Fetal bovine serum induces sustained, but reversible, epithelial-mesenchymal transition in the BEAS-2B cell line.

BEAS-2B is a non-malignant, immortalized human cell line that has been used extensively as a model of lung epithelium. Despite ATCC recommendations to culture BEAS-2B in defined, serum-free media, many publications describe culturing BEAS-2B in fetal bovine serum (FBS)-containing media. The objective of this study was to define the effects of FBS on BEAS-2B cells. FBS exposure resulted in increased nuclear levels of transcription factors responsible for regulating epithelial-mesenchymal transition (EMT), increased cell invasiveness and increased anchorage-independent growth. FBS-exposed BEAS-2B cells exhibited a decrease of the epithelial markers, E-cadherin and claudin-1 at the mRNA and protein levels, along with a corresponding increase of the mesenchymal marker, vimentin, at the protein level. Fractionation studies implicated an active moiety in FBS with a molecular weight larger than 30 kD. The mesenchymal phenotype was persistent provided FBS exposure was maintained. Upon FBS removal, both epithelial and mesenchymal markers began to revert toward an epithelial phenotype. Transforming growth factor ß1 (TGFß1) exposure to BEAS-2B recapitulated some key features of FBS-induced EMT. Our data suggest that FBS-exposed BEAS-2B cells do not accurately model the epithelial phenotype. Interpretation of data from BEAS-2B should include careful consideration of the effect of culture conditions.

Use of dust fall filters as passive samplers for metal concentrations in air for communities near contaminated mine tailings.

Mine tailings are a source of metal exposures in many rural communities. Multiple air samples are necessary to assess the extent of exposures and factors contributing to these exposures. However, air sampling equipment is costly and requires trained personnel to obtain measurements, limiting the number of samples that can be collected. Simple, low-cost methods are needed to allow for increased sample collection. The objective of our study was to assess if dust fall filters can serve as passive air samplers and be used to characterize potential exposures in a community near contaminated mine tailings. We placed filters in cylinders, concurrently with active indoor air samplers, in 10 occupied homes. We calculated an estimated flow rate by dividing the mass on each dust fall filter by the bulk air concentration and the sampling duration. The mean estimated flow rate for dust fall filters was significantly different during sampling periods with precipitation. The estimated flow rate was used to estimate metal concentration in the air of these homes, as well as in 31 additional homes in another rural community impacted by contaminated mine tailings. The estimated air concentrations had a significant linear association with the measured air concentrations for beryllium, manganese and arsenic (p < 0.05), whose primary source in indoor air is resuspended soil from outdoors. In the second rural community, our estimated metal concentrations in air were comparable to active air sampling measurements taken previously. This passive air sampler is a simple low-cost method to assess potential exposures near contaminated mining sites.

Genetic regulation of rejection and survival following human lung transplantation by the innate immune receptor CD14.

We have developed the hypothesis that genetic polymorphisms which alter the expression or function of innate immune receptors contribute to the marked interindividual differences in the onset and severity of lung transplant rejection. In this analysis, we considered the effects of a common promotor polymorphism of the lipopolysaccharide receptor CD14 associated with increased transcriptional activity upon the development of posttransplant rejection and graft survival. Genotyping was performed in 226 lung transplant recipients well characterized with regards to clinical outcomes. An earlier onset of acute rejection, bronchiolitis obliterans syndrome (BOS) and worse posttransplant graft survival due to greater BOS related deaths was evident in patients with the CD14 -159 TT genotype (TT). The adverse effect upon graft survival of the TT genotype remained significant in a multivariate Cox model (Hazard Ratio 1.65, 95% CI, 1.03-2.64, p-value = 0.04) after adjusting for other important covariates. Furthermore, TT patients have significantly greater sCD14, TNF-alpha and IFN-gamma in the peripheral blood implying a heightened state of innate immune activation drives the development of increased post-transplant rejection. Inhibition of innate immune activation through CD14 represents a novel and potentially important therapeutic target to prevent post-transplant rejection and improve outcomes after human lung transplantation.

5-Aza-2'-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation.

The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2'-deoxycytidine (5-aza-CdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, post-transcriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

Association between exposure to farming, allergies and genetic variation in CARD4/NOD1.

