Nabothian cyst content: A potential pitfall for the diagnosis of invasive cancer on Pap test cytology.

Nabothian cysts are common, benign findings of the squamocolumnar junction of the adult cervix. These cysts are filled with mucus and can also contain proteinaceous material, neutrophils, or neutrophil debris. Nabothian cysts can be broken by the spatula during smear taking, may stick to the brush and be smeared onto slides in conventional cytology or dissolved in the preserving solution for liquid-based cytology (LBC) preparations. The granular content of Nabothian cysts may be mistaken for the tumor diathesis (TD) pattern associated with invasive carcinoma. In the case described, the patient presented a high-grade squamous intraepithelial lesion associated with granular material (Nabothian cyst content) that we considered erroneously on LBC to be TD-like material, thus, raising the suspicion of invasive carcinoma. To the best of our knowledge, this is the first report showing that Nabothian cyst content may present a potential pitfall in the diagnosis of invasive carcinoma on LBC.

Strain-dependent high-level expression of a transgene for manganese superoxide dismutase is associated with growth retardation and decreased fertility.

Manganese superoxide dismutase (MnSOD) is essential in protecting mitochondria against the damaging effects of superoxide radicals (O(2)(*-)), and increased expression of MnSOD protects cells and transgenic animals from various forms of oxidative stress. In addition, increased levels of MnSOD have been shown to slow down cell growth and induce differentiation. To study the effects of high MnSOD levels in vivo, we generated a series of transgenic mice using a mouse genomic sequence under control of the endogenous promoter. Four transgenic lines produced by pronuclear DNA injection exhibited up to 2-fold elevated MnSOD levels in brain and heart. However, using an embryonic stem cell approach, a line having 10-fold elevated MnSOD levels in the brain and 6- to 7-fold elevated levels in the heart and kidney was generated. Surprisingly, the genetic background of this transgenic line influenced the expression level of the transgene, with DBA/2 (D2) and C57BL/6 (B6) mice exhibiting low- and high-level transgene expression, respectively. This difference was the result of an increased transcription rate of the transgene. High-level MnSOD expression in B6 animals was associated with small size, male infertility, and decreased female fertility. These features are absent on the D2 background and indicate that high levels of MnSOD activity may interfere with normal growth and fertility.

Overexpression of manganese superoxide dismutase (MnSOD) in whole lung or alveolar type II cells of MnSOD transgenic mice does not provide intrinsic lung irradiation protection.

Intratracheal (IT) injection of the transgene for human manganese superoxide dismutase in plasmid/liposome (SOD2-PL) complex prior to irradiation protects C57BL/6J mice from whole lung irradiation-induced organizing alveolitis/fibrosis. Transgene mRNA was detected in alveolar type II (AT-II) and tracheobronchial tree cells explanted to culture 48 hours after gene therapy. To determine whether constitutive overexpression of murine MnSOD (Sod2) in whole lung or surfactant promoter-restricted AT-II cells (SP1)-SOD2 mice would provide intrinsic radioresistance, transgenic mice of two strains were compared with age-matched controls. Other groups of surfactant promoter-restricted (SP1)-SOD2 transgenic mice or control FeVB/NHsd mice received IT SOD2-PL gene therapy prior to irradiation. There was no significant intrinsic lung protection in either strain of MnSOD transgenic mice. The SP1-SOD2 transgenic mice were protected from lung damage by IT injection of the human SOD2-PL complex 24 hours prior to irradiation. Thus, overexpression of either human SOD2 or murine Sod2 in the lungs of transgenic mice does not provide intrinsic lung irradiation protection. The overexpression of SOD2 in the SP1-SOD2 mice may have made the mice more sensitive to irradiation.

The use of transgenic and mutant mice to study oxygen free radical metabolism.

