Canonical WNT signalling determines lineage specificity in Wilms tumour.

Wilms tumours (WTs) have two distinct types of histology with or without ectopic mesenchymal elements, suggesting that WTs arise from either the mesenchymal or epithelial nephrogenic lineages. Regardless of the presence or absence of CTNNB1 mutations, nuclear accumulation of beta-catenin is often observed in WTs with ectopic mesenchymal elements. Here, we addressed the relationship between the WNT-signalling pathway and lineage in WTs by examining CTNNB1 and WT1 mutations, nuclear accumulation of beta-catenin, tumour histology and gene expression profiles. In addition, we screened for mutations in WTX, which has been proposed to be a negative regulator of the canonical WNT-signalling pathway. Unsupervised clustering analysis identified two classes of tumours: mesenchymal lineage WNT-dependent tumours, and epithelial lineage WNT-independent tumours. In contrast to the mesenchymal lineage specificity of CTNNB1 mutations, WTX mutations were surprisingly observed in both lineages. WTX-mutant WTs with ectopic mesenchymal elements had nuclear accumulation of beta-catenin, upregulation of WNT target genes and an association with CTNNB1 mutations in exon 7 or 8. However, epithelial lineage WTs with WTX mutations had no indications of active WNT signalling, suggesting that the involvement of WTX in the WNT-signalling pathway may be lineage dependent, and that WTX may have an alternative function to its role in the canonical WNT-signalling pathway.

Novel urothelium specific gene expression identified by differential display reverse transcriptase-polymerase chain reaction.

PURPOSE: Understanding the molecular basis of differential gene expression among different tissues at various developmental stages and in neoplastic transformation is an important biological goal. The potential clinical applications of this improved understanding are more precise diagnosis of disease, prediction of prognosis, novel targeted therapies and prediction of response to therapy. MATERIALS AND METHODS: Differential display reverse transcriptase-polymerase chain reaction was used to compare gene expression in bovine urothelium to that in autologous lung, esophagus, liver and spleen. Products that appeared to have urothelial specific expression were sequenced and assessed for homology with known sequences. Ribonuclease protection assays were used to further confirm the expression pattern. RESULTS: A total of 32 discrete cDNAs were identified, including 3 products from genes known to be urothelium specific in their expression, 16 with significant homology to bovine, human or mouse expressed sequence tags and 5 with no sequence homology to any currently available sequence. Urothelium specific mRNA expression was confirmed for 3 genes by ribonuclease protection assays and one (Udd06) was further characterized as a urea transporter. CONCLUSIONS: The use of differential display reverse transcriptase-polymerase chain reaction and other complementary techniques for parallel gene expression analysis will permit the complete characterization of the urothelial transcriptome and help identify potential molecular targets for rationally targeted therapy.

Transcriptional control of the human urothelial-specific gene, uroplakin Ia.

The transcriptional control elements of tissue-specific genes may be exploited in the design of therapeutic constructs for use in human gene therapy. The uroplakins are a family of four proteins which form the asymmetric unit membrane of the urothelium. We have cloned the human uroplakin Ia gene and defined its genomic structure and transcriptional start site. Using quantitative RT-PCR in an extended panel of normal tissues, we have demonstrated highly urothelial-specific expression of this gene. A Dual-Luciferase assay was used to assess the transcriptional activity of a variety of promoter fragments of the human uroplakin Ia gene. A highly specific promoter fragment (consisting of 2147 bp of 5'-flanking sequence, intron 1 and the 5' UTR) was identified which regulated urothelial-specific expression in vitro. The human uroplakin Ia promoter identified has potential use in future gene therapy strategies to restrict transgene expression to the urothelium.

Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate.

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.

Isolation of Blastomyces dermatitidis in soil associated with a large outbreak of blastomycosis in Wisconsin.

In investigating six cases of blastomycosis in two school groups that had separately visited an environmental camp in northern Wisconsin in June 1984, we identified a large outbreak of the disease and isolated Blastomyces dermatitidis from soil at a beaver pond near the camp. Of 89 elementary-school children and 10 adults from the two groups, 48 (51 percent) of the 95 evaluated in September had blastomycosis. Of the cases, 26 (54 percent) were symptomatic (the median incubation period was 45 days; range, 21 to 106 days). No cases were identified in 10 groups that visited the camp two weeks before or after these two groups. A review of camp itineraries, a questionnaire survey, and environmental investigation showed that blastomycosis occurred in two of four groups that visited a beaver pond and in none of eight groups that did not. Walking on the beaver lodge (P = 0.008) and picking up items from its soil (P = 0.05) were associated with illness. Cultures of soil from the beaver lodge and decomposed wood near the beaver dam yielded B. dermatitidis. We conclude that B. dermatitidis in the soil can be a reservoir for human infection.

Recurrent urban histoplasmosis, Indianapolis, Indiana, 1980-1981.

In January 1981, informal surveillance of acute histoplasmosis in Indianapolis, Indiana, revealed a marked increase in disease activity for the last quarter of 1980. Fifty-one patients with onset of acute histoplasmosis during this period were identified through review of hospital admissions, emergency room visits, and serologic records at Indianapolis hospitals and the Indiana State Board of Health. In a retrospective case-control study, the authors found a significant association between developing acute histoplasmosis during this period and working or attending classes in a 2 sq mi (5.2 sq km) area encompassing the Indiana University-Purdue University campus (p = 0.015, Fisher's exact test). A review of construction activities on or near the campus during the epidemic period suggested that the probable source of infection was excavation activity for a large new indoor swimming complex (natatorium). Skin tests and serosurveys of students on campus by a newly developed radioimmunoassay for histoplasmal immunoglobulin M antibodies supported the association of infection with exposure to this site (p less than 0.05).

