Tetsuya Toyozaki

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. Copyright

Levels of soluble Fas ligand in myocarditis

Serum levels of soluble Fas ligand (sFasL) increased with the severity of congestive heart failure (p <0.01), and the percentages of apoptotic myocytes detected by in situ DNA nick-end labeling were significantly higher in the patients with increased levels of sFasL than in those with normal levels of sFasL (p <0.05). These findings indicated that sFasL may play an important role in pathogenesis of myocarditis.

Synchronous and metachronous lung carcinomas: Molecular evidence for multicentricity

The present study Is aimed to evaluate the genetic evidence for multicentricity of synchronous and metachronous multiple lung carcinomas. Nineteen cases of synchronous multlple lung carcinomas and 11 cases of metachronous multiple lung carcinomas were analyzed for p53 protein overexpression by lmmunohistochemlstry (DO‐7) and for genetic abnormality of the p53 gene by loss of heterozygosity (LOH) at chromosome 17p and by poly‐merase chaln reaction (PCR)‐singlestrand conformation polymorphism (SSCP) analysis. They were also analyzed for K‐ras mutation. DNA from three patients was also sequenced by the dideoxy sequencing method to confirm the presence of mutations and determine the base substitutions. Different spectrums of genetic changes, which were evaluated by a combination of p53 mutation, LOH at chromosome 17p and p53 overexpression, were observed in 11 of 19 cases of synchronous multiple lung carcinomas (57.9%) In the present study. Slmllarly, five of 11 cases of metachronous multiple lung carcinomas (45.4%) showed a different pattern of genetic changes. The present data suggest that some of the multiple carcinomas have dlfferent clonal orlgins, although their histological types are identical, and support the use of genetic markers In the differential diagnosis between metastasis and second primary carcinoma of the lung.

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.

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.

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. Copyright

Levels of Soluble Fas in Patients With Myocarditis, Heart Failure of Unknown Origin, and in Healthy Volunteers 1

This study showed that serum levels of sFas were elevated in patients with myocarditis, and that this elevation was correlated with sIL-2R level as a marker of T-cell activation. Therefore, sFas levels may be associated with T-cell activation in patients with myocarditis, and elevation of sFas may inhibit apoptosis in activated T cells, leading to persistent cell-mediated destruction of myocytes in myocarditis.

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.

Prognostic Utility of Myocardial Blood Flow Assessed by N-13 Ammonia Positron Emission Tomography in Patients With Idiopathic Dilated Cardiomyopathy

Previous studies in patients with idiopathic dilated cardiomyopathy (IDC) have suggested that myocardial perfusion is impaired and spatially heterogeneous in such cases. Our objective was to identify any association between an abnormality in myocardial perfusion and the prognosis of patients with IDC. We collected data on N-13 ammonia positron emission tomography (PET) studies performed in 26 patients with IDC (9 nonsurvivors, 17 survivors) and in 8 normal control subjects. Regional myocardial blood flow (rMBF) was quantified using N-13 ammonia positron emission tomography and the Simple flow model. The spatial heterogeneity of myocardial perfusion was assessed by calculating the coefficient of variance of rMBF. Mean rMBF of the survivors was significantly lower (0.54 +/- 0.13 ml/min/g) than that of control subjects (0.66 +/- 0.06 ml/min/g) (p = 0.03 vs control), but did not differ significantly between nonsurvivors (0.58 +/- 0.15 ml/min/g) and control subjects. The coefficient of variance of rMBF was significantly higher in nonsurvivors than in either survivors or control subjects (0.24 +/- 0.08 vs 0.15 +/- 0.08, p = 0.007, and 0.16 +/- 0.05, p = 0.03, respectively). The probability of 3-year survival (Kaplan-Meier method) was 33.0% in subjects whose coefficient of variance of rMBF was above the median compared with 90.0% in subjects whose coefficient of variance of rMBF was below the median (p = 0.01). The probability of 3-year survival did not differ among subjects whose mean rMBF was above versus below the median (61.5% vs 62.9%, respectively). The results suggest that the prognosis of patients with IDC is associated with the spatial heterogeneity of myocardial perfusion, not with initial absolute rMBF.

Expression of intercellular adhesion molecule-1 on cardiac myocytes for myocarditis before and during immunosuppressive therapy

Right ventricular endomyocardial biopsies were performed in 15 patients with unexplained cardiac dysfunction, and the expression of intercellular adhesion molecule-1 (ICAM-1) on the myocardium was examined using the streptavidin-biotin complex method. Three of 15 patients (20%) had histologic evidence of myocarditis, and 2 of 15 patients (13%) had borderline myocarditis. The patients with biopsy-proven myocarditis received immunosuppressive therapy (prednisolone 60 mg/day). Two of the 3 patients demonstrated a substantial increase (> 10%) in percent fractional shortening and left ventricular ejection fraction during therapy. Subsequent biopsies revealed ongoing myocarditis in 1 patient, and resolved myocarditis in the other. The remaining patient had persistent cardiac dysfunction, but the subsequent biopsy revealed resolving myocarditis. ICAM-1 immunoreactivity was observed on cardiac myocytes, vascular endothelial cells, and interstitial cells of the patients with myocarditis. However, in patients without myocarditis, ICAM-1 immunoreactivity was observed only on vascular endothelial cells and interstitial cells. ICAM-1 immunoreactivity was still observed on cardiac myocytes in the patients with ongoing or resolving myocarditis during immunosuppressive therapy, but was not detected in the patient with resolved myocarditis. This study demonstrates that ICAM-1 is expressed on cardiac myocytes of patients with myocarditis and persistent cardiac dysfunction despite immunosuppressive therapy. The persistent expression of ICAM-1 may cause chronic inflammation of the myocardium.