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Neutrophils express functional toll-like receptor2 (TLR2) and 4 (TLR4) that are crucial for the production of inflammatory cytokines. Here, we show that dimethylsulfoxide-induced neutrophil-like differentiation of promyelocytic HL-60 cells (dHL-60) results in cells that respond to TLR2 and TLR4 ligands similarly to primary neutrophils. Consistent with the increased responsiveness of the cells to TLR2 ligand, the TLR2 gene was strongly up-regulated in dHL-60 cells. On the other hand, increased surface expression of LPS receptor complex, TLR4/MD2/CD14, was observed without affecting TLR4 gene expression. Thus, the data demonstrate a higher responsiveness of dHL-60 cells to TLR2 and TLR4 ligands because of increased TLR2 and MD2/CD14 gene expression.

Oligonucleotides can mediate sequence-specific gene modification that results in the correction and/or alteration of genomic DNA. There is evidence to suggest that the polymerase chain reaction (PCR)-based analytical methods usually used to analyze oligonucleotide-mediated modification can generate artifacts. To investigate the conditions under which a PCR artifact can be generated and eliminated when analyzing small fragment homologous replacement (SHFR)-mediated modification, cells homozygous for the DeltaF508 mutation (CFBE41o-) were mixed with small DNA fragments (SDFs) containing the wild-type CFTR (wt-CFTR) sequence. An artifact could be generated after wild-type allele-specific PCR (wtAS-PCR) if the genomic DNA was not gel purified. Without gel purification, the amount of SDF/cell required to generate the artifact was dependent to the AS primer pairs used. When the genomic DNA was gel purified, no artifact could be detected with any of the wtAS-PCR primers whether the SDF was mixed with the cells or transfected into the cells. Furthermore, treatment of cellular mRNA with DNase was sufficient to eliminate potential artifacts in the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, it is critical to gel purify genomic DNA and DNase treat mRNA when analyzing SFHR-mediated modification by PCR.

Cystic fibrosis (CF) is the most common lethal inherited disorder caused by mutation in the gene encoding CF transmembrane regulator (CFTR). The clinical course of CF is characterized by recurrent pulmonary infections and chronic inflammation. Here, we show that toll-like receptor-2 (TLR2) expression and response were strongly enhanced in the human CF bronchial epithelial cell line, CFBE41o-. Treatment of the cells with 5-azacytidine decreased the promoter methylation within TLR2 proximal promoter and increased endogenous expression of TLR2 in non-CF 16HBE14o- cells, suggesting that TLR2 expression is epigenetically regulated by CpG methylation. Moreover, bisulfite sequence analysis revealed that TLR2 promoters were highly demethylated in CFBE41o- cells, implying that decreased methylation of the TLR2 promoter is responsible for CF-related up-regulation of TLR2. Finally, stable expression of WT-CFTR in CFBE41o- cells (CFBE41o-/WT-CFTR cells) reduced TLR2 expression and the response to its ligand peptidoglycan (PGN), implying a causal relationship between CFTR dysfunction and TLR2 up-regulation. Consistent with reduced expression of TLR2 in CFBE41o-/WT-CFTR cells, CpG methylation was increased in CFBE41o-/WT-CFTR cells. Taken together, our results demonstrate that TLR2 expression is epigenetically up-regulated in CF bronchial epithelial cells and suggest that TLR2 overexpression or prolonged activation of TLR2 signaling might be critical in CF pathogenesis.

We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).

The generation of a pig model of cystic fibrosis (CF) is a multistep process. Initial steps in this process involved the design and cloning of a small DNA fragment (SDF) or large oligodeoxynucleotide (LODN) that contains the F508del mutation and a silent restriction fragment length polymorphism causing mutation. This SDF/LODN was transfected into wild-type (WT) pig fetal fibroblast with the intention of modifying the pig genomic DNA by small fragment homologous replacement (SFHR). The targeted deletion (F508del) was detected in a subpopulation of transfected cells by allele-specific polymerase chain reaction (AS-PCR).

