Comparison of three different brushing techniques to isolate and culture primary nasal epithelial cells from human subjects.

PURPOSE Primary nasal epithelial cells are used for diagnostic purposes in clinical routine and have been shown to be good surrogate models for bronchial epithelial cells in studies of airway inflammation and remodeling. We aimed at comparing different instruments allowing isolation of nasal epithelial cells. METHODS Primary airway epithelial cell cultures were established using cells acquired from the inferior surface of the middle turbinate of both nostrils. Three different instruments to isolate nasal cells were used: homemade cytology brush, nasal swab, and curette. Cell count, viability, time until a confluent cell layer was reached, and success rate in establishing cell cultures were evaluated. A standard numeric pain intensity scale was used to assess the acceptability of each instrument. RESULTS Sixty healthy adults (median with interquartile range [IQR] age of 31 [26-37] years) participated in the study. Higher number of cells (×10(5) cells/ml) was obtained using brushes (9.8 [5.9-33.5]) compared to swabs (2.4 [1.5-3.9], p < 0.0001) and curettes (5.5 [4.4-6.9], p < 0.01). Cell viability was similar between groups. Cells obtained by brushes had the fastest growth rate, and the success rate in establishing primary cell cultures was highest with brushes (90% vs. 65% for swabs and 70% for curettes). Pain was highest with curettes (VAS score 4.0 [3.0-5.0] out of 10). The epithelial phenotype of the cultures was confirmed through cytokeratin and E-cadherin staining. CONCLUSIONS All three types of instruments allow collection and growth of human nasal epithelial cells with good acceptability to study participants. The most efficient instrument is the nasal brush.

The Inflammatory Response of Primary Bovine Mammary Epithelial Cells to Staphylococcus aureus Strains Is Linked to the Bacterial Phenotype

Staphylococcus aureus is a major mastitis-causing pathogen in dairy cows. The latex agglutination-based Staphaurex test allows bovine S. aureus strains to be grouped into Staphaurex latex agglutination test (SLAT)-negative [SLAT(-)] and SLAT-positive [SLAT(+)] isolates. Virulence and resistance gene profiles within SLAT(-) isolates are highly similar, but differ largely from those of SLAT(+) isolates. Notably, specific genetic changes in important virulence factors were detected in SLAT(-) isolates. Based on the molecular data, it is assumed that SLAT(+) strains are more virulent than SLAT(-) strains. The objective of this study was to investigate if SLAT(-) and SLAT(+) strains can differentially induce an immune response with regard to their adhesive capacity to epithelial cells in the mammary gland and in turn, could play a role in the course of mastitis. Primary bovine mammary epithelial cells (bMEC) were challenged with suspensions of heat inactivated SLAT(+) (n = 3) and SLAT(-) (n = 3) strains isolated from clinical bovine mastitis cases. After 1, 6, and 24 h, cells were harvested and mRNA expression of inflammatory mediators (TNF-α, IL-1β, IL-8, RANTES, SAA, lactoferrin, GM-CSF, COX-2, and TLR-2) was evaluated by reverse transcription and quantitative PCR. Transcription (ΔΔCT) of most measured factors was induced in challenged bMEC for 6 and 24 h. Interestingly, relative mRNA levels were higher (P<0.05) in response to SLAT(+) compared to SLAT(-) strains. In addition, adhesion assays on bMEC also showed significant differences between SLAT(+) and SLAT(-) strains. The present study clearly shows that these two S. aureus strain types cause a differential immune response of bMEC and exhibit differences in their adhesion capacity in vitro. This could reflect differences in the severity of mastitis that the different strain types may induce.

