[Post-treatment assessment of Helicobacter pylori eradication, by polymerase chain reaction in gastric biopsies]. / Evaluación de la erradicación postratamiento del Helicobacter pylori por reaccion en cadena de la polimerasa en biopsias gástricas.

BACKGROUND: The pre-treatment detection of H.p. in the stomach of patients is easily achieved with routine methods. Conversely, with conventional methods, it is difficult to detect the presence of H.p. after treatment. OBJECTIVE: To estimate the actual percentage of successfully treated patients by using a more sensitive and specific technique (PCR) in the same biopsies where standard methods were negative for H.p. MATERIALS AND METHODS: We selected 97 treated patients (31 Gastric Ulcers/66 Duodenal Ulcers, 62 male/35 female, age: 49 +/- 14 years), in whom success of treatment was defined by histological means and CLO Test. In the same gastric biopsies H.p-DNA PCR was performed. Different therapeutic schemes were utilized, but all included Proton Pump Inhibitors + ATB. Eight weeks after the end of the treatment, without medication, the patients were controlled as follows: 5 biopsies per patient, 2 of antrum, 2 of corpus (in different zones) and 1 for CLO Test. H.p. eradication was defined on histological grounds (gastric biopsy histology: 10% formaldehide buffer fixation, paraffin inclusion, Giemsa, HE staining and inmunohistochemistry), CLO Test (Delta West Pty. Ltd. Bentley, Australia) and by the absence of H.p.-DNA by PCR (amplification of a 296 bp of the species-specific antigen of H.p. and visualization of the amplified product in agarose gel with Ethidium Bromide and UV light). RESULTS: [table: see text] CONCLUSIONS: The higher sensitivity of PCR (10(3) fold more than conventional methods) allowed us in this group of patients to detect 13% of false eradication. It would be necessary to follow up this group of patients in order to know whether they develop or not clinical symptoms and/or histological evidence of disease. If such a case PCR could become an important tool for treatment evaluation.

A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement.

The new, fluorescent Ca2+ indicator, fura-2, promises to expand our understanding of the role of subcellular changes in Ca2+ underlying cell function. During an investigation of the role of Ca2+ in the polarization response of human polymorphonuclear leukocytes to formyl-methionyl-leucyl-phenylalanine, we found that fura-2 trapped by cells incubated with the acetoxy-methyl ester of fura-2, F2-AM, yielded measurements of Ca2+ that were depressed at rest and during the response to formyl-methionyl-leucyl-phenylalanine. Fura-2, trapped by the cells, exhibited a spectrum in the presence of saturating Ca2+ that differed from that of fura-2 free acid. We have shown that the cellular fluorescence can be spectrally decomposed into two components: one with Ca2+ sensitivity identical to fully deesterified fura-2, and another which is Ca2+-insensitive. The Ca2+-insensitive component appears to be more fluorescent than F2-AM as well as spectrally different from F2-AM. The insensitive form probably results from incomplete deesterification of F2-AM by the cells. In order to accurately measure Ca2+ in polymorphonuclear leukocytes, it is imperative to check for the presence of Ca2+-insensitive fluorescence. The contribution of Ca2+-insensitive fura-2 fluorescence can be assessed routinely from spectral data obtained by calibration of intracellular fura-2 with known [Ca2+] using ionomycin. The end-of-experiment calibration step not only ensures accurate [Ca2+] measurements in polymorphonuclear leukocytes and in other cell types that display Ca2+-insensitive, contaminating fluorescence but also yields the spectral characteristics of the insensitive species.