Diagnostic utility of tenascin-C for evaluation of the activity of human acute myocarditis.

Tenascin-C (TN-C) is an extracellular matrix protein that is expressed transiently in close association with tissue remodelling in various body sites. In the heart, TN-C is only present during early stages of development, is not expressed in the normal adult, but reappears in pathological states. The purpose of this study was to analyse the expression of TN-C in myocardial tissue from myocarditis patients, and to evaluate the diagnostic value of immunostaining for TN-C in the assessment of inflammatory activity in biopsy specimens. A total of 113 biopsy specimens obtained from 32 patients with a clinical diagnosis of acute myocarditis were examined by immunohistochemistry and in situ hybridization for TN-C. The immunostaining was semi-quantified and compared with histological diagnosis according to the Dallas criteria. Furthermore, serial biopsies from 22 patients were taken during convalescence, and sequential changes in TN-C levels were analysed. Expression of TN-C was specifically detected in endomyocardial biopsy specimens from patients with active-stage inflammation, and disappeared in healed stages. The degree of expression of TN-C correlated with the severity of histological lesions. These data suggest that TN-C reflects disease activity in cases of human myocarditis. Immunostaining for TN-C could enhance the sensitivity and accuracy of diagnosis using biopsy specimens.

Pioglitazone, a peroxisome proliferator-activated receptor gamma activator, ameliorates experimental autoimmune myocarditis by modulating Th1/Th2 balance.

OBJECTIVES: The aim of this study is to investigate the effect of Peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand on experimental autoimmune myocarditis (EAM). BACKGROUND: Rat EAM model resembles the giant cell myocarditis in human. Recent studies have suggested that Th1 cytokines are involved in the initiation and progression of EAM, whereas Th2 cytokines are associated with the remission. PPAR gamma, which is a member of nuclear hormone receptor superfamily, has been known to affect not only glucose homeostasis but also immune responses, by regulating the Th1/Th2 balance. METHODS: Lewis rats were immunized with cardiac myosin and divided into three groups: Group N, normal control rats (n = 16); Group E, EAM rats (n = 17); and Group P, EAM rats treated with a PPAR gamma activator pioglitazone (n = 20). RESULTS: Cardiac dysfunction and remodeling were inhibited in Group P compared to Group E. Heart weight/body weight ratio and the degree of inflammation and fibrosis were significantly lower in Group P than in Group E. The mRNA levels of macrophage inflammatory protein-1alpha (MIP-1alpha), which plays an important role in the recruitment of inflammatory cells in the early stage of EAM, were upregulated in the heart of Group E, but not in the heart of Group P. Furthermore, the treatment with pioglitazone decreased the expression levels of proinflamatory cytokine (TNF alpha and IL-1beta) genes and Th1 cytokine (IFN-gamma) genes, and increased the expression levels of Th2 cytokine (IL-4) gene. CONCLUSIONS: PPAR gamma ligands may have beneficial effects on myocarditis by inhibiting MIP-1alpha expression and modulating the Th1/Th2 balance.

Genetic alterations in early-stage pulmonary large cell neuroendocrine carcinoma.

BACKGROUND: Small cell lung carcinoma (SCLC) and pulmonary large cell neuroendocrine carcinoma (LCNEC) are high-grade malignant neuroendocrine tumors. Histologic differentiation between SCLC and LCNEC is difficult in some cases and to the authors' knowledge, genetic alterations associated with LCNEC have not been identified. Therefore, the authors studied genetic alterations found in LCNEC and compared them with those of SCLC and classic large cell carcinoma (CLCC). METHODS: Twenty-two patients with UICC TNM Stage I LCNEC, 12 patients with Stage I CLCC, and 11 patients with SCLC with limited disease were studied. All tumors were resected completely. Loss of heterozygosity (LOH) of the tumor cells was detected using fluorescent primers. Methylation status of the p16 gene and expression of the p53 protein, retinoblastoma protein, and p16 protein were evaluated immunohistochemically. RESULTS: LOH at TP53 and 13q14 was observed in most patients. The prevalence of LOH at D3S1295, D3S1234, and D5S407 was significantly higher in patients with LCNEC and SCLC than in patients with CLCC. The prevalence of LOH at D5S422 was higher in patients with CLCC and in patients with SCLC than in patients with LCNEC. Expression of the p16 protein was observed more frequently in SCLC than in CLCC or LCNEC. Hypermethylation of the p16 gene was observed more frequently in LCNEC than in SCLC. Patients with allelic losses at D3S1234 and D10S1686 had poorer prognoses compared with patients without allelic losses at these sites. CONCLUSIONS: Genetic alterations of LCNEC were akin to those of SCLC. However, allelic losses at 5q and abnormalities in the p16 gene may differentiate LCNEC from SCLC.