Biochanin A and prunetin improve epithelial barrier function in intestinal CaCo-2 cells via downregulation of ERK, NF-κB, and tyrosine phosphorylation

The single-layered gut epithelium represents the primary line of defense against environmental stressors; thereby monolayer integrity and tightness are essentially required to maintain gut health and function. To date only a few plant-derived phytochemicals have been described as affecting intestinal barrier function. We investigated the impact of 28 secondary plant compounds on the barrier function of intestinal epithelial CaCo-2/TC-7 cells via transepithelial electrical resistance (TEER) measurements. Apart from genistein, the compounds that had the biggest effect in the TEER measurements were biochanin A and prunetin. These isoflavones improved barrier tightness by 36 and 60%, respectively, compared to the untreated control. Furthermore, both isoflavones significantly attenuated TNFα-dependent barrier disruption, thereby maintaining a high barrier resistance comparable to nonstressed cells. In docking analyses exploring the putative interaction with the tyrosine kinase EGFR, these novel modulators of barrier tightness showed very similar values compared to the known tyrosine kinase inhibitor genistein. Both biochanin A and prunetin were also identified as potent reducers of NF-κB and ERK activation, zonula occludens 1 tyrosine phosphorylation, and metalloproteinase-mediated shedding activity, which may account for the barrier-improving ability of these isoflavones.

Accumulation of FOXP3+T-cells in the tumor microenvironment is associated with an epithelial-mesenchymal-transition-type tumor budding phenotype and is an independent prognostic factor in surgically resected pancreatic ductal adenocarcinoma.

Here we explore the role of the interplay between host immune response and epithelial-mesenchymal-transition (EMT)-Type tumor-budding on the outcome of pancreatic adenocarcinoma (PDAC).CD4+, CD8+, and FOXP3+T-cells as well as iNOS+ (M1) and CD163+- macrophages (M2) were assessed on multipunch tissue-microarrays containing 120 well-characterized PDACs, precursor lesions (PanINs) and corresponding normal tissue. Counts were normalized for the percentage of tumor/spot and associated with the clinico-pathological features, including peritumoral (PTB) and intratumoral (ITB) EMT-Type tumor-budding and outcome.Increased FOXP3+T-cell-counts and CD163-macrophages and decreased CD8+T-cell-counts were observed in PDACs compared with normal tissues and PanINs (p < 0.0001). Increased peritumoral FOXP3+T-cell-counts correlated significantly with venous invasion, distant metastasis, R1-status, high-grade ITB, PTB and independently with reduced survival. Increased intratumoral FOXP3+T-cells correlated with lymphatic invasion, N1-stage, PTB and marginally with adverse outcome. High peritumoral CD163-counts correlated with venous invasion, PTB and ITB. High intratumoral CD163-counts correlated with higher T-stage and PTB.PDAC-microenvironment displays a tumor-favoring immune-cell composition especially in the immediate environment of the tumor-buds that promotes further growth and indicates a close interaction of the immune response with the EMT-process. Increased peritumoral FOXP3+T-cell density is identified as an independent adverse prognostic factor in PDAC. Patients with phenotypically aggressive PDACs may profit from targeted immunotherapy against FOXP3.

The immune response of bovine mammary epithelial cells to live or heat-inactivated Mycoplasma bovis

Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1β, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites.

Comparison of innate immune responses towards rhinovirus infection of primary nasal and bronchial epithelial cells.

BACKGROUND AND OBJECTIVE Rhinoviruses (RV) replicate in both upper and lower airway epithelial cells. We evaluated the possibility of using nasal epithelial cells (NEC) as surrogate of bronchial epithelial cells (BEC) for RV pathogenesis cell culture studies. METHODS We used primary paired NEC and BEC cultures established from healthy subjects and compared the replication of RV belonging to the major (RV16) and minor (RV1B) group, and the cellular antiviral and proinflammatory cytokine responses towards these viruses. We related antiviral and pro-inflammatory responses of NEC isolated from CF and COPD patients with those of BEC. RESULTS RV16 replication and major group surface receptor (ICAM-1) expression were higher in healthy NEC compared with BEC (P < 0.05); RV1B replication and minor group surface receptor (LDLR) expression were similar. Healthy NEC and BEC produced similar levels of IFN-β and IFN-λ2/3 upon RV infection or after simulation with poly(IC). IL-8 production was similar between healthy NEC and BEC. IL-6 release at baseline (P < 0.01) and upon infection with RV16 (P < 0.05) and poly(IC) stimulation (P < 0.05) was higher in NEC. RV1B viral load in NEC was related to RV1B viral load in BEC (r = 0.49, P = 0.01). There was a good correlation of IFN levels between NEC and BEC (r = 0.66, P = 0.0004 after RV1B infection). IL-8 production in NEC was related to IL-8 production in BEC (r = 0.48, P = 0.02 after RV1B infection). CONCLUSION NEC are a suitable alternative cellular system to BEC to study the pathophysiology of RV infections and particularly to investigate IFN responses induced by RV infection.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

