Method to characterize inorganic particulates in lung tissue biopsies using field emission scanning electron microscopy.

Humans accumulate large numbers of inorganic particles in their lungs over a lifetime. Whether this causes or contributes to debilitating disease over a normal lifespan depends on the type and concentration of the particles. We developed and tested a protocol for in situ characterization of the types and distribution of inorganic particles in biopsied lung tissue from three human groups using field emission scanning electron microscopy (FE-SEM) combined with energy dispersive spectroscopy (EDS). Many distinct particle types were recognized among the 13 000 particles analyzed. Silica, feldspars, clays, titanium dioxides, iron oxides and phosphates were the most common constituents in all samples. Particles were classified into three general groups: endogenous, which form naturally in the body; exogenic particles, natural earth materials; and anthropogenic particles, attributed to industrial sources. These in situ results were compared with those using conventional sodium hypochlorite tissue digestion and particle filtration. With the exception of clays and phosphates, the relative abundances of most common particle types were similar in both approaches. Nonetheless, the digestion/filtration method was determined to alter the texture and relative abundances of some particle types. SEM/EDS analysis of digestion filters could be automated in contrast to the more time intensive in situ analyses.

Organizing pneumonia and lymphoplasmacytic inflammation predict treatment response in idiopathic pulmonary fibrosis.

AIMS: To identify individual histopathological features within usual interstitial pneumonia pattern that predict responsiveness to immunosuppressive therapy. METHODS AND RESULTS: Fifty-six retrospectively confirmed usual interstitial pneumonia pattern surgical lung biopsy specimens from subjects with idiopathic pulmonary fibrosis treated with corticosteroid and cytotoxic therapy were included. Eleven prospectively defined histopathological features were evaluated by two expert pulmonary pathologists. Regression analysis identified predictors of response to therapy, as defined by the change in percent predicted forced vital capacity over 6 months. Additional end-points were change in dyspnoea score over 6 months, and survival time. Improvement in percent predicted forced vital capacity was associated with lymphoplasmacytic inflammation, while worsening of percent predicted forced vital capacity was associated with the presence of organizing pneumonia and fibroblast foci. Worsening dyspnoea was associated with fibroblast foci. Survival time was associated with age and baseline percent predicted forced vital capacity, but not with any individual histopathological feature. CONCLUSIONS: In pathological usual interstitial pneumonia pattern, the presence of lymphoplasmacytic inflammation predicts responsiveness to immunomodulatory therapy, while airspace organization predicts lack of response.

Biphasic regulation of breast cancer cell growth by progesterone: role of the cyclin-dependent kinase inhibitors, p21 and p27(Kip1).

Depending on the tissue, progesterone is classified as a proliferative or a differentiative hormone. To explain this paradox, and to simplify analysis of its effects, we used a breast cancer cell line (T47D-YB) that constitutively expresses the B isoform of progesterone receptors. These cells are resistant to the proliferative effects of epidermal growth factor (EGF). Progesterone treatment accelerates T47D-YB cells through the first mitotic cell cycle, but arrests them in late G1 of the second cycle. This arrest is accompanied by decreased levels of cyclins D1, D3, and E, disappearance of cyclins A and B, and sequential induction of the cyclin-dependent kinase (cdk) inhibitors p21 and p27(Kip1). The retinoblastoma protein is hypophosphorylated and extensively down-regulated. The activity of the cell cycle-dependent protein kinase, cdk2, is regulated biphasically by progesterone: it increases initially, then decreases. This is consistent with the biphasic proliferative increase followed by arrest produced by one pulse of progesterone. A second treatment with progesterone cannot restart proliferation despite adequate levels of transcriptionally competent PR. Instead, a second progesterone dose delays the fall of p21 and enhances the rise of p27(Kip1), thereby intensifying the progesterone resistance in an autoinhibitory loop. However, during the progesterone-induced arrest, the cell cycling machinery is poised to restart. The first dose of progesterone increases the levels of EGF receptors and transiently sensitizes the cells to the proliferative effects of EGF. We conclude that progesterone is neither inherently proliferative nor antiproliferative, but that it is capable of stimulating or inhibiting cell growth depending on whether treatment is transient or continuous. We also suggest that the G1 arrest after progesterone treatment is accompanied by cellular changes that permit other, possibly tissue-specific, factors to influence the final proliferative or differentiative state.

Surprises with antiprogestins: novel mechanisms of progesterone receptor action.

When hormone antagonists have inappropriate agonist-like effects, the clinical consequences are grave. We describe novel molecular mechanisms by which antiprogestin-occupied progesterone receptors behave like agonists. These mechanisms include agonist-like transcriptional effects that do not require receptor binding to DNA at progesterone response elements, or that result from cross-talk between progesterone receptors and other signalling pathways. We discuss the complex structural organization of progesterone receptors, and demonstrate that the B receptor isoform has a unique third activation domain that may confer agonist-like properties in the presence of antiprogestins, whereas the A receptor isoform is a dominant-negative inhibitor. We argue that these novel mechanisms play a role in the apparent hormone resistance of breast cancers and the variable tissue-specific responses to antagonists.

New T47D breast cancer cell lines for the independent study of progesterone B- and A-receptors: only antiprogestin-occupied B-receptors are switched to transcriptional agonists by cAMP.

Because progesterone antagonists are growth inhibitors, they are in Phase III clinical trials for the treatment of breast cancer. However, when cellular cAMP levels are elevated, some antiprogestins inappropriately activate transcription. We have proposed that hormone "resistance" may result from such unintended stimulation of breast cancer by antagonists. In transient expression systems, the two natural isoforms of human progesterone receptors (PR), B-receptors and truncated A-receptors, have dissimilar effects on agonist-mediated transcription. We show here that in the presence of 8-Br-cAMP, antiprogestin-occupied B-receptors but not A-receptors become transcriptional activators. Therefore, we developed new model systems to study each PR isoform independently in a breast cancer setting: (a) a stable PR-negative monoclonal subline (T47D-Y) of PR-positive T47D breast cancer cells was selected by flow cytometric PR screening. T47D-Y cells are PR-negative by immunoassays, by ligand binding assay, by growth resistance to progestins, by failure to bind a progesterone response element (PRE) in vitro, and by failure to transactivate PRE-regulated promoters; and (b) T47D-Y cells were stably transfected with expression vectors encoding one or the other PR isoform, and two monoclonal cell lines were selected that express either B-receptors (T47D-YB) or A-receptors (T47D-YA) at levels equal to those seen in natural T47D cells. The ectopically expressed receptors are properly phosphorylated, and like endogenously expressed receptors, they undergo ligand-dependent down-regulation. The expected B:B or A:A homodimers are present in cell extracts from each cell line, but A:B heterodimers are missing in both. In the presence of agonists, cAMP-dependent, transcriptional synergism of PRE-regulated promoters is seen in both cell lines. By contrast, in the presence of the antiprogestins RU486 or ZK112993, inappropriate transactivation occurs in YB cells but not in YA cells. The class of antiprogestins represented by ZK98299, which blocks PR binding to DNA, does not activate transcription in either cell line. We propose that these new cell lines are physiological models for the study of PR isoform-specific antiprogestin resistance in breast cancer.