Diagnostic and prognostic significance of cyclin A expression in low-grade astrocytomas: comparison with astrogliosis and high-grade tumours.

AIM: Definitive distinction between low-grade astrocytoma and astrogliosis is a long-standing difficulty due to their similar histopathological characteristics. To clarify differences in biological significance, this study focused on various components of the cell cycle machinery and proliferation as key parameters, comparing expression in astrogliosis, as well as low- and high-grade astrocytomas. METHODS: The expression of p16, p21 and p27, and cyclin A, cyclin D1, cyclin E, Rb and Ki-67 was immunohistochemically examined in 40 cases of astrogliosis and 48 cases of low-grade astrocytomas (grade II), as well as 50 high-grade tumours (grades III and IV). The results were also compared with survival data for the astrocytomas. RESULTS: Cell proliferation determined by Ki-67 immunoreactivity did not differ between astrogliosis and low-grade tumours. Average labelling indices (LIs) for p16, p21, Rb, cyclin A and cyclin E showed a stepwise increase from astrogliosis, through low- to high-grade astrocytomas, indicating the possibility that over 9%, 6% and 4% of LIs for p16, p21 and cyclin A, respectively, may be useful predictors in the case of the latter, in contrast to significant decrease in p27 LIs. Significantly higher mean LI values for cyclin D1 were also evident in astrogliosis (12.42) as compared with astrocytomas (low grade, 2.26; high grade, 4.60). Positive correlations between LIs for Rb and Ki-67 were observed with astrogliosis and low- but not high-grade tumours. In addition, high cyclin A LI values were independently associated with poor outcome in low-grade tumours. CONCLUSION: These findings provide evidence that expression of cell-cycle-related molecules may be a reliable parameter for differential diagnosis of low-grade astrocytomas and astrogliosis. Moreover, detection of cyclin A appears to be useful for predicting behaviour of low-grade astrocytomas.

Spontaneous rupture of a left gastroepiploic artery aneurysm in a patient with autosomal-dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) has been known to be associated with a variety of vascular diseases. We present a hemodialysis patient with ADPKD who died of a massive intraperitoneal hemorrhage caused by the spontaneous rupture of a left gastroepiploic artery aneurysm. A 64-year-old male was admitted to our hospital with acute upper abdominal pain and hemorrhagic shock. An abdominal angiography showed three aneurysms and the source of hemorrhage was assumed to be the left gastroepiploic artery aneurysm. The patient died of severe metabolic acidosis and disseminated intravascular coagulation (DIC) on the second hospital day. At autopsy, there was massive bleeding into the abdominal cavity, and pathological examination of the left gastroepiploic artery aneurysm revealed a dissecting aneurysm. This is the first case describing a rupture of a gastroepiploic aneurysm in a patient with ADPKD.

High microvascular density is correlated with high VEGF, iNOS and COX-2 expression in penetrating growth-type early gastric carcinomas.

AIMS: Early gastric carcinomas have two characteristic growth types, superficial spreading (SUP) and penetrating (PEN). Higher mucosal apoptotic activity and lower p21(WAF1/CIP1) expression and submucosal low proliferative activity have been shown in the former, compared with the latter. In order to cast light on whether angiogenesis contributes to these growth patterns, the present immunohistochemical study was performed with cancer tissues. METHODS AND RESULTS: Of a total of 807 early gastric carcinomas, 30 PEN and 33 SUP type submucosal invasive carcinoma cases were immunohistochemically compared. CD34 positivity, microvascular density (MVD), and expression of vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS), but not cyclooxygenase 2 (COX-2) were higher in cancer cells in both mucosal and submucosal layers in PEN than in SUP (P < 0.05). Submucosal MVD in PEN type was greater (P < 0.01) in cases with high than with low Ki67 labelling. Significant correlations were shown between MVD and VEGF, iNOS and COX-2, and VEGF and iNOS expression in the PEN type, but only a weak correlation between iNOS and COX-2 expression was evident with the SUP type. CONCLUSIONS: Increased MVD in PEN type has an intimate causal relationship to angiogenic factors, high VEGF and iNOS expression. The SUP type, in contrast, has characteristics of low angiogenesis.

Significant correlations of E-cadherin, catenin, and CD44 variant form expression with carcinoma cell differentiation and prognosis of extrahepatic bile duct carcinomas.

