic aortas (aortoiliac occlusive disease [AOD]) and healthy abdominal aortas as controls. Methods: Immunohistochemical staining was applied with antibodies for the aminoterminal propeptides of type I (PINP) and type III (PIIINP) procollagens, which represent newly synthesized type I and III pN-collagens. In addition, an antibody against the aminoterminal telopeptide of type III collagen (IIINTP) was used as a means of detecting maturely cross-linked type III collagen fibrils. Results: The newly synthesized type III procollagen detected by means of PIIINP staining was concentrated in the media in aneurysmal aortas, whereas type I pN-collagen was localized in the intima in both AAAs and AODs. The healthy aortas showed no immunoreactivity for either PIIINP or PINP. The cross-linked type III collagen, detected by means of IIINTP staining, stained transmurally in all study groups, but appeared more abundant in the media in AAAs. Conclusion: Our results strongly suggest that the metabolism of type III collagen is enhanced in AAAs. Intensive type III pN-collagen staining was present mainly in the media layer in AAAs, suggesting a role of type III collagen in aneurysm formation, whereas type I pN-collagen was present in the intima in both AAAs and AODs, suggesting that type I collagen synthesis is a fibroproliferative response related to the atherosclerotic process. The increased type III pN-collagen in AAAs may result in impaired fibril formation and, thus, in decreased tensile strength of aneurysmal tissue. (J Vasc Surg 2000;32:1201-7.)

Mutations in BRCA1 and BRCA2 account for a large portion of the inherited predisposition to breast and ovarian cancer. It was recently discovered that mutations in these two genes are less common in the Finnish population than expected. Because the genetic background of breast cancer, in particular, is largely obscure, it became necessary to search for mutations in other susceptibility genes. Because seven of our BRCA1 and BRCA2 mutation-negative families fulfilled the criteria of either Li-Fraumeni syndrome (LFS) or Li-Fraumeni-like syndrome (LFL), we decided to screen them for germ-line TP53 mutations in exons 5-8 using a dual-temperature single-strand conformation polymorphism assay (SSCP). Two missense mutations (Asn235Ser and Tyr220Cys) were identified. The clinical significance of these findings was evaluated by comparison to previously reported germ-line TP53 mutation data, and by using the tumor loss of heterozygosity (LOH) analysis. In addition, an immunohistochemical analysis of tumor specimens from mutation-positive individuals was performed. Our results suggest that the observed missense mutations confer susceptibility to cancer, and that germ-line TP53 mutations would therefore explain an additional fraction of hereditary breast cancer in Finland.

Purpose: This study was performed to test whether tenascin, a large oligomeric glycoprotein of the extracellular matrix, is present in AAA disease and whether it could play a pathophysiologic role in the development of this disease. Methods: Tenascin immunoreactivity was investigated from samples of the aneurysmal walls of 17 patients with AAAs, and the results were compared with the results of those of six patients with aortoiliac occlusive disease (AOD) and one normal control patient. To study the source of tenascin mRNA synthesis, some tissue samples were also examined with a tenascin RNA probe by in situ hybridization. Results: The difference in immunoreactivity between the AODs and AAAs was especially prominent in the adventitial layer, where the specimens from AAAs displayed strong diffuse and reticular immunostaining. In AAAs the immunostaining was clearly associated with the degree of mononuclear inflammatory cell infiltrate and with the neovascularization of the adventitial layer. Conclusion: Tenascin expression is evident in AAA disease and is distinctly associated with mononuclear inflammatory cells. The adhesive properties of tenascin may offer a relevant explanation for the mechanism by which monocytes transmigrate into the aortic wall. The definitive role of tenascin in AAA process may be more complex, however, and will necessitate further investigation. (J Vasc Surg 1997;26:670-5.)

The expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was studied in eight malignant fibrous histiocytomas (MFH) and in eight dermatofibromas (DF) using in situ hybridization methods (ISH). Immunohistochemical stainings were also performed using corresponding antibodies to TIMP-1 and TIMP-2. In ISH the neoplastic cells of MFHs showed a high level of expression for both TIMP-1 and TIMP-2 mRNAs. The cells usually expressed similarly both TIMPs, except for osteoclast-like giant cells, which showed a distinct signal for TIMP-2 but not for TIMP-1. A distinctly lower level of both TIMP-1 and TIMP-2 mRNAs was seen in DFs. Immunohistochemical stainings were concordant with the results obtained by ISH. The findings suggest that the behavior of MFHs and DFs is not directly or solely dependent on the quantity of type IV collagenase inhibitors. The increased TIMP synthesis in MFHs might represent a chaotic response of malignant cells to increased matrix degradation. Alternatively, it may reflect a deranged communication between type IV collagenases and TIMPs in malignant tissues.

Summary Nine cases of malignant fibrous histiocytomas (MFH) were examined immunohistochemically in frozen sections with six different monoclonal antibodies to histiomonocytic and related cells (EBM11, HAM-56, KB90, antibodies to dendritic reticulum cells, HLADR and LCA). Ten other soft tissue sarcomas, two desmoid tumors, twelve carcinomas, three seminomas and four lymphomas were studied for comparison. All cases of MFH showed positivity for histiomonocytic cell antigens. In six cases, the positive cells could be clearly interpreted to be infiltrating non-neoplastic cells. However, immunoreactivity for multiple histiocytic markers (EBM11, HAM-56, KB90, HLADR) was seen in tumor cells in three cases of MFH. In one of these cases, the positivity could be verified with KP1, an antibody to histiomonocytic cells applied in formalin fixed and paraffin embedded tissue. None of the tumors was positive with the antibody to dendritic reticulum cells or LCA. In the series of non-histiocytic tumors, no cases showed widespread positivity for multiple histiocytic markers. Our results suggest that in relation to true histiomonocytic differentiation MFH might be a heterogeneous group of tumors. The widespread immunoreactivity for multiple histiocytic markers in some cases may indicate a true histiomonocytic differentiation in some MFHs.

Purpose The prognosis of pharyngeal carcinoma is poorest of all head and neck cancers and the survival rate has not improved in the last two decades. Epithelial-mesenchymal transition (EMT) is a complex process during which epithelial cells lose their arrangement and attain motile and migratory characteristics. It is a crucial process in tumorigenesis enabling tumor cells to invade and metastasize. Transcription factor SIP1 is a key regulator of EMT. The objective of this study was to evaluate the significance of SIP1 in pharyngeal squamous cell carcinoma (PSCC) progression and prognosis. Materials and methods Our material consists of clinicopathological information, follow-up data and tissue samples of 109 patients diagnosed with PSCC. Immunohistochemistry was used to evaluate the expression of SIP1 in tumor epithelial cell, stromal cell and endothelial cell nuclei. Results Tumors with positive epithelial SIP1 immunostaining were more advanced (SIII-IV, p = 0.02) and had more lymph node metastases (p = 0.04). There were also more often second primaries diagnosed in that group (p = 0.048). Tumors with positive stromal staining of SIP1 relapsed more often than SIP1 negative tumors (p = 0.007). Patients with Sip1 epithelial or stromal immunopositive tumors had significantly lower 5-year disease specific and overall survival than patients with SIP1 negative tumors (p = 0.012 and p = 0.003 for epithelial reactivity, p = 0.018 and p = 0.003 for stromal reactivity respectively). Also in Cox proportional hazards model SIP1 epithelial immunoreactivity was an independent prognostic factor for disease specific survival (p = 0.045) together with Karnofsky performance status score and T-class (p Conclusion SIP1 expression in tumor epithelial or stromal nuclei is related to cancer progression and is an important prognostic factor in PSCC. Sip1 may have a role as a new biomarker indicating aggressive tumors with poor prognosis.