Qian, Xiang

AbstractWe examined the specific cell types in normal human pituitaries that expressed PRL receptor (PRL-R) messenger ribonucleic acid (mRNA) by combined in situ hybridization and immunohistochemistry. The distribution of PRL-R mRNA in 28 pituitary adenomas was examined by in situ hybridization and reverse transcription-PCR in 12 cases of adenomas. In another set of experiments, 34 PRL adenomas from men, women, and bromocriptine-treated patients were analyzed for PRL-R by in situ hybridization.In the normal pituitary, PRL- and LH-producing cells had significantly more mean grain counts per cell and higher percentages of cells positive for PRL-R than GH and TSH cells. PRL-R mRNA was present in all groups of adenomas by in situ hybridization and reverse transcription-PCR. PRL adenomas had a significantly higher density of labeling compared to other adenoma types. Although there was no difference in the levels of PRL-R mRNA in PRL adenomas from men and premenopausal and postmenopausal women, patients treated with bromocriptine before pituitary surgery had significantly lower levels of PRL-R compared to all other groups. These results indicate that in the normal pituitary, PRL and LH cells have the highest level of PRL-R mRNA, whereas PRL adenomas have significantly higher levels of PRL-R mRNA than other types of adenomas, and bromocriptine treatment decreases the levels of PRL-R mRNA in PRL adenomas.

Abstract Galectin-3 (Gal-3), a β-galactoside-binding protein is expressed in a specific cell–type manner in pituitary tumors. Here we questioned the mechanism of Gal-3 expression in pituitary tumors, by using methylation-specific PCR and DNA sequence analyses to analyze the methylation status of the promoter region of the LGALS3 gene. DNA analysis of a human pituitary tumor, breast carcinoma cell lines, and thyroid carcinoma cell lines showed that in cells expressing Gal-3 protein, the LGALS3 gene was unmethylated, whereas in Gal-3 null cells, the promoter of the LGALS3 gene was methylated. Treatment of cells with 30 μmol/L 5-aza-2′-deoxycytidine induced Gal-3 mRNA and protein expression. Among pituitary tumors, 30% (7/23), mainly in follicle-stimulating hormone/luteinizing hormone–producing (38%) and null cell (57%) adenomas, the promoter of the LGALS3 was found to be methylated and silenced, although prolactin- and adrenocorticotropic hormone–producing tumors, which were unmethylated, expressed the Gal-3 protein. These results show for the first time that Gal-3 expression is regulated in part by promoter methylation in pituitary as well as in other tumors. Because it is functionally involved in cancer progression and metastasis, Gal-3 may serve as a possible therapeutic target in the treatment of pituitary tumors.

ABSTRACT We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila , and by Warthin-Starry (WS) staining. The results were compared to the “gold standard” method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.