NFkappaB and STAT binding elements participate in regulation of MUC1 expression in normal and transformed mammary epithelial cells

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in several cancers of epithelial origin, including those of breast, pancreas, lung, ovary, and colon. Functions of MUC1 include protection of mucosal epithelium, modulation of cellular adhesion, and signal transduction. Aberrantly increased expression of MUC1 in cancer cells promotes tumor progression through adaptation of these functions. Some regulatory elements participating in MUC1 transcription have been described, but the mechanisms responsible for overexpression are largely unknown. A region of MUC1 5′ flanking sequence containing two conserved potential cytokine response elements, an NFκB site at −589/−580 and a STAT binding element (SBE) at −503/−495, has been implicated in high level expression in breast and pancreatic cancer cell lines. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. ^ T47D breast cancer cells and normal human mammary epithelial cells (HMEC) were used to determine the roles of the κB site and SBE in basal and stimulated expression of MUC1. Treatment of T47D cells and HMEC with interferon-γ (IFNγ) alone enhanced MUC1 expression at the level of transcription, and the effect of IFNγ was further stimulated by tumor necrosis factor-α (TNFα). MUC1 responsiveness to these cytokines was modest in T47D cells but clearly evident in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that the κB site at −589/−580 and the SBE at −503/−495 and were required for cooperative stimulation by TNFα and IFNγ. Electrophoretic mobility shift assays (EMSA) revealed that the synergy was mediated not by cooperative binding of transcription factors but by the independent actions of STAT1α and NFκB p65 on their respective binding sites. Independent mutations in the κB site and SBE abrogated cytokine responsiveness and reduced basal MUC1 promoter activity by 45–50%. However, only the κB site appeared to be constitutively activated in T47D cells, in part by NFκB p65. These findings implicate two cytokine response elements in the 5 ′ flanking region of MUC1, specifically a κB site and a STAT binding element, in overexpression of MUC1 in breast cancer cells. ^

Ontogeny and regulation of interleukin-7 expressing thymic epithelial cells

T cell development is a multistage process of differentiation that depends on proper thymocyte-thymic epithelial cell (TEC) interactions. Epithelial cells in the thymus are organized in a three-dimensional network that provides support and signals for thymocyte maturation. Concurrently, proper TEC differentiation in the adult thymus relies on thymocyte-derived signals. TECs produce interleukin-7 (IL-7), a non-redundant cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We have identified IL-7 expressing TECs throughout ontogeny and in the adult thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the thymic primordium by embryonic day 11.5, in a Foxn1 independent pathway. Marked changes occur in the localization and regulation of IL-7 expressing TECs during development. Whereas IL-7 expressing TECs are present throughout the early thymic rudiment, the majority of IL-7 producing TECs are concentrated in the adult thymic medulla. By analyzing mouse strains that sustain blocks at different stages of thymocyte development, we show that IL-7 expression is initiated independently of hematopoietic-derived signals during thymic organogenesis. However, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Furthermore, distinct thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium. Intraperitoneal injection of Recombination Activating Gene deficient mice (RAG-2−/−) with anti-CD3ϵ monoclonal antibody (mAb) induces CD4− 8− double negative thymocytes to undergo β-selection and differentiate into CD4+8+ cells. Analysis of the thymic stromal compartment reveals that progression through β-selection renders thymocytes competent to alter the pattern of IL-7 expression in the cortical TEC compartment. RAG-2−/− mice do not generate mature T cells and therefore the RAG-2−/− thymus is devoid of organized medullary regions. Histological examination of RAG-2−/− thymus following anti-CD3ϵ stimulation reveals the emergence of mature thymic medullary regions, as assessed by H & E staining and expression of thymic stromal medullary markers. Stromal medullary reorganization occurs in the absence of T cell receptor αβ expression, suggesting that activation of RAG-2−/− thymocytes by CD3ϵ ligation generates thymocyte-derived signals that induce thymic epithelial reorganization, generating a mature medullary compartment. This model provides a tool to assess the mechanisms underlying thymic medullary development. ^

