Actin assembly is a crucial factor for superoxide anion generation from adherent human eosinophils

BACKGROUND: Cellular adhesion is crucial for eosinophil effector functions. OBJECTIVE: We sought to elucidate the role of the actin cytoskeleton in cellular adhesion and superoxide anion generation by human eosinophils. METHODS: Eosinophils were stimulated with platelet-activating factor (PAF) or complement component 5a on human serum albumin-coated plates with or without an actin-polymerization inhibitor, cytochalasin B (CB), or cytochalasin D (CD). Superoxide anion generation was measured on the basis of reduction of absorbance associated with cytochrome c.2 Eosinophil adhesion was assessed on the basis of eosinophil protein X content in adherent cells. Transient stimulus-induced increase of intracellular calcium and translocation of protein kinase C (PKC) betaII, PKC delta, PKC zeta, and p47 phagocyte oxidase (a component of nicotinamide adenine dinucleotide phosphate oxidase) were also investigated. RESULTS: CB, CD, or antibodies against CD18 (the beta2 chain of integrin, alphaMbeta2) inhibited stimulus-induced eosinophil superoxide anion generation. Stimulus-induced eosinophil adhesion was unaltered by CB, whereas it was significantly suppressed by CD or anti-CD18 antibodies. Transient PAF-induced intracellular calcium increase was also unaffected by CB or CD, but stimulus-induced eosinophil shape changes and translocation of PKCs and p47 phagocyte oxidase to the cell membrane region were completely inhibited by CB. PAF-induced eosinophil degranulation was inhibited by CB, CD, or anti-CD18 antibodies, whereas complement component 5-induced degranulation was not suppressed by CB. CONCLUSION: By itself, beta2 integrin-dependent cellular adhesion is not sufficient for promoting eosinophil effector function. Adequate actin assembly is required for eosinophil adhesion and also for full superoxide anion generation in eosinophils.     

Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis

The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.     

Expression of CD10 by stromal cells during colorectal tumor development

CD10 is a cell surface metalloprotease expressed by a variety of normal cell types, including lymphoid precursor cells, germinal center B lymphocytes, and some epithelial cells. We noticed that stromal cells of some cancers are positive for CD10. In this study, we investigated the role of CD10 produced by the stromal cells of colorectal neoplasms in the progression of colorectal neoplasms. Immunohistochemical examination of CD10 and p53 was performed in 169 colorectal epithelial neoplasms representing various stages of carcinogenesis. The results were correlated with the morphologic characteristics of the neoplasms. There was no expression of CD10 in the stromal cells of normal colorectal tissue. CD10-positive stromal cells were present adjacent to the tumor cells in 16 of 73 adenomas with mild or moderate dysplasia. More frequent expression of CD10 by the stromal cells was detected in adenomas with severe dysplasia (12 of 17), intramucosal carcinomas (10 of 16), and invasive carcinomas (50 of 63) than in adenomas with mild or moderate dysplasia (P < 0.0001). Expression of CD10 by > 10% of the stromal cells was detected only within the area of the invasive growth front of invasive carcinomas, not in adenomas and in only 1 of the intramucosal carcinomas. The difference between invasive and non invasive tumors was significant (P < 0.0001). The stromal expression of CD10 was significantly associated with the accumulation of p53 and a larger tumor size. These results indicate that CD10 expression is an integral part of colorectal carcinogenesis. CD10 expression seems to contribute to the invasion and thus probably facilitates metastasis.     

Atrial natriuretic peptide in human urine

Summary A highly sensitive radioimmunoassay to measure atrial natriuretic peptide (ANP) concentration in urine has been established, and its clinical usefulness is presented. ANP in urine was stable at 4° C for several days and was easily measured by our radioimmunoassay. The average ANP excretion in 65 healthy persons was 25.0±1.4 ng/day (mean ± SEM) and the fractional excretion of ANP was 0.7±0.05%. In 14 patients with congestive heart failure, the average ANP excretion was 119.2±29.4 ng/day, which decreased to 53.3±11.0 after successful treatment.Abbreviations ANP atrial natriuretic peptide - hANP human atrial natriuretic peptide - RIA radioimmunoasay - NSB non specific bound - FE_ANP the fractional excretion of atrial natriuretic peptide - FE_Na the fractional excretion of sodium - SIADH the syngrome of inappropriate secretion of antidiuretic hormone     

Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis

Insulin-like growth factor-1 receptor (IGF-1R) has been implicated in regulation in tumor growth. The results of previous studies performed by radioimmunoassay are conflicting, and the prognostic significance of IGF-1R expression in primary breast cancer is still controversial. IGF-1R expression was evaluated in formalin-fixed, paraffin-embedded tissue of 210 primary breast cancer patients by using anti-IGF-1R antibody. The clinicopathologic variables and 5-year disease-free survival were studied, and their correlations between IGF-1R expressions were investigated. IGF-1R overexpression was observed in 43.8% of tumors. IGF-1R overexpression had no correlation with prognosis or with other clinicopathologic parameters, such as age, tumor size, nodal status, histologic grade, hormone receptor status, and human epidermal growth factor 2 status. Though its prognostic value in breast cancer is limited, immunohistochemical evaluation of IGF-1R by using this monoclonal antibody may be useful in translational research using archived material.     