Cultured airway epithelial cells are widely used in cystic fibrosis (CF) research as in vitro models that mimic the in vivo manifestations of the disease and help to define a specific cellular phenotype. Recently, a number of in vitro studies have used an airway adenocarcinoma cell line, Calu-3 that expresses submucosal gland cell features and significant levels of the wild-type CFTR mRNA and protein. We further characterized previously described CF tracheobronchial gland cell lines, CFSMEo- and 6CFSMEo- and determined that these cell lines are compound heterozygotes for the F508del and Q2X mutations, produce vestigial amounts of CFTR mRNA, and do not express detectable CFTR protein. Electrophysiologically, both cell lines are characteristically CF in that they lack cAMP-induced Cl- currents. In this study the cell lines are evaluated in the context of their role as the CF correlate to the Calu-3 cells. Together these cell systems provide defined culture systems to study the biology and pathology of CF. These airway epithelial cell lines may also be a useful negative protein control for numerous studies involving gene therapy by cDNA complementation or gene targeting.

An ultimate goal of gene therapy is development of efficient methods to correct mutant genomic sequences in pathologic cells. In this study SFHR; an oligonucleotide-based gene targeting strategy; was used to convert A|[srarr]| T in codon 6 of the |[beta]|-globin gene of CD34+ Lin- Hematopoietic Stem/Progenitor Cells (HSPCs) isolated from full term umbilical cord blood. HSPCs were transfected with a defined number of a 559-bp small DNA fragment (SDF) using the AMAXA Nucleofection/Electroporation system. After growing the transfected cells in stem cell media containing EPO for different time intervals up to 32 days, RNA was extracted and DNase I-treated before further analysis. RFLP analysis of a 430-bp PCR product generated from mRNA-derived cDNA with the DdeI enzyme indicated |[beta]|A- to |[beta]|S-globin conversion. Sequencing of the 430-bp amplicon also showed the conversion A|[srarr]| T. These data are consistent with previous studies showing both conversion of |[beta]|S- to |[beta]|A-globin in SC1 cells and |[beta]|A- to |[beta]|S-globin in HSPCs after electroporation and microinjection of SDF, respectively. These studies represent a critical next step in developing SFHR as a therapy for sickle cell disease, in that , they demonstrate long-term SFHR-mediated modification of |[beta]|-globin following mass transfection by electroporation of HSPCs. This work is supported by an NIH Fellowship Training Grant DK007636 and the CPMC Research Foundation.

Top of pageAbstract The development of immortalized airway epithelial cells has been a significant contribution to the elucidation of the biochemical and molecular mechanisms that underlie cystic fibrosis (CF) pathology and in the development of CF therapies. The different cell systems have been employed in genetic complementation studies as well as the characterization of CF transmembrane conductance regulator (CFTR) related metabolic pathways. An important feature of CF cells complemented with wild-type (WT)-CFTR is reversal of the cAMP-dependent Cl ion transport defect in these cells. Such complementation studies have led to the understanding of numerous relationships between CFTR function and specific biochemical pathways. However, there is very little known about the affect of the degree of CFTR expression on CFTR related functions. In particular, the level of vector-driven WT-CFTR expression has not been well compared to |[Delta]|F508-CFTR expression driven by the same expression vector in airway epithelial cells. Furthermore, many of the complemented CF cells have been complemented with CFTR cDNA that is only comprised of the |[sim]|4.7 kB open reading frame (ORF) rather that the entire |[sim]|6.2 kB CFTR cDNA comprised of not only the ORF, but also the 3' and 5 ' untranslated regions (UTR). The studies presented describe CF airway epithelial cell culture systems that have been complement with the 4.7 kB and 6.2 kB WT-CFTR as well as the 4.7 kB |[Delta]|F508-CFTR under the regulation of a cytomeglovirus (CMV) promoter and cloned into an episomal expression vector. Several different cell lines that are either homozygous for the |[Delta]|F508 mutation or are compound heterozygotes for the |[Delta]|F508 and Q2X alleles have been developed and evaluated for their ion transport characteristics as it relates to their CFTR expression levels. Of particular note, is the relationship between the degree of |[Delta]|F508-CFTR expression and level of the levels cAMP-dependent Cl transport observed. In one set of cells complemented with the |[Delta]|F508-CFTR that is expressed at levels 5-10 fold higher than those observed for WT-CFTR (6.2kB), appeared to result in |[sim]|25% of cAMP-dependent Cl transport observed for the WT-CFTR. The correlation between the expression of the 6.2 kB or the 4.7 kB WT-CFTR and ion transport appear, at present, to be associated with the plasmid number in the cells. Further studies are underway to more fully correlate the degree of CFTR expression with the ion transport characteristics of the complemented cells. These studies will be important in assessing the degree of transgene expression required to achieve and maintain wild-type function in mutant cells.