Activation of dendritic cells from patients with epithelial ovarian cancer

Dendritic cells (DCs) are the most potent antigen-presenting cells for inducing immune responses to tumor cells. Lin−HLA-DR + DC populations in peripheral blood mononuclear cells (PBMCs) and in ascites mononuclear leukocytes (MNLs) of patients with epithelial ovarian cancer (EOC) are phenotypically immature. Lin−HLA-DR + DCs from PBMCs of normal subjects and EOC patients and MNLs from ascites cells of patients were examined for specific cell surface markers or indicators of differentiation or activation. Separating Lin− HLA-DR+ DCs into subsets based on their HLA-DR intensity provided an additional method for identifying the two major lineages of DCs, myeloid and plasmacytoid. The activation potential of these DCs following exposure to the maturation agents CD40 ligand (CD40L) and lipopolysaccharide (LPS) was examined by measurement of IL-12 and IL-10 concentrations in DC culture supernatants in addition to their ability to stimulate allogeneic T cells. DCs from PBMCs of normal subjects and EOC patients and DCs isolated from ascites MNLs of EOC patients were separated into subsets based on CD11c and CD123 cell surface marker expression identifying the major DC types. These subsets were then compared with cells sorted on the basis of HLA-DR intensity. The in vivo behavior of DCs and DC subsets in peripheral blood and ascites following treatment of peritoneal carcinoma patients with the growth factor fins-like tyrosine kinase 3 ligand (Flt3L) was also examined. Increases in proportions and total numbers of DCs from peripheral blood and ascites were associated with increased secretion of IL-12 and IL-10 following in vitro activation of cultured DCs. There were differences between DCs from PBMCs and ascites and between DC subsets in expression of cell surface markers, cytokine profile, and the ability of Lin−HLA-DR + cells to stimulate proliferation of allogeneic T cells from EOC patients. These Lin−HLA-DR+ cells have certain functional properties that suggest that they could have the potential to facilitate an adaptive anti-tumor immune response. ^

THE p63 ISOFORM ∆Np63α INHIBITS EPITHELIAL – MESENCHYMAL TRANSITION BY PROMOTING THE EXPRESSION OF MIR-205 IN HUMAN BLADDER CANCER CELLS

