A DNA-based assay for toxic chemicals in wastewater.

Chemical toxicants, particularly metal ions, are a major contaminant in global waterways. Live-organism bioassays used to monitor chemical toxicants commonly involve measurements of activity or survival of a freshwater cladoceran (Ceriodaphnia dubia) or light emitted by the marine bacterium Vibrio fischeri, used in the commercial Microtox® bioassay. Here we describe a novel molecule-based assay system employing DNA as the chemical biosensor. Metals bind to DNA, causing structural changes that expel a bound (intercalated) fluorescent reporter dye. Analyses of test data using 48 wastewater samples potentially contaminated by metal ions show that the DNA-dye assay results correlate with those from C. dubia and Microtox bioassays. All three assays exhibit additive, antagonistic, and synergistic responses that cannot be predicted by knowing individual metal concentrations. Analyses of metals in these samples imply the presence of chemical toxicants other than metal ions. The DNA-dye assay is robust, has a 12-month shelf life, and is only slightly affected by sample pH in the range 4 to 9. The assay is completed in a matter of minutes, and its portability makes it well suited as a screening assay for use in the field. We conclude that the DNA-dye test is a surrogate bioassay suitable for screening chemical toxicity.

Urine cytology in the evaluation of urological malignancy revisited: is it still necessary?

OBJECTIVE: We aim to determine if urine cytology was still necessary as a routine part of the evaluation for the presence of urological malignancy and to evaluate its cost effectiveness. METHODS: Urine cytology reports over a 6-year period (2000-2005) were retrieved from our institution's pathology department database. Patients with urine cytology positive for malignant cells were identified. We retrospectively reviewed the charts of these patients for age, sex, flexible cystoscopy and radiological imaging results. The cost of urine cytology was retrieved from the pathology department. RESULTS: There were a total of 2,568 urine cytological examinations. Of these, 25 were positive for malignant cells. There were 19 male (76%) and 6 female (24%) patients with a mean age of 72 years (range: 49-97). In 21 patients with positive cytology, a bladder tumor was identified at flexible cystoscopy and/or imaging studies. For a positive cytology yield of 1%, EUR 210,000 was spent. CONCLUSIONS: Routine urine cytology was not cost effective and did not add to the diagnostic yield beyond cystoscopy and diagnostic imaging. It may be omitted in the initial evaluation of urological malignancy.

Number and size of lymph nodes recovered from dukes B rectal cancers: correlation with prognosis and histologic antitumor immune response.

PURPOSE: In rectal cancer variation in lymph node recovery influences the detection of nodal metastases and prognosis among Dukes B (Stage II) cases. However, the possible prognostic importance of node size and inherent patient/tumor characteristics in determining node recovery has not been studied. METHODS: We examined 269 Dukes B (Stage II) rectal tumors, with a mean of 12 nodes per case. Primary tumor characteristics were correlated with the number and size of recovered nodes. Clinical follow-up permitted determination of long-term survival. RESULTS: The five-year survival of 94 Dukes B cases with nine or fewer nodes was 69.4 percent vs. 87.6 percent in 175 cases with ten or more nodes (P = 0.001). Lymph nodes were smaller in patients dying of recurrence; among 130 Dukes B patients whose mean node diameter was <4 mm, survival was 73.3 vs. 88 percent when mean nodal diameter was > or =4 mm. The number and size of recovered nodes was related to patient age, histologic antitumor immune response, and tumor growth pattern. By combining the number and size of nodes, a poor prognosis subgroup of 98 Dukes B patients with relatively few large nodes (no more than 5 measuring > or =4 mm) was identified with a five-year survival of 65.6 percent compared with 89.6 percent for the remaining 158 Dukes B cases (P < 0.0001). CONCLUSIONS: In Dukes B rectal tumors, the number and size of lymph nodes are related to inherent patient and tumor characteristics and permit the identification of Dukes B cases at increased risk of recurrence. A valid comparison of nodal sampling efficiency between centers necessitates measuring and counting harvested lymph nodes.

Gastric motor dysfunction: is eosinophilic mural gastritis a causative factor?

Delayed gastric emptying caused either by gastric motor dysfunction or by gastroparesis is a profoundly debilitating disorder. When unresponsive to medical therapy, patients may undergo radical surgery including near-total gastro-oesophageal, with varied symptomatic improvement. We describe two patients who presented with symptoms consistent with gastro-oesophageal reflux, unresponsive to medical management. After fundoplication both developed symptoms of profound gastric motor dysfunction and subsequently proceeded to near-total gastro-oesophageal with symptomatic improvement. Histological examination of both excised gastric specimens revealed eosinophilic mural gastritis. To our knowledge, these are the first cases to demonstrate the association of mural eosinophilia and symptomatic gastric motor dysfunction. We propose that patients with gastric motor dysfunction, refractory to medical management, progress to laparoscopy and mural biopsy before gastrectomy. This would allow histological analysis of the gastric wall, and in the event of a positive finding of mural eosinophilic gastritis would allow a trial of medical therapy that could include an eosinophilic stabilizer such as the leukotriene D4 receptor antagonist montelukast or intravenous corticosteroid therapy, which may alleviate the symptoms.

Bacterial DNA within granulomas of patients with Crohn's disease--detection by laser capture microdissection and PCR.

OBJECTIVES: We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohn's disease. While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects. The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohn's disease. METHODS: Archival tissue from 15 surgical cases of Crohn's disease and 10 non-Crohn's granulomatous bowel disease controls were examined. Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E. coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene. RESULTS: E. coli DNA was detected in microdissected granulomas in 12/15 Crohn's disease patients and in 1/10 non-Crohn's control granulomas (p < 0.001). Also, E. coli DNA was detected in 8/15 Crohn's full-thickness sections and in 4/10 control full-thickness sections. CONCLUSIONS: E. coli DNA may be detected more frequently in Crohn's granulomas than in other non-Crohn's bowel granulomas. This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells. In light of previous detection of M. paratuberculosis DNA in Crohn's granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohn's disease.