BACKGROUND: Caspase recruitment domain protein (CARD) 4 has been recently identified as an intracellular pattern recognition receptor that interacts with muropeptides found in common Gram-negative bacteria. We therefore aimed to explore whether the previously observed inverse association between exposure to microbial products and asthma and allergies in childhood is modified by genetic variation in CARD4. METHODS: We genotyped 668 children [mean age 9.3 (SD 1.5) years] enrolled in the cross-sectional ALEX study for seven haplotype tagging single nucleotide polymorphisms in CARD4. We studied the association of asthma, hay fever and allergen-specific serum immunoglobulin E with exposure to a farming environment and with levels of endotoxin and muramic acid measured in house dust samples. We tested whether these associations differed between the genotypes of the polymorphisms under study. RESULTS: A strong protective effect of a farming environment on allergies was only found in children homozygous for the T allele in CARD4/-21596, but not in children carrying the minor allele (C). Among the former, farmers' children had a significantly lower frequency of sensitization against pollen (5.8%), hay fever (1.7%) and atopic asthma symptoms (1.7%) compared with children not living on a farm (19.4%, 13.0% and 7.6%, P<0.01, <0.01 and <0.05, respectively). Conversely, no significant difference in prevalence of these phenotypes by farming status was found among children with a C allele in CARD4/-21596 (14.3%, 7.1% and 8.0%vs 16.5%, 9.0% and 5.7%, respectively). CONCLUSION: Polymorphisms in CARD4 significantly modify the protective effect of exposure to a farming environment.

The impact of CD14 polymorphisms on the development of soluble CD14 levels during infancy.

CD14 is a receptor involved in the recognition of lipopolysaccharide and other bacterial wall components that may be involved in the balance between infectious and allergic disease and the early polarization towards TH1. Our group has shown an association between polymorphisms in the 5' flanking region of the CD14 gene and plasma soluble CD14 (sCD14) levels at 11 years of age. However, whether this association is present at birth and in infancy remains to be determined. In this study, we measured sCD14 levels in plasma from the umbilical cord (n = 387) and at 3 months (n = 357) and 1 year (n = 312) of age in non-selected healthy infants to assess their relationship with CD14 genotypes at -4190, -2838, -1720 and -260 (relative to translation start site). There was no relation of CD14 genotypes with sCD14 at birth. However, there was a significant association between CD14 genotypes and sCD14 as early as 3 months. Longitudinal analysis suggests that CD14 polymorphisms modulate sCD14 levels up to 1 year of age. This association early in life may have an impact on TH1 polarization and subsequent protection against allergic disease.

Toll-like receptor 6 gene (TLR6): single-nucleotide polymorphism frequencies and preliminary association with the diagnosis of asthma.

Toll-like receptor 6 (TLR6) is one of a series of highly conserved innate immune receptors. We resequenced TLR6 in DNA samples from 24 African Americans, 23 European Americans, and 24 Hispanic Americans, identifying 53 SNPs, 22 with an allele frequency >5%. Significant differences in SNP frequencies among the three populations were noted. In all, 11 SNPs caused amino-acid changes, including one with a frequency >5% in all three populations. Utilizing this SNP (Ser249Pro), we performed exploratory nested case-control disease-association studies, including one involving 56 African Americans with asthma and 93 African American controls. The minor allele of this SNP was associated with decreased risk for asthma (odds ratio 0.38, 95% CI 0.16-0.87, P=0.01), an effect consistent with the known biology of the toll-like receptors. Although replication of this finding in other, larger samples is needed, variation in TLR6 may have relevance to the pathogenesis of immunologically mediated diseases.

Effects of acute and chronic arsenic exposure of human-derived keratinocytes in an In Vitro human skin equivalent system: a novel model of human arsenicism.

An organotypic culture (OTC) of a human keratinocyte cell line (HaCaT) over a human fibroblast-embedded collagen gel was used to model human epidermis in arsenicism, a syndrome that currently lacks valid experimental models. Keratinocytes were exposed acutely or chronically to a mixture of arsenate (0.5 muM), monomethylarsonic acid (MMA; 0.5 muM) and dimethylarsinic acid (DMA; 1.5 muM), or to the individual components of the mixture. OTCs were assayed for microscopic morphology, the proliferating cell marker, Ki-67, labelling and cytokeratin expression. Acute exposures resulted in an epidermal phenotype that accurately modelled early human lesions, including hyperkeratosis, acanthosis and keratin 16 induction. Chronic exposures resulted in a de-differentiated epidermal phenotype with focal nests of keratinocytes growing into the collagen gel. The keratin 8 18 pair was induced by either acute or chronic arsenic exposure, as was the proliferating cell marker, Ki-67. Exposure of keratinocytes to individual arsenic compounds demonstrated that all arsenic mixture-induced changes could be duplicated by exposure to arsenate alone. In contrast, MMA and DMA were inactive. This study establishes OTC as a useful model of arsenicism, and implicates inorganic arsenic as the ultimate carcinogen.

Inhibition of cell-mediated cytolysis and P-glycoprotein function in natural killer cells by verapamil isomers and cyclosporine A analogs.