p63, a p53 family member, is a transcription factor that has complex roles in cancer. This study focuses on the role of the ∆Np63α isoform in bladder cancer (BC). Epithelial – mesenchymal transition (EMT) is a physiological process that plays an important part in metastasis and drug resistance. At the molecular level, EMT is characterized by the loss of the epithelial marker E-cadherin, and the acquisition of the transcriptional repressors of E-cadherin (ZEB1, ZEB2, TWIST, SNAI1 and SNAI2). Recent publications highlight the role of microRNAs belonging to the miR-200 family and miR-205 in preventing EMT through suppression of ZEB1 and ZEB2. p53, the homologue of p63, is implicated in regulating EMT by modulating the expression of miR-200c; however, the mechanisms underlying miR-205 control remain unclear. Here we show that ∆Np63α regulates the transcription of miR-205 and controls EMT in human BC cells. We observed a strong correlation between the expression of ∆Np63α, miR-205 and E-cadherin in a panel of BC cell lines (n=28) and also in bladder primary tumors from a cohort of patients (n=98). A remarkably inverse correlation is observed between ∆Np63α and ZEB1/2 in cell lines. Stable knockdown (KD) ∆Np63α in UC6, an “epithelial” BC cell line, decreased the expression of miR-205 and induced ZEB1/2 expression, the effects that were reversed by expression of exogenous miR-205. Moreover, overexpressing ∆Np63α in UC3, a “messenchymal” BC cell line, brought about opposite results, an increase in miR-205 expression and a reduction in ZEB1/2 expression. Modulation of ∆Np63α expression resulted in a parallel change in the expression of miR-205 and miR-205 “host” gene (miR-205HG). Nuclear run-on and chromatin immunoprecipitation experiments demonstrated that ∆Np63α regulates the transcription of miR-205 through controlling the recruitment of RNA Polymerase II to the promoter of miR-205HG. Interestingly, high miR-205 expression correlated with poor clinical outcome in BC patients, consistent with our recent publication highlighting the enrichment of ∆Np63 in a lethal subset of muscle invasive BC. In summary, our data present the important roles of ∆Np63α in preventing EMT mediated by miR-205. Our study also identifies miR-205 as a potential molecular marker to predict clinical outcome in BC patients.

Growth regulatory role of R -PTPmu in normal and neoplastic epithelial cells

Cellular oncogenes and tumor suppressor genes regulate cellular adhesion and proliferation, two important events in malignant transformation. Even though receptor-like protein tyrosine phosphatases (R-PTPs) can influence these events, their role in malignant transformation has not been studied. The major goal of this study was to determine whether downregulation of R-PTP$\mu$ expression in lung epithelial cells is associated with or causal to neoplastic transformation. Examination of R-PTP$\mu$ expression in normal and carcinoma cells demonstrated that lung epithelial cells expressed R-PTP$\mu$ whereas lung carcinoma cells did not, and that incubation with TGF-$\alpha$ and HGF induced a two fold increase in R-PTP$\mu$ mRNA expression. To associate the expression of R-PTP$\mu$ with neoplastic transformation, we transfected lung epithelial cells with the H-ras oncogene. Transformation resulted in the activation of the MAPK signal transduction pathway, the hyperphosphorylation of c-met, and the production of HGF. Upon analysis of R-PTP$\mu$ expression, we observed a significant decrease in R-PTP$\mu$ mRNA and protein levels suggesting that transformation can directly or indirectly downregulate the expression of R-PTP$\mu.$ TGF-$\beta$ reversed the H-ras transformed phenotype, an event directly correlated with upregulation of R-PTP$\mu.$ To provide a casual relationship between R-PTP$\mu$ and cessation of tumor cell growth, we transfected carcinoma cells with the wild type R-PTP$\mu$ cDNA. Transiently expressing cells were selected by FACS using the mAb 3D7 and plated into individual wells. Carcinoma cells positive for R-PTP$\mu$ expression did not grow into colonies whereas non-R-PTP$\mu$ expressing carcinoma cells did, suggesting that expression of R-PTP$\mu$ arrested cell growth. To better understand the growth arrest induced by R-PTP$\mu$, we transfected the H-ras transformed lung epithelial cell line (MvLu-1-ras) with R-PTP$\mu$ (MvLu-1-ras/R-PTP$\mu$). Examination of growth factor receptor phosphorylation revealed significant inhibition of c-met and EGF-R. Furthermore, these cells underwent apoptosis in the absence of serum. Taken together the data demonstrate that the downregulation of R-PTP$\mu$ expression is an important step in neoplastic transformation of lung epithelial cells and that its presence can induce apoptosis and inhibit the signaling of c-met and EGF-R, two major growth factor receptors in lung carcinoma. In conclusion, the expression of R-PTP$\mu$ is inversely correlated with neoplastic transformation, growth and survival of tumor cells. ^