Fatal infectious mononucleosis with evidence suggestive of the development of B cell lymphoma.

A 4-year-old girl presented to a local hospital in August 1999 with fever and cervical lymphadenopathy. A diagnosis of Epstein-Barr virus (EBV) infection was made and the patient was treated with corticosteroids. One month later she developed dyspnea secondary to tonsilar swelling, and underwent tonsillectomy and adenoidectomy. Her dyspnea increased, however, and by mid September she required mechanical ventilation. Six weeks later, she was transferred to Chiba Children's Hospital (Chiba, Japan). Despite vigorous treatment, she died within four weeks of admission. At autopsy, microscopic examination revealed numerous histiocytes with frequent hemophagocytosis in her lungs, liver, spleen, thymus, and lymph nodes. The tentative diagnosis was EBV-associated hemophagocytic syndrome (EBVAHS). A proliferation of atypical lymphocytes was observed in the lymph nodes, the majority of which stained positive with CD79a antibody. A whitish nodule, 8 mm in diameter, was noted in her right ovary. It consisted of a proliferation of pleomorphic lymphoid cells expressing CD79a antigen. In situ hybridization detected EBV RNA within CD79a antigen-positive cells in the lungs, spleen, thymus, bone marrow, lymph nodes, and the right ovary. Polymerase chain reaction analysis of DNA from the ovarian nodule demonstrated a monoclonal rearrangement of the immunoglobulin heavy chain gene indicating that it consisted of a clone of B lymphocytes. We suggest that EBVAHS develops into polyclonal and monoclonal lymphoproliferative disorder in a short period, and that EBVAHS is a preneoplastic condition that may result in B cell lymphoma.

Pulmonary large cell carcinoma with rhabdoid phenotype.

A 70-year-old woman presented with a coin lesion in her left lung. The tumor was well circumscribed and had a large area of central necrosis with a thin rim of viable tumor cells. It showed a solid growth pattern of polygonal cells with eosinophilic intracytoplasmic inclusion bodies. Immunohistochemically, the tumor cells were positive for vimentin, neural cell adhesion molecule, neuron-specific enolase, and vascular endothelial growth factor. Electron microscopy revealed intracytoplasmic inclusion bodies consisting of whorled intermediate filaments. Based on histological and immunohistochemical findings, the patient was diagnosed as having pulmonary large cell carcinoma with rhabdoid phenotype (LCCRP). The patient was in stage IA, and the histological findings may be the prototype of pure LCCRP. The tumor recurred after 6 years, and the second tumor had more apparent intracytoplasmic inclusion bodies. It is worthwhile detecting and recognizing the significance of these intracytoplasmic inclusions because of the poor prognosis of this tumor.

Proliferative activity and apoptosis in thymic epithelial neoplasms.

The classification of thymic epithelial tumors is controversial because prediction of the biological behavior of these tumors from their morphologic appearance is difficult. The aim of this study was to evaluate the proliferative activity and rate of apoptosis of thymic epithelial tumors classified according to World Health Organization histological classification. We also attempted to determine the importance of a number of proapoptotic factors in these processes. We investigated 46 surgically resected thymic epithelial tumors (8 Type A, 8 Type AB, 7 Type B1, 7 Type B2, 6 Type B3, and 10 Type C). Immunohistochemical staining was performed to determine the tumor expression of p53 protein, Bax, Bcl-2, and survivin. In addition, the Ki-67 labeling index (LI) and apoptotic index (AI) of these tumors were evaluated. Type C thymoma had a higher LI (16.55 +/- 12.12%) than did the other histological subtypes. Stage IV thymoma (12.36 +/- 9.99%) had a higher LI than did Stage I tumor. The AI was significantly elevated in Type B1 thymoma (1.47 +/- 0.55%). Overexpression of p53 protein was observed in Type B3 and C thymomas. p53 protein-positive tumors had a higher LI than did p53 protein-negative tumors (P <.0001). Bcl-2 expression was observed in Type A, AB, and C thymomas. Bcl-2-positive thymoma had a lower AI than did Bcl-2-negative thymoma (P =.0157). These results suggest that overexpression of p53 protein is associated with a higher tumor proliferative activity and that Bcl-2 acts as an inhibitor of apoptosis in thymoma. Bcl-2 and p53 protein expression may be useful markers in differentiating thymoma subtypes.