Detection of two Blastomyces dermatitidis serotypes by exoantigen analysis.

We evaluated the diagnostic value of antibody to Blastomyces dermatitidis A precipitin arcs for identifying B. dermatitidis cultures by the exoantigen technique with 88 isolates from the United States and Canada, 12 from Africa, and 1 each from India and Israel. In addition, we studied 190 mycelial-form isolates of other fungi which could be confused antigenically or morphologically with B. dermatitidis. Antigen extracts from all of the North American isolates, the Indian and Israeli isolates, and one of the African isolates reacted with the B. dermatitidis A antibody. The anti-A serum did not react with antigens from the other 11 African isolates or with antigens from any of the 190 heterologous fungi. Studies with unadsorbed and adsorbed antisera to selected North American and African isolates of B. dermatitidis showed that the North American isolates not only produced the distinct A antigen, but also formed another antigen that was common to all 11 of the antigenically less complex African isolates. The results of this study indicate that at least two serotypes of B. dermatitidis exist. All of the North American, Indian, and Israeli isolates and one of the African isolates contained the A antigen and were designated serotype 1. Most of the African isolates (11 of 12) constituted a second serotype that was deficient in A but showed an antigen designated K.

A winter outbreak of acute histoplasmosis in northern Michigan.

In January 1980, an outbreak of 138 cases of acute pulmonary disease occurred among employees of a Michigan limestone quarry. The source of exposure was a vessel repair building (relative risk = 5.8) and the time of exposure was January 10 (relative risk = 2.4). Work activities associated with the specific place and time suggested exposure to a pulley which had been stored in a ring-billed gull nesting area as the cause of illness. Histoplasma capsulatum was recovered from the nesting area, the pulley, and the sputum of several patients. Early serologic testing confirmed the diagnosis of acute pulmonary histoplasmosis.

Decontamination of a Histoplasma capsulatum-infested bird roost in Illinois.

Histoplasma capsulatum was isolated from 27 of 34 soil specimens collected from a 3.23-hectare (8-acr) estate in illinois that had been the site of a bird roost for more than 10 yr. When the estate was sold to a developer for for the construction of a shopping center, it was feared that disturbing the roost would cause airborne transmission of spores of H. Capsulatum and thus create a public health hazard for the workmen and surrounding community. To prevent this, the bird roost was decontaminated with the application of 3,785 ml (1 gallon) of 3% formalin per square foot of soil. Safety precautions were taken to minimize the inhalation of dust and formalin by the decontamination team. Thirty-five soil specimens collected after application of the formalin were negative for H. capsulatum. No cases of histoplasmosis occurred among the decontamination workers, construction crews, or the general population. The total cost of the decontamination project was $75,000.

Conventional vs. formulated foods in school lunches. II. Cost of food served, eaten, and wasted.

School lunches containing all conventional foods were designed to provide one-third or one-half the recommended allowances for elementary students. The same nutrient levels were planned in meals containing some formulated items. Each type of meal was served for five days, and total food costs and costs of food served, consumed, and wasted were calculated. At the same nutritional level, mean preparation costs of partially formulated meals were lower than for meals composed of all conventional foods. Meals providing one-half the allowances and containing formulated items cost slightly less than totally conventional meals which provided one-third of the allowances. Cost of waste was lower with partially formulated meals.

Conventional vs. formulated foods in school lunches. I. Comparison of students' food and nutrient intakes.

School lunches containing all conventional foods were designed to provide either one-third or one-half the Recommended Dietary Allowances for elementary students. The same nutrient goals were used in planning meals containing some formulated foods. Goals and specified portion sizes for first through third graders were distinct from those for fourth through sixth graders, and data from the two groups were maintained separately. Nutrients in meals and plate waste were analyzed to compare nutrient intake among the treatments. Formulated meals weighed less than control and conventional meals, and students ate higher percentages of both the weight and the nutrients served in formulated meals. Nutrient intake was higher when half the allowances was served, but waste was also higher.

Major nutrients in the Type A lunch. II. Amounts consumed by students.

Plate waste from Type A school lunches was collected during the period that fifth, seventh, and tenth grade students ate lunch. Twelve elementary, three junior high, and seven high schools distributed among three geographic regions of North Carolina were included in the sample. Nutrients served and nutrients in plate waste were determined by laboratory analyses of two five-day composites of meals and of waste from each school. Nutrients consumed were determined by difference. Relative to the Type A goal, protein intake was highest; riboflavin and vitamin A values were consumed in satisfactory (one-third or more of the allowances) amounts. Younger students consumed adequate iron and calcium, and calcium intake approached adequacy among older students. High school students consumed only 69 per cent of their goal for iron and 75 per cent of the goal of energy value; among the three groups, however, this was the highest percentage of the goal for energy value consumed. Ascorbic acid intake was least satisfactory for fifth and seventh grade students. Overall, fifth graders consumed a significantly (P less than .01) lower percentage of the nutrients served than the two older groups. With a few exceptions, students consumed 80 to 90 per cent of the various nutrients which were served: 62 to 66 per cent of the vitamin A value served was consumed; fifth grade students consumed only 77 per cent of the iron served; and the two younger groups consumed only 69 per cent of the ascorbic acid served. Although the most marked regional differences in nutrients consumed occurred with vitamin A and ascorbic acid, overall elementary students in the eastern region consumed a significantly (P less than .01) higher percentage of nutrients served than did those in other two regions.