Effectiveness of 16S ribosomal DNA real-time PCR and sequencing for diagnosing bacterial keratitis

Graefe's Archive for Clinical and Experimental Ophthalmology - To evaluate the efficacy of real-time PCR for 16S ribosomal DNA (16S r-DNA) and sequencing for diagnosing microbial keratitis. We...     

Induction of Extrahepatic Biliary Carcinoma by N-Nitrosobis(2-oxopropyl)amine in Hamsters Given Cholecystoduodenostomy with Dissection of the Common Duct

The methods we used to produce a carcinoma in the extrahepatic bile duct and gallbladder in hamsters are described along with the characteristics of the induced tumors. Female Syrian golden hamsters were first subjected to Cholecystoduodenostomy with dissection of the extrahepatic bile duct on the distal end of the common duct (CDDB) and were, 4 weeks later, treated with weekly subcutaneous injections of N-nitrosobis(2-oxopropyl)amine (BOP) at a dose of 10 mg/kg body weight for 9 weeks. The animals were killed at the 12th, 16th and 20th week after the initiation of BOP treatment. Extrahepatic bile duct carcinoma developed in 16%, 24% and 41% and gallbladder carcinoma occurred in 58%, 81% and 82% of the hamsters, respectively, at the corresponding times of killing. The incidences were significantly higher than those in sham-operated controls ( P <0.01). The induced extrahepatic bile duct carcinomas were predominantly of the polypoid type and gallbladder carcinomas were of the papillary type in growth form, being morphologically similar to early stage biliary carcinoma in humans. Immunohistochemical staining using bromodeoxynridine and anti-bromo-deoxyuridine monoclonal antibody demonstrated that the CDDB procedure greatly accelerated the cell kinetic activity of the biliary epithelium, and this was considered to be a major factor promoting the development of biliary carcinomas in this hamster model. In conclusion, this new model provides a high incidence of tumor development at the extrahepatic biliary tract and is expected to be useful for clarifying the characteristics of this highly malignant tumor.     

Diagnosis of Kala-Azar by Nested PCR Based on Amplification of the Leishmania Mini-Exon Gene

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.     

Endolymphatic perfusion with EGTA-acetoxymethyl ester inhibits asphyxia- and furosemide-induced decrease in endocochlear potential in guinea pigs

We examined the effect of the Ca(2+) concentration in the endolymph ([Ca](e)) or in the endolymphatic surface cells ([Ca](i)) on the endocochlear potential (EP) by using an endolymphatic or perilymphatic perfusion technique, respectively. (i) A large increase in [Ca](e) up to approximately 10(-3) M with a fall in the EP was induced by transient asphyxia ( approximately 2 min) or by the intravenous administration of furosemide (60 mg/kg), and a significant correlation was obtained between the EP and p[Ca](e) (= -log [Ca](e), r = 0.998). (ii) Perfusion of the endolymph with 10 mM EGTA for 5 min neither produced any significant change in the EP nor altered the asphyxia-induced change in EP (DeltaEP(asp)), suggesting that neither [Ca](e) nor the Ca(2+) concentration gradient across the stria vascularis contributed directly to the generation of the EP in the condition of low [Ca](e). In contrast, endolymphatic perfusion with high Ca(2+) (more than 10 mM) produced a decrease in EP and a significant correlation was obtained between the EP and the Ca(2+) concentration of perfusion solution (r = 0.982), suggesting that Ca(2+) permeability may exist across the stria vascularis. (iii) The administration of a Ca(2+) chelator, EGTA-acetoxymethyl ester (AM, 0.3 mM), to the endolymph, which produced a gradual increase in EP, suppressed significantly, by 60-80%, DeltaEP(asp) or furosemide-induced changes in EP. In contrast, perilymphatic administration of 0.5 mM EGTA-AM caused no significant suppression of the DeltaEP(asp). These findings suggest that [Ca](i) plays an important role in generating/maintaining a large positive EP.     

Metabolism and distribution in the rat of peak E substance,a constituent inl-tryptophan product implicated in eosinophilia-myalgia syndrome

Peak E substance, 1,1-ethylidenebis[tryptophan], a contaminant found inl-tryptophan tablets, has been suggested as a causative agent for eosinophilia-myalgia syndrome (EMS). Peak E substance (50 mg/kg) was administered perorally to Wistar rats to determine its metabolism and distribution. A purification procedure using Bond Elut C₈ cartridges followed by HPLC was developed for the determination of peak E substance. The plasma concentration of peak E substance was 136 ng/ml at 1 h, and urinary excretion was 717 ng at 5 h and 10342 ng for 5–24 h, showing slow excretion of peak E substance into urine. The amount of peak E substance in the contents of the large intestine at 5 h, however, was 3136 g, much greater than urinary excretion for 24 h, indicating considerable transfer of peak E substance to large intestine without decomposition by gastric fluid in the stomach. We have detected for the first time not only the occurrence of peak E substance in plasma and urine, but also 1-methyl-tetrahydro--carboline-3-carboxylic acid (MTCA) in blood and organs of rats treated with peak E substance, thereby suggesting MTCA as one of the the metabolites of peak E substance. The amount of MTCA in the contents of the large intestine as well as in urine of rats treated with peak E substance was significantly greater than inl-tryptophantreated rats (50 mg/kg p.o.), demonstrating that MTCA was more readily produced from peak E substance than froml-tryptophan. Finally, we propose acetaldehydeinduced production of MTCA from peak E substance.