Evidence of neoangiogenesis and an increase in the number of proliferating cells within the bronchial epithelium of smokers.

BACKGROUND: Normal bronchial epithelium gradually acquires cellular and genetic changes that result in the formation of invasive tumors. The objective of this study was to evaluate the degree of proliferative change and the amount of neovascularization in both normal and preneoplastic lesions in smokers who were at high risk for developing lung carcinoma. METHODS: The authors studied bronchial biopsy specimens from 7 nonsmokers and 52 smokers. Immunohistochemical staining of the specimens with antibodies for the presence of p53 protein, Ki-67 and CD34 antigens, and vascular endothelial growth factor was performed. The proliferation index (PI) was assessed by immunohistochemical staining for Ki-67 antigen. RESULTS: Overexpression of p53 protein was observed frequently in regions of squamous dysplasia and in squamous cell carcinoma tissue. The PI of normal epithelium from smokers was increased compared with nonsmokers, and the difference was statistically significant (P < 0.05). The microvessel count (MC) in normal mucosa obtained from smokers was higher compared with the MC in normal mucosa obtained from nonsmokers (P < 0.05). A significant difference in MC also was observed between regions of squamous metaplasia or dysplasia with projections of capillary loops into the bronchial mucosa and similar lesions without capillary loops (P < 0.005); however, there was no difference in either the PI or the incidence of p53 overexpression between these groups. CONCLUSIONS: These results show that smoking appears to induce both a proliferative response and neovascularization in bronchial mucosa. The projection of capillary loops into the bronchial mucosa also may be a result of neovascularization occurring within the lamina propria of the bronchial wall.

Antibody binding to fas ligand attenuates inflammatory cell infiltration and cytokine secretion, leading to reduction of myocardial infarct areas and reperfusion injury.

Fas ligand (FasL) induces apoptotic cell death when bound to Fas antigen. The engagement of FasL has anti-inflammatory effects through the prevention of cell proliferation and cytokine secretion. However, the role of FasL in myocardial ischemia/reperfusion (MI/R) injury is unclear. We examined the expression of FasL mRNA in the myocardium of MI/R rats by ligating the left coronary artery for 30 minutes and allowing reperfusion to occur for 0, 1, 3, and 24 hours. The expression of FasL mRNA was enhanced 1 hour after reperfusion, and enhanced levels were consistently seen after 24 hours of reperfusion. FasL immunostaining was observed on neutrophils, macrophages, T cells, and vascular endothelial cells. We then assessed the potential role of FasL in the cell proliferation and cytokine production seen in MI/R injury after 24 hours of reperfusion. Rats were divided into three groups; Group A, without treatment; Group B, treated with nonspecific rabbit IgG; and Group C, treated with anti-FasL antibody. Anti-FasL antibody or rabbit IgG were administered intravenously before coronary artery occlusion. In Group C, interleukin-1beta and interleukin-2 mRNA levels were decreased, and neutrophil and T cell accumulation was attenuated. The infarct area determined by triphenyltetrazolium chloride staining was significantly smaller in Group C (18 +/- 4%) than in Group A (34 +/- 2%) or Group B (33 +/- 4%) (p< 0.0001). However, there was no significant difference in the prevalence of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling-positive cardiomyocytes among the three groups. These findings suggest that the cardioprotective effect of anti-FasL antibody is due to its anti-inflammatory action, rather than antiapoptotic action. The Fas/FasL system may be involved in the development of MI/R injury.

Detection of experimental autoimmune myocarditis in rats by 111In monoclonal antibody specific for tenascin-C.