To clarify the relation between alteration of expression of cell adhesion molecules and progression of extrahepatic bile duct carcinomas. 55 cases were immunohistochemically examined for E-cadherin, alpha-catenin, beta-catenin, and CD44, with additional reverse transcription-polymerase chain reaction and Southern blotting hybridization (RT-PCR/SBH) assays. Levels of E-cadherin, alpha-catenin, and beta-catenin proteins were lower in carcinomas than in normal mucosa, while CD44 variants 3 and 6 were upregulated. Well-differentiated carcinoma showed higher expression of E-cadherin and alpha-catenin than moderately to poorly differentiated types. Macroscopically papillary lesions had higher expression of E-cadherin than their nonpapillary counterparts. RT-PCR/SBH for CD44 revealed the CD44 variant form to be more prevalent in carcinoma than in normal mucosa, correlating with the immunohistochemical results, and with more exon variety. The Cox proportional hazards test identified histologic type and E-cadherin expression as prognostic factors. Among the examined molecules, E-cadherin was especially related to papillary mass formation and a good prognosis.

Zygosporin D and two new cytochalasins produced by the fungus Metarrhizium anisopliae.

Zygosporin D (3) and two new cytochalasins (4 and 5) were isolated from the culture filtrate of the fungus Metarrhizium anisopliae and characterized on the basis of their spectral data and chemical conversions. The new cytochalasins, 4 and 5, were determined to be deacetylcytochalasin C and (6R,16S,18R,21R)-18,21-dihydroxy-16, 18-dimethyl-10-phenyl[11]cytochalasa-13(E),19(E)-diene-1,7,17-trio ne, respectively. Of these three cytochalasins, only zygosporin D is an effective inhibitor of shoot elongation of rice seedlings.

Systemic aspergillosis caused by an aflatoxin-producing strain of Aspergillus flavus.

The first case of human systemic infection by an Aspergillus flavus isolate demonstrated to produce aflatoxins in vitro and in vivo is described. The patient, a 41-year-old man with acute myelogenous leukaemia, developed a complication of suspected pulmonary Aspergillus infection during remission induction therapy. Antifungal chemotherapy brought about a considerable degree of improvement, but remission of the underlying disease was not attained. Bone marrow transplantation was also not effective. The patient showed recovery from neutropenia but died despite aggressive antifungal chemotherapy. The autopsy revealed lesions in the lungs, myocardium, kidneys, brain, thyroid gland and skin due to a suspected Aspergillus sp. A fungus isolated from the right lung and the skin lesions was identified as A. flavus. Aflatoxins B1, B2 and M1 were detected in culture filtrates of the isolated A. flavus, and in an extract of lung lesions. These aflatoxins are considered to have played an important role in damaging the immune system of the patient through their toxic effects.

Isolation and characterization of a major allergenic component (gp55) of Aspergillus fumigatus.

IgE class antibodies specific for antigens in a water-soluble extract of Aspergillus fumigatus (strain NHL-5759) were analyzed by immunoblotting with sera from patients with allergic bronchopulmonary aspergillosis. All the sera tested were reactive with a major 50 to 60 kd protein in the extract. This allergen, designated gp55, was purified by gel filtration and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen was found to be present in the water-soluble extract in the form of a complex composed of approximately eight molecules of gp55. The carbohydrate and phosphate content of the purified antigen were 23.1% and 0.46%, respectively. The molar ratio of mannose to galactose residues was 2.76:1, and the protein was glycosylated predominantly with N-linked oligosaccharides. The serologic activity of the gp55 antigen was abolished by treatment with nonspecific protease (Pronase) but not by treatment with sodium metaperiodate or endoglycosidases. Thus the major antigenic site of the glycoprotein is located within its peptide moiety. The antigen itself displayed no chymotryptic or tryptic activity. The amino acid sequence of the 20 N-terminal residues of the antigen (ATPHEPVFFSWDAGAVTSFP) is different from that of any other protein previously reported.

Sensitive determination of zearalenone and alpha-zearalenol in barley and Job's-tears by liquid chromatography with fluorescence detection.

A sensitive and reliable method for liquid chromatographic (LC) determination of zearalenone and alpha-zearalenol in barley and Job's-tears was investigated. The method by which these toxins were determined involves addition of an internal standard (zearalenone 6'-oxime) to barley and Job's-tears samples. Extracts from grain samples were cleaned up by passage through chromatography on piperidinohydroxypropyl Sephadex LH-20 as a lipophilic gel. Individual toxins were resolved by LC on a reversed-phase (ODS) column with fluorescence detection. The detection limit is estimated to be 0.2 ng for zearalenone and alpha-zearalenol standards. Known amounts of zearalenone and alpha-zearalenol (25-1250 ng) were added to a barley sample (5 g). Average recoveries for alpha-zearalenol and zearalenone, respectively, ranged from 96 to 102% (mean CV, 3.6%) and from 96 to 103% (mean CV, 3.3%). This method is applicable to determination of alpha-zearalenol and zearalenone in barley and Job's tears with satisfactory sensitivity and accuracy.