We have previously shown that among normal leukocytes, CD56+ and CD8+ cells express relatively high levels of P-glycoprotein (P-gp), a transmembrane efflux pump. While the physiologic significance of P-gp expression in leukocytes is unknown, the relatively high levels of P-gp in CD56+ and CD8+ cells suggest that P-gp may function in cell-mediated cytolysis. To explore this possibility we examined the effect of four inhibitors of P-gp efflux [(R)-verapamil (R-ver), (S)-verapamil (S-ver), cyclosporine A (CsA), and PSC833 (PSC)] on both the inhibition of natural killer cell (NK) function and on P-gp efflux. NK function was assayed by measuring the lysis of 51Cr-labeled K562 target cells in the presence and absence of inhibitors. All four P-gp efflux inhibitors inhibited NK-mediated cytolysis in a dose-dependent manner. The stereoisomers of verapamil were more potent inhibitors of cell-mediated cytolysis than the cyclosporines CsA and PSC. In contrast, CsA and PSC were more potent as inhibitors of P-gp-mediated rhodamine 123 dye efflux than the verapamil isomers. Both CsA and PSC maximally inhibited P-gp efflux at 3 microM, but only minimally inhibited cell-mediated cytolysis. The verapamil compounds demonstrated closer correlation between efflux inhibition of NK-mediated cytolysis. The data support a role for P-gp in NK-mediated cytolysis; however, these studies also suggest that the NK cytolytic process is multifaceted and that inhibition of the P-gp-mediated efflux mechanism only partially abrogates this process.

Analysis of multidrug resistance-associated protein (MRP) messenger RNA in normal and malignant hematopoietic cells.

The multidrug resistance-associated protein (MRP) gene is a member of the ATP-binding cassette transporter gene superfamily and may be partially responsible for clinical drug resistance. Reverse transcriptase-polymerase chain reaction was used to measure MRP mRNA in normal hematopoietic cells from bone marrow and peripheral blood as well as patients with high risk acute myelocytic leukemia and multiple myeloma. All normal peripheral blood cells, regardless of cell lineage (CD4, CD8, CD14, CD15, CD19, CD56), expressed a similar basal level of MRP mRNA. Specimens from bone marrow containing mixed lineages also expressed a similar basal level of MRP expression. In patients with acute myelocytic leukemia, 10 of 12 (83%) of the specimens had detectable MRP mRNA, but the level of expression was similar to that of normal blood cells and low compared to a cell line known to overexpress MRP (H69/AR). All myeloma patients (12 of 12) had detectable MRP mRNA expression at levels comparable to normal peripheral blood and bone marrow cells. We conclude that MRP is commonly expressed in normal hematopoietic cells as well as certain hematopoietic malignancies. The therapeutic relevance of MRP expression is unknown, but these studies emphasize the importance of measuring MRP expression in normal cells as a point of reference and comparison for detection in malignant cells. We also recommend obtaining sequential specimens from patients, which may reveal an increased expression of MRP from baseline as the disease progresses and becomes resistant.

P-glycoprotein expression and function in circulating blood cells from normal volunteers.

In contrast to its clearly defined role as a multidrug efflux pump in neoplastic cells, the physiologic function of P-glycoprotein (P-gly) in normal cells is unclear. Recent reports identifying P-gly in normal blood and bone marrow suggest that hematopoietic development or function may be dependent on P-gly. To understand the normal function of P-gly in the blood, its level of expression and function must first be quantitated relative to a known standard. In this study, P-gly, MDR1 gene expression, and P-gly function were quantitated in normal leukocytes. P-gly and MDR1 expression were analyzed in individual leukocyte lineages (T-helper, T-suppressor, monocyte, granulocyte, B-lymphocyte, NK cell) from normal volunteers. P-gly on the cell surface was detected by fluorescent double-labeling for lineage (CD4, CD8, CD14, CD15, CD19, CD56, respectively) and P-gly (MRK16) with analysis by flow cytometry and in some cases immunoblot analysis. MDR1 mRNA analysis on purified lineages was performed using quantitative reverse transcription-polymerase chain reaction. P-gly function was determined for each lineage using dual-labeling for lineage and P-gly substrate (rhodamine 123). The P-gly expressing human myeloma cell line, 8226/Dox6, was used as a reference of comparison for levels of P-gly, MDR1 mRNA, and function. CD56+ cells expressed the highest levels of MDR1 mRNA followed by CD8+ > CD4+ approximately equal to CD15+ > CD19+ > CD14+, with percentage values relative to Dox6 of 49%, 17%, 8%, 8%, 4%, and 2%, respectively. The assays for P-gly immunofluorescence and function correlated well with mRNA analysis except for CD15+ cells (granulocytes), which showed a moderate MDR1 mRNA level with a lack of both function and surface P-gly staining. Granulocyte membranes did show P-gly on immunoblot analysis when probed with either C219 or JSB1. We conclude that (1) P-gly and the MDR1 mRNA are expressed in normal leukocytes, (2) this P-gly expression is lineage specific with relatively high levels among CD56+ cells, and (3) the expression of P-gly in granulocytes is not associated with transport of the P-gly substrate, rhodamine 123, out of the cell.