BACKGROUND: Although the identification of inflammatory infiltrates in endomyocardial biopsy specimens is necessary for the definite diagnosis of myocarditis, the biopsy test is invasive and is not sensitive. Therefore, a new diagnostic technique for the early and noninvasive evaluation of myocarditis has been awaited. Expression of tenascin-C (TNC), one of the oligometric extracellular glycoproteins, is induced in various pathological states, including inflammation, suggesting that TNC can be a molecular marker of myocarditis. METHODS AND RESULTS: An 111In anti-TNC monoclonal antibody Fab' fragment was injected intravenously into rats with experimental autoimmune myocarditis (EAM), and the biodistribution of this radiotracer was measured. Rapid clearance of radioactivity from the blood was observed in both EAM and control rats (<1% at 6 hours after injection). Myocardial uptake of the tracer was much higher in EAM rats than in control rats (7.54-, 4.39-, and 3.51-fold at 6, 24, and 48 hours after injection, respectively). By autoradiography, high radioactivities were clearly observed in the regions indicative of inflammation in EAM rats. Single-photon emission CT imaging demonstrated the focal myocardial uptake of 111In anti-TNC Fab' in vivo. CONCLUSIONS: Radiolabeled anti-TNC Fab' may be useful for the noninvasive diagnosis of myocarditis.

Tenascin-C is a useful marker for disease activity in myocarditis.

Tenascin-C (TNC) is an extracellular matrix protein which appears at active sites of tissue remodelling during embryogenesis or cancer invasion. In normal heart, TNC is only present during the early stages of development but reappears in pathological states. This study examined the diagnostic value of TNC for assessing disease activity of myocarditis. Expression of TNC was examined in myosin-induced autoimmune myocarditis mouse models. Sequential changes in amount, localization and the producing cells were analysed by reverse transcriptase-polymerase chain reaction, western blotting, immunohistochemistry and in situ hybridization and compared with the histological picture. The expression of TNC was upregulated at a very early stage of myocarditis. Immunostaining was detectable before cell infiltration and myocytolysis became histologically apparent, remained during the active stage while cell infiltration and necrosis continued, and disappeared in scar tissue with healing. TNC immunostaining was always observed at the periphery of necrotic or degenerating cardiomyocytes in foci of inflammation, the expression level correlating with histological evidence of inflammatory activity. Interstitial fibroblasts were the major source of TNC, expressing the large isoform containing alternative splicing sites. These data demonstrate that TNC is a useful marker for evaluation of disease activity in myocarditis.

Prognostic significance of neuroendocrine differentiation in adenocarcinoma of the lung.

BACKGROUND: The relative incidence of adenocarcinoma of the lung is increasing and some patients with lung carcinoma, detected at an early stage, still develop recurrent disease despite complete resection of the tumor. Recently, neuroendocrine differentiation in large cell carcinoma of the lung has been reported to be of prognostic significance. Therefore, we have evaluated the prognostic significance of neuroendocrine differentiation in adenocarcinoma of the lung. METHODS: A total of 90 resected specimens of adenocarcinoma of the lung measuring 3 cm or less (T1 N0 M0 or T2 N0 M0) were reviewed histologically and immunohistochemical staining was performed to determine the degree of neuroendocrine differentiation. RESULTS: Seven adenocarcinomas exhibited neuroendocrine differentiation in 10% or more of tumor cells. The disease-free survival rate for these patients was significantly lower than that of patients with tumors exhibiting neuroendocrine differentiation in less than 10% of tumor cells or with absent neuroendocrine differentiation (p < 0.0005). Other conventional pathologic factors such as vascular invasion (p < 0.0005), lymphatic invasion (p < 0.05), and pleural involvement (p < 0.05) were also of prognostic significance. In multivariate analysis, the presence of 10% or more neuroendocrine marker-positive tumor cells, vascular invasion, and lymphatic invasion were found to be significantly adverse prognostic factors (p = 0.0162, p = 0.0111, and p = 0.0173, respectively). CONCLUSIONS: Neuroendocrine differentiation of tumor cells is a prognostic factor in lung adenocarcinoma. It is suggested that the identification of neuroendocrine differentiation as well as vascular invasion by tumor in small peripheral adenocarcinoma of the lung may predict the prognosis of these patients.