Radioimmunoassay of nivalenol in barley.

Antibodies against nivalenol (NIV) tetraacetate (Tetra-Ac-NIV) were prepared by immunizing rabbits with a hemisuccinate derivative of 8-hydroxy-3,4,7,15-tetraacetyl-12, 13-epoxytrichothece-9-en conjugated to bovine serum albumin. A radioimmunoassay system with one of these sera was developed to measure NIV contamination in barley. The detection limit for Tetra-Ac-NIV was about 0.5 ng/ml. The relative cross-reactivities of the antiserum with Tetra-Ac-NIV, acetyl T-2 toxin, and scirpenol triacetate, which were determined by the competitive radioimmunoassay, were 1, 0.78, and 0.56, respectively. Other derivatives showed no cross-reactivity. For the determination of NIV in a barley sample, NIV was extracted from the sample with acetonitrile-water (7:3), defatted with hexane, and then acetylated with acetic anhydride to form Tetra-Ac-NIV. The reaction mixture was loaded onto a C18 cartridge to remove excess reagents and impurities. Tetra-Ac-NIV was eluted from the cartridge with 50% methanol in water, and the eluate was subjected to radioimmunoassay. Analysis of six naturally contaminated barley samples for NIV revealed that radioimmunoassay results agreed well with gas chromatographic analyses.

Production and characterization of monoclonal antibodies to nivalenol tetraacetate and their application to enzyme-linked immunoassay of nivalenol.

Three monoclonal antibodies were obtained by the fusion of mouse myeloma cells with splenocytes isolated from BALB/c mice that had been immunized with 8-hydroxy-3,4,7,15-tetraacetyl-nivalenol hemiglutarate covalently bound to bovine serum albumin. These anti-nivalenol tetraacetate monoclonal antibodies were of the IgG type and highly specific to nivalenol tetraacetate, with an apparent association constant of about 10(8)M-1. The relative cross-reactivities of one monoclonal antibody with nivalenol tetraacetate, acetyl T-2 toxin, and scirpenol triacetate were found to be 1.0, 0.02 and 0.03, respectively. Other derivatives showed no cross-reactivity at all. An indirect enzyme-linked immunosorbent assay (ELISA) based on the competitive binding principle was developed using the antibody from clone D18.102.59. The sensitivity of the system was about 0.1 ng of nivalenol tetraacetate per assay. Comparison of nivalenol levels detected in naturally contaminated barley samples by competitive indirect ELISA and gas chromatography (GC) showed good agreement, indicating that the antibody is useful for the measurement of nivalenol in naturally contaminated cereals and grains.

Occurrence of trichothecin in wheat.

Trichothecin, a toxic metabolite of Trichothecium roseum, was detected in 3 of 13 wheat samples examined at levels of 560, 290, and 270 ng/g. In addition, all the T. roseum isolates from the wheat produced the toxin on wheat.

Chemotaxonomy of Gibberella zeae with special reference to production of trichothecenes and zearalenone.

By adopting a single-spore isolation technique, 113 isolates of Gibberella zeae, the perfect stage of Fusarium graminearum, were isolated from rice stubbles in barley and wheat fields and tested for production of trichothecenes and zearalenone on rice grains. Of the isolates, 93% produced the trichothecenes, and they could be subdivided into two chemotaxonomic groups: nivalenol and fusarenon-X producers and deoxynivalenol and 3-acetyldeoxynivalenol producers. No cross production of these two types of trichothecenes was observed in these isolates. Zearalenone was detected in 68% of the isolates, but no clear relationship could be observed regarding its position with respect to the two chemotaxonomic groups.

Mite fauna and fungal flora in house dust from homes of asthmatic children.

Mite fauna and fungal flora in the house dust from homes of asthmatic children with positive and negative skin test to house dust allergen and non-asthmatic controls were examined. There was no conspicuous difference in mite species distribution among the three groups. Pyroglyphid mites dominate the mite fauna in house dust more than half of which being Dermatophagoides: D. pteronyssinus and D. farinae. There was no statistically significant difference in numbers between the two species and either species could dominate depending on the conditions of the individual houses. The average number of acarina in 0.5 g of fine dust did not differ statistically among the three groups; however, mite number per square meter floor differed between patients with positive skin test and negative skin test. The results suggest that house-cleaning might influence the possible sensitization of children. The genetic distribution of mould fungi in house dust was largely similar to that of airborne fungi. The average number of fungal colonies detected in 0.5 g of dust did not differ statistically among the three groups. Wallemia with its minute spores may cause sensitization but has so far been insufficiently investigated.