To distinguish the role of Mn superoxide dismutase (MnSOD) from that of cytoplasmic CuZn superoxide dismutase (CuZnSOD), the mouse MnSOD gene (Sod2) was inactivated by homologous recombination. Sod2 -/- mice on a CD1 (outbred) genetic background die within the first 10 days of life (mean, 5.4 days) with a complex phenotype that includes dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, metabolic acidosis and ketosis, and a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required to maintain the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide. On the other hand, Lebovitz et al. reported an independently derived MnSod null mouse (Sod2tmlLeb) on a mixed C57BL/6 and 129Sv background with a different phenotype. Because a difference in genetic background is the most likely explanation for the phenotypic differences, the two mutant lines were crossed into different genetic backgrounds for further analyses. To study the phenotype of Sod2tmlLeb mice CD1 background, the Sod2tmlLeb mice were crossed to CD1 for two generations before the -/+ mice were intercrossed to generate -/- mice. The life span distribution of CD1 < Sod2-/- > Leb was shifted to the left, indicating a shortened life span on the CD1 background. Furthermore, the CD1 < Sod2-/- > Leb mice develop metabolic acidosis at an early stage as was observed with CD1 < Sod2-/- > Cje. When Sod2tmlCje was placed on C57BL/6J (B6) background, the -/- mice were found to die either during midgestation or within the first 4 days after birth. However, when the B6 < Sod2 -/+ > Cje were crossed with DBA/2J (D2) for the generation of B6D2F2 < Sod2-/- > Cje mice, an entirely different phenotype, similar to that described by Lebovitz et al., was observed. The F2 Sod -/- mice were able to survive up to 18 days, and the animals that lived for more than 15 days displayed neurological abnormalities including ataxia and seizures. Their hearts were not as severely affected as were those of the CD1 mice, and neurological degeneration rather than heart defect appears to be the cause of death.

Antisense manganese superoxide dismutase mRNA inhibits the antiviral action of interferon-gamma and interferon-alpha.

Manganese superoxide dismutase (MnSOD) is induced by interferon-gamma (IFN-gamma) in various cell lines. To determine whether MnSOD plays a role in the antiviral action of IFN-gamma, we employed an antisense strategy to inhibit the expression of MnSOD in the human melanoma cell line, A375. Three antisense-containing clones that exhibited reduced induction of MnSOD were investigated with respect to their response to the antiviral protective effects of IFN-gamma and IFN-alpha. We observed a striking decrease in the ability of IFN-gamma to protect antisense clones from vesicular stomatitis virus infection (VSV). The IFN-alpha induced antiviral state was also impaired, but to a lesser degree than was observed with IFN-gamma. We excluded the possibility that these effects were caused by a higher sensitivity of the antisense cells to VSV itself and found that the antisense clones actually were less sensitive to VSV. Therefore, we conclude that MnSOD is involved in the establishment of the IFN-gamma-induced antiviral state and to a lesser degree in the antiviral actions of IFN-gamma.

Superoxide radical and iron modulate aconitase activity in mammalian cells.

Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-..

Analysis of human immunodeficiency virus type 1 promoter insertion in vivo.

Our study reports the occurrence of HIV-1 insertional activation events in vivo. Using a previously described PCR assay, small aliquots of broncho-alveolar lavage (BAL) cells obtained from AIDS patients were analyzed. Nine percent (5/54) of the aliquots contained proviral-host sequence transcripts indicating HIV-1 promoter insertion, whereas multiply spliced HIV-1 mRNAs were found in 28% (15/54) of the aliquots. In four of five events, insertions affect distinct cellular transcription units expressed in a T-cell line. To establish a ratio between provirus integration and promoter insertion events, an in vitro infection study was performed and transcripts containing HIV-1 and K-ras or CD4 gene sequences, respectively, were monitored. Given the randomness of retrovirus integration, 170 sense-oriented HIV integrations into these gene loci were predicted to occur. Three distinct promoter insertion events were observed, indicating that 1.8% of integrated proviruses transcribed adjacent genes. Based on this result and a mean of 257 proviral copies per 10(6) BAL cells, we would expect to observe 25 promoter insertion events in our in vivo study. That only five events were found may be due to the lower transcriptional activity of HIV-1 in vivo than that in cell cultures.

HIV-1 promotor insertion revealed by selective detection of chimeric provirus-host gene transcripts.

To study host gene activation by retroviral promotor insertion, a polymerase chain reaction (PCR) assay was developed. This method allows a sensitive and selective detection of chimeric provirus-host gene transcripts, hallmarks of insertional activation events, which does not rely on an induction of tumor cell growth. We analysed HIV-1 infected cells of a CD4+ T-cell line (H9), infected peripheral blood mononuclear cells and cells in broncho-alveolar washes of AIDS patients. In each case, a variety of chimeric mRNA molecules were detected using a PCR amplification reaction and 5' primers specific to the HIV-1 LTR and 3' primers specific to poly A of mRNA. In infected H9 lymphocytes, a mRNA was identified encoding a putative protein of 145 amino-acids that was not expressed in uninfected H9 cells. This shows for the first time that HIV-1 can activate transcription of host cellular genes by promotor insertion in a fashion similar to slow-transforming avian and murine retroviruses.