ClC-3B, a novel ClC-3 splicing variant that interacts with EBP50 and facilitates expression of CFTR-regulated ORCC.

We have cloned ClC-3B, a novel alternative splicing variant of ClC-3 (ClC-3A) that is expressed predominantly in epithelial cells. ClC-3B has a different, slightly longer C-terminal end than ClC-3A and contains a consensus motif for binding to the second PDZ (PSD95/Dlg/ZO-1) domain of the epithelium-specific scaffolding protein EBP50. Both in vitro and in vivo binding assays demonstrate interaction between ClC-3B and EBP50. C127 mouse mammary epithelial cells transfected with ClC-3B alone showed diffuse immunoreactivity for ClC-3B in the cytoplasmic region. In contrast, when EBP50 was cotransfected with ClC-3B, strong immunoreactivity for ClC-3B appeared at the leading edges of membrane ruffles. Patch-clamp experiments revealed that cotransfection of ClC-3B and EBP50 resulted in a remarkable increase in outwardly rectifying Cl- channel (ORCC) activities at the leading edges of membrane ruffles in C127 cells. The electrophysiological properties of the ClC-3B-induced ORCCs are similar to those of ORCCs described in native epithelial cells. When cystic fibrosis transmembrane conductance regulator (CFTR) was cotransfected with ClC-3B and EBP50, ClC-3B-dependent ORCCs were activated via the protein kinase A-dependent pathway. These findings indicate that ClC-3B is itself a CFTR-regulated ORCC molecule or its activator.

Alpha-fetoprotein-producing lung carcinoma: report of three cases.

Three cases of alpha-fetoprotein (AFP)-producing lung carcinoma were studied histologically and immunohistochemically. Samples were obtained from two men and one woman who ranged in age from 64 to 71 years. Serum AFP levels for the three samples were 9826, 74.4 and 24.3 ng/mL. One case was classified as stage IIIA and two as stage IIIB. Two cases were diagnosed as large cell neuroendocrine carcinoma, and AFP expression was detected immunohistochemically. One of these samples showed differentiation to a hepatoid carcinoma, while the other was combined with a squamous cell carcinoma. The remaining case was a squamous cell carcinoma, and AFP was detected in only some of the tumor cells. All patients died within 2 years. The Ki-67 labeling indices of the AFP-producing pulmonary carcinomas (30.2 +/- 4.6%) were significantly higher than those of AFP-negative pulmonary carcinomas (P < 0.05). The high proliferative activity, advanced stage at presentation, vascular endothelial growth factor expression and vascular invasion observed in these tumors may explain the poor prognosis of AFP-producing lung carcinomas.

Systemic production of vascular endothelial growth factor and fms-like tyrosine kinase-1 receptor in acute Kawasaki disease.

BACKGROUND: Increased vascular permeability is an important event during the initial process of Kawasaki disease (KD). One potential responsible candidate for the induction of vascular hyperpermeability is vascular endothelial growth factor (VEGF). METHODS AND RESULTS: We investigated the expression of VEGF and its receptors (flt-1, KDR) in acute KD tissues at 7 days to 5 weeks of illness. Neuropilin-1, which enhances the binding of VEGF(165) to KDR, was also studied. Abundant expression of VEGF and flt-1 was documented immunohistochemically in many organs from acute KD, including heart and lung. VEGF and flt-1 were colocalized in all vessels that showed edema. These molecules resided in endothelium and vascular media and also in migrating smooth muscle cells in neointima and infiltrating macrophages. Compared with controls, coronary vessels of acute KD had upregulation of VEGF and flt-1 but not KDR or neuropilin-1. KDR was expressed by vessels at 7 days of illness but not later in the illness. Plasma proteins were more extensively bound to the extracellular matrix in coronary vessels in acute KD than controls. Furthermore, elevation of serum VEGF levels was correlated with low serum albumin in acute KD (n=220, r=-0.53, P<0.001). CONCLUSIONS: These findings suggest that VEGF and flt-1 are upregulated in blood vessels in many organs of acute KD. Expression of KDR was limited to the early stage of acute KD. The roles of VEGF in acute KD may involve promotion of vascular permeability and macrophage activation. Low serum albumin may indicate overproduction of VEGF in acute KD.