Nuclear accumulation of beta-catenin in intestinal-type gastric carcinoma: correlation with early tumor invasion

Mutation of the adenomatous polyposis coli gene, which is known to be an early event in the carcinogenesis of intestinal-type gastric carcinoma, leads to accumulation of beta-catenin. In addition, beta-catenin has been found to activate down stream signaling molecules in the wingless/Wnt pathway. In this study, the clinical significance of nuclear accumulation of beta-catenin was evaluated in gastric carcinoma. Immunohistochemical staining showed nuclear localization in 16 (12%) of 139 (94 intestinal-type and 45 diffuse-type) gastric carcinomas, and all 16 lesions with nuclear staining were intestinal-type adenocarcinomas. Of the 16 cases, 15 were in the early clinical stage. In the remaining case, the lesion had invaded the subserosal layer and showed strong nuclear staining at the invasive front. In 14 of the 16 cases with nuclear localization, there were no abnormal mobility shifts detected using polymerase chain reaction-single strand conformational polymorphism analysis. This was confirmed using direct sequencing analysis, which revealed the wild-type sequence in the 12 cases tested. Nuclear accumulation of beta-catenin did not correlate with lymph node metastasis or 5-year survival. These findings suggest that high intranuclear levels of beta-catenin protein play an important role in early tumor growth and may function in initiation of invasive processes in intestinal-type gastric carcinoma.     

IMMUNOHISTOCHEMICAL STUDY OF PANCREATOBLASTOMA

Three cases of pancreatoblastoma in children were examined immunohis-tochemically and the results were compared with those of pancreatic duct carcinoma in adults. The pancreatoblastoma demonstrated positive reactions to α-fetoprotein (AFP) (67%: 2/3), α-1-antitrypsin (AAT) (100%: 3/3), carcinoembryonic antigen (CEA) (67% : 2/3) and keratin (33% : 1/3), although CEA was only weakly positive in both cases. On the other hand, adult pancreatic duct carcinoma showed positive reactions as follows; AFP: 3% (1/29), AAT: 21% (6/29), CEA: 97% (28/29) and keratin: 93% (27/29). Also, endocrine substances including insulin, glucagon and somatostatin were all negative in the pancreatoblastomas. Two cases of pancreatoblastoma which were immunohistochemically positive for AFP also showed elevation of the serum AFP level clinically. The different expressive pattern of oncofetal antigens in pancreatoblastoma as compared with pancreatic duct carcinoma in adults may provide further supporting evidence for the embryonic nature of pancreatoblastoma, and suggests that such a pattern might be used as a tumor marker for pancreatoblastoma. ACTA PATHOL. JPN. 37 : 1581-1590, 1987.     

Hyperplastic epithelial foci in honeycomb lesions in idiopathic pulmonary fibrosis

Seventy-two cases of idiopathic pulmonary fibrosis (IPF) were examined from 2856 consecutive autopsy cases at the Japanese Red Cross Medical Center in Tokyo from 1973-1996. Primary lung cancer had arisen in 31 of 72 cases of IPF (43%), significantly higher than the incidence in cases without IPF (8.1%) and in the cases with non-IPF chronic lung diseases (11.9%). Hyperplastic epithelial foci in the honeycomb lesions of IPF cases were significantly more prominent in the lower than in the upper lobe, in cases with or without lung cancer, and they were more prominent in the lower lobe of IPF with than in those without cancer. The length of hyperplastic epithelial foci in the lower lobe of IPF cases was longer than that in interstitial pneumonia-associated with collagen vascular diseases. There was a higher PCNA labeling index of hyperplastic epithelial foci in IPF cases than in cases of interstitial pneumonia-associated with collagen vascular diseases. The PCNA labeling index was almost the same between smokers and nonsmokers with IPF. Overexpression of p53 was observed in hyperplastic epithelial foci in honeycomb lesion of IPF. DNA ploidy analysis of hyperplastic epithelial foci in the paraffin sections of 12 IPF cases revealed aneuploidy patterns in eight cases. These results strongly suggest that accelerated cell proliferation occurs in the honeycomb lesion of IPF, and that regenerative epithelia becomes susceptible to carcinogenic agents in addition to the smoking effect.     

Identification and characterization of temperature-sensitive mild mutations in three Japanese patients with nonsevere forms of very-long-chain acyl-CoA dehydrogenase deficiency

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is clinically classified into severe, intermediate, and myopathic forms. We identified mutations in three unrelated Japanese patients with VLCAD deficiency: two with the myopathic form and one with the intermediate form, all compound heterozygotes of K264E/M437V, A416T/1798delA, and P89S/IVS16-3delAA, respectively. We characterized four missense mutations, K264E, M437V, A416T, and P89S, by transisent expression analysis, using SV40-transformed fibroblasts derived from a VLCAD-null patient, as recipient cells. In transient expression of the wild-type VLCAD cDNA, VLCAD activity at 30 degrees C was higher than at 37 degrees C. Moreover, this temperature-sensitive character is more evident in all the mutant proteins tested than in wild type. Based on characterization of the five missense mutations identified in four Japanese patients, including data on one patient with the myopathic form previously reported, patients with the nonsevere forms (intermediate or myopathic forms) have missense mutations with residual activities in at least one allele. Expression analysis at 30 degrees C may be more useful for evaluating these missense mutations, compared with that at 37 degrees C.