Sample Size Considerations for GEE Analyses of Three‐Level Cluster Randomized Trials

Summary Cluster randomized trials in health care may involve three instead of two levels, for instance, in trials where different interventions to improve quality of care are compared. In such trials, the intervention is implemented in health care units (“clusters”) and aims at changing the behavior of health care professionals working in this unit (“subjects”), while the effects are measured at the patient level (“evaluations”). Within the generalized estimating equations approach, we derive a sample size formula that accounts for two levels of clustering: that of subjects within clusters and that of evaluations within subjects. The formula reveals that sample size is inflated, relative to a design with completely independent evaluations, by a multiplicative term that can be expressed as a product of two variance inflation factors, one that quantifies the impact of within‐subject correlation of evaluations on the variance of subject‐level means and the other that quantifies the impact of the correlation between subject‐level means on the variance of the cluster means. Power levels as predicted by the sample size formula agreed well with the simulated power for more than 10 clusters in total, when data were analyzed using bias‐corrected estimating equations for the correlation parameters in combination with the model‐based covariance estimator or the sandwich estimator with a finite sample correction.     

Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization

During epithelial cell migration, membrane ruffles can be visualized by phase contrast microscopy as dark waves arising at the leading edge of lamellipodia that move centripetally toward the main cell body. Despite the common use of the term membrane ruffles, their structure, molecular composition, and the mechanisms leading to their formation remained largely unknown. We show here that membrane ruffles differ from the underlying cell lamella by more densely packed bundles of actin filaments that are enriched in the actin cross-linkers filamin and ezrin, pointing to a specific bundling process based on these cross-linkers. The accumulation of phosphorylated, that is, inactivated, cofilin in membrane ruffles suggests that they are compartments of inhibited actin filament turnover. High Rac1 and low RhoA activities were found under conditions of suboptimal integrin-ligand interaction correlating with low lamellipodia persistence, inefficient migration, and high ruffling rates. Based on these findings, we define membrane ruffles as distinct compartments of specific composition that form as a consequence of inefficient lamellipodia adhesion.     

Cross sectional study on cytokine production (TNF-α, IL-8) in German coalminers with progressive massive fibrosis and in control miners using a rapid wholeblood assay

Whole-blood release of tumour necrosis factor (TNF-α) and interleukin 8 (IL-8) was studied in 26 German ex-coalminers with progressive massive fibrosis (≥A, ILO 1980; cases) and 26 ex-miners free of pneumoconiosis (≤ 0/1; controls) using a simple wholeblood assay. Cases and controls were matched individually by age and duration of coalmine dust exposure (5-year window). Whole-blood cytokine release was determined (blinded to case control status) in incubations without additions (spontaneous) and with endotoxin (LPS, 3 ng ml^-1) or with coalmine dust (CMD; 5 mg ml^-1). CMD-stimulated TNF-α release was significantly increased and LPS-induced IL-8 release was significantly decreased in cases (matched t-tests: p < 0.01). No effect of duration of exposure was detectable in an unmatched analysis. No clear relationship with lung function parameters independent from case/control-status was observed, although a possible positive association with central airway resistance was indicated by multiple regression for both CMD stimulated TNF-α and LPS-stimulated IL-8. This study on individually matched coalminers validates previous findings on monocyte TNF release as a marker for pneumoconiosis using a method (whole-blood assay) that is more feasible for epidemiological studies. The different response of TNF-α and IL-8 may be useful in studying the occurrence of different endpoints like pneumoconiosis and lung function decrease.     

Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H₂O₂ is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, and more in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.     

Inhibition of the mitochondrial respiratory chain function abrogates quartz induced DNA damage in lung epithelial cells

Respirable quartz dust has been classified as a human carcinogen by the International Agency for Research on Cancer. The aim of our study was to investigate the mechanisms of DNA damage by DQ12 quartz in RLE-6TN rat lung epithelial type II cells (RLE). Transmission electron microscopy and flow-cytometry analysis showed a rapid particle uptake (30 min to 4 h) of quartz by the RLE cells, but particles were not found within the cell nuclei. This suggests that DNA strand breakage and induction of 8-hydroxydeoxyguanosine - as also observed in these cells during these treatment intervals - did not result from direct physical interactions between particles and DNA, or from short-lived particle surface-derived reactive oxygen species. DNA damage by quartz was significantly reduced in the presence of the mitochondrial inhibitors rotenone and antimycin-A. In the absence of quartz, these inhibitors did not affect DNA damage, but they reduced cellular oxygen consumption. No signs of apoptosis were observed by quartz. Flow-cytometry analysis indicated that the reduced DNA damage by rotenone was not due to a possible mitochondria-mediated reduction of particle uptake by the RLE cells. Further proof of concept for the role of mitochondria was shown by the failure of quartz to elicit DNA damage in mitochondria-depleted 143B (rho-0) osteosarcoma cells, at concentrations where it elicited DNA damage in the parental 143B cell line. In conclusion, our data show that respirable quartz particles can elicit oxidative DNA damage in vitro without entering the nuclei of type II cells, which are considered to be important target cells in quartz carcinogenesis. Furthermore, our observations indicate that such indirect DNA damage involves the mitochondrial electron transport chain function, by an as-yet-to-be elucidated mechanism.     

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Structural and compositional analysis of the keratinocyte migration track

Slowly migrating cells such as fibroblasts leave behind a "migration track," which has been assumed not to occur in fast-moving cells such as keratinocytes. Here we show that keratinocytes left behind "migration tracks" of cellular remnants consisting of membranous patches or macroaggregates that were anchored to a meshwork of extracellular matrix proteins consisting of collagen type IV, fibronectin, laminin, and laminin 5. According to their origin and localisation, two types of macroaggregates could be distinguished : (1) Spherical and elongated tubular structures (diameter about 50-110 nm) both of which were arranged like "pearls on a string" and that apparently derived from fragmentation of retracting fibres. (2) Spherical structures (diameter about 50 nm) left behind in the gaps between the retracting fibres and presumably derived from former focal adhesion sites. Both types of macroaggregates did not contain cytoplasmic proteins but carried on their surface adhesion proteins, particularly high amounts of integrins : type 1 macroaggregates contained alpha3beta1-integrins, whereas type 2 macroaggregates contained other types of integrins such as alpha6beta4-integrins. Modulation of keratinocyte adhesion by using poly-L-lysine coated cover slips resulted in an increased application of inhibitory beta1-antibodies and slightly reduced migration velocity and track formation. Within 24 h of migration, we observed a migration velocity-dependent loss of cellular beta1-integrin by macroaggregate formation of about 11% for fast and about 4% for slowly migrating keratinocytes. The physiological role of the migration track is unclear. However, with its multiple adhesion sites it may serve as a provisional basement membrane during reepithelialization of epidermal wounds.     

Group‐specific polymerase chain reaction amplification of SSU rRNA‐encoding gene fragments from 12 microbial taxa

Starting from an alignment of all known representatives in GenBank, we designed group specific primers targeting SSU rRNA‐encoding sequences of 12 microbial taxa known to contain insect pathogens and symbionts. We tested the specificity of these primers using representative species of all 12 groups as control templates. Polymerase chain reaction amplification conditions were modified until only group‐specific templates yielded a positive signal. The presented primer pairs thus allow for the amplification of SSU rRNA‐encoding sequences representing specific microbial groups directly from the environment (a social insect host in our study). We discuss possible applications of the identified molecular tools.     

Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H2O2 